DNA polymerase variants

Abstract

The present disclosure relates to engineered DNA polymerase polypeptides and compositions thereof, and polynucleotides encoding the engineered DNA polymerase polypeptides. The present disclosure also provides methods of using the engineered DNA polymerase polypeptides, or compositions thereof, for diagnostic and other purposes.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 383,053, filed on November 9, 2022, which is incorporated herein by reference.

[0003] Reference to a sequence listing, table or computer program

[0004] The sequence listing, having a file size of 4,056,133 bytes, created on November 8, 2023, and filed concurrently herewith as the file name CX9-227WO1_ST26.xml, is part of the specification and is incorporated herein by reference. Technical Field

[0005] The present disclosure provides engineered DNA polymerase polypeptides and compositions thereof, as well as polynucleotides encoding engineered DNA polymerase polypeptides. The present disclosure also provides methods of using recombinant DNA polymerases or compositions thereof for diagnostics, molecular biology tools, and other purposes. Background Art

[0006] DNA polymerase is the enzyme that synthesizes DNA from deoxyribonucleotide.These enzymes are essential for dna replication.There are various types of DNA polymerases, which show different properties and are found in different types of organisms.The polymerase obtained from thermophilic organisms has been widely used in various in vitro methods, including but not limited to polymerase chain reaction (PCR), nucleic acid sequencing and other diagnosis, molecular biology and forensic application.Although there are numerous commercially available thermostable DNA polymerases, such as Taq and Pfu DNA polymerases, this area still needs to have a thermostable enzyme that improves properties, such as sensitivity, processability and / or fidelity of enhanced properties. Summary of the Invention

[0007] The present disclosure provides engineered DNA polymerase polypeptides and compositions thereof, as well as polynucleotides encoding engineered DNA polymerase polypeptides. The present disclosure also provides methods of using the engineered DNA polymerase polypeptides and compositions thereof for diagnostic and other purposes.

[0008] In one aspect, the present disclosure provides an engineered DNA polymerase or a functional fragment thereof, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 220, 226, 272, 328, 546, 710 or 1208, or to a reference sequence corresponding to SEQ ID NO: 2, 220, 226, 272, 328, 546, 710 or 1208, wherein the amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 220, 226, 272, 328, 546, 710 or 1208, or to a reference sequence corresponding to SEQ ID NO: One or more mutations of the reference sequence of NO: 2, 220, 226, 272, 328, 546, 710 or 1208.

[0009] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2.

[0010] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, 226, 272, 328, 546, 710, or 1208, or a reference sequence corresponding to SEQ ID NO: 220, 226, 272, 328, 546, 710, or 1208, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0011] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 4-1378, or to a reference sequence corresponding to an even numbered SEQ ID NO: 4-1378, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO: 2.

[0012] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises mutations at at least the following amino acid positions: 2, 3, 21, 39, 51, 59, 66, 68, 72, 73, 78, 79, 87, 124, 147, 178, 181, 185, 199, 216, 224, 233, 239, 241, 251, 257, 262, 271, 275, 288, 291, 303, 316, 317, 327, 328, 336, 338, 341, 342, 351, 356, 359, 362, 365, 368, 370, 371, 372. 2、373、374、376、377、378、379、380、381、382、388、390、392、393、395、396、398、399、402、403、404、409、410、412、413、416、417、418、420、425、426、427、429、430、431、432、434、435、437、438、442、443、447、448、449、450、451、453、453、454、457、461、465、466、469、473、 474, 475, 477, 478, 479, 482, 483, 488, 490, 503, 508, 514, 515, 516, 526, 527, 528, 529, 536, 541, 542, 544, 545, 550, 554, 557, 558, 559, 562, 569, 575, 577, 578, 579, 589, 594, 601, 603, 604, 612, 613, 641, 644, 651, 656, 660, 662, 663, 664, 667, 671, 677, 685, 687, 691, 69 792, 798, 804, 807, 808, 809, 812, 815, 820, 824, 825 or 831 , or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0013] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation at amino acid position 478, 515, 526, 527, or 528, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0014] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation at amino acid position 402, 478, 515, 526, 527, 528, 559, 604, 660, or 760, or a combination thereof, wherein the positions are relative to SEQ ID NO: 2.

[0015] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation at amino acid position 478, 515, 526, 527, 528, 559, or 760, or a combination thereof, wherein the positions are relative to SEQ ID NO: 2.

[0016] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation at amino acid position 147, 257, 291, 395, 402, 474, 475, 478, 514, 515, 526, 527, 528, 536, 559, 604, 660, 662, 687, 745, or 760, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0017] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation or set of mutations at the following amino acid positions: 541 / 554 / 557, 516 / 541 / 550 / 554 / 557, 478 / 514 / 526 / 527, 478 / 516 / 526 / 527 / 529 / 541, 288 / 478 / 514 / 516 / 541 / 557, 288 / 478 / 516 / 541 / 550 / 554 / 557, 550 / 554 / 557, 478 / 399 / 514 / 516, 399 / 516 / 526 / 528 / 529 / 554 / 557, 478 / 514 / 541 / 550 / 554 / 557. 1. 288 / 399 / 514 / 554 / 557, 478 / 514 / 554 / 557, 288 / 478 / 526 / 550 / 554, 478 / 541 / 550 / 557, 541, 288 / 514 / 526 / 541, 554 / 557, 399 / 478, 288 / 514 / 554 / 557 57, 478 / 514 / 516 / 526 / 527 / 529 / 557, 399 / 541 / 550 / 554, 516 / 529 / 541 / 554 / 557, 478 / 541 / 554 / 557, 288 / 478 / 526 / 528 / 529 / 554 / 557, 288 / 514 / 516 / 5 29 / 541、478 / 514 / 516 / 541、478 / 514 / 557、399 / 541 / 557、399 / 541、288 / 399 / 516 / 527 / 541 / 557、288 / 478 / 514 / 541、288 / 514 / 516、664 / 698、399 / 478 / 516 / 541、399 / 528 / 550、399 / 478 / 514 / 541 / 557、288 / 478 / 514 / 516、224 / 664、478 / 526 / 527 / 528、288 / 478 / 514 / 516 / 541 / 550 / 554、478 / 514 / 541 / 554、 478 / 516, 478 / 514 / 516 / 541 / 554 / 557, 288 / 399 / 478 / 516 / 541, 288 / 478 / 514 / 554 / 557, 288 / 478 / 516, 288 / 399 / 516 / 554 / 557, 550 / 554, 288 / 550 / 554 、288 / 399 / 478 / 516 / 550 / 554、399 / 514 / 541 / 550 / 554、288 / 478 / 514 / 554、199、288 / 399 / 529 / 541、399 / 478 / 554、516 / 528 / 541 / 554 / 557、39 / 199 / 698、399 / 478 / 526 / 528 / 529、288 / 399 / 514 / 541 / 550 / 554、288 / 541、288 / 478 / 514 / 550 / 554 / 557、288 / 399 / 478 / 516、526 / 529 / 554 / 557、288 / 478 / 557、288 / 478 / 514 / 550 / 554、478 / 541 / 550 / 554、516 / 526 / 528、288 / 526 / 528 / 550 / 554 / 557 、199 / 664 / 698、514 / 526 / 528 / 529 / 550 / 557、288、399 / 554 / 557、288 / 554 / 557、399 / 514 / 541、288 / 478 / 514 / 527 / 554 / 557、399 / 478 / 516 / 541 / 554、87 / 541 / 554、288 / 550 / 554 / 557、527 / 529 / 541、528 / 529 / 554、478 / 554 / 557、478 / 514 / 516 / 541 / 550 / 554 / 557、199 / 216 / 644 / 664、396 / 478 / 514 / 554 / 557 / 694、241 / 644 / 663 / 664、399 / 478 / 554 / 557、288 / 399 / 550、288 / 516 / 526 / 527 / 528 / 557、399 / 526 / 527 / 528 / 529、288 / 478 / 526、399 / 478 / 514 / 516 / 526 / 527 / 528 / 529 / or 478 / 514 / 526 / 527 / 528, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0018] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation or set of mutations at an amino acid position of the mutations or sets of mutations provided in Tables 4.1, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 9.3, 10.1, 10.2, 11.1, and 11.2, wherein the positions are relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation or set of mutations at an amino acid position of the mutations or sets of mutations provided in Tables 4.1, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 9.3, 10.1, 10.2, 11.1, and 11.2, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

[0019] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, 226, 272, 328, 546, 710 or 1208, or to a reference sequence corresponding to SEQ ID NO: 220, 226, 272, 328, 546, 710 or 1208, wherein the amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, 226, 272, 328, 546, 710 or 1208, or to a reference sequence corresponding to SEQ ID NO: One or more mutations of the reference sequence of NO: 220, 226, 272, 328, 546, 710 or 1208.

[0020] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 4-1378, or a reference sequence corresponding to an even numbered SEQ ID NO: 2-1378, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, 226, 272, 328, 546, 710, or relative to the reference sequence corresponding to SEQ ID NO: 220, 226, 272, 328, 546, 710.

[0021] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises mutations at at least the following amino acid positions: 2, 3, 21, 39, 51, 59, 66, 68, 72, 73, 78, 79, 87, 124, 147, 178, 181, 185, 199, 216, 224, 233, 239, 241, 251, 257, 262, 271, 275, 288, 291, 303, 316, 317, 327, 328, 336, 338, 341, 342, 351, 356, 359, 362, 365, 368, 370, 371, 372. 2、373、374、376、377、378、379、380、381、382、388、390、392、393、395、396、398、399、402、403、404、409、410、412、413、416、417、418、420、425、426、427、429、430、431、432、434、435、437、438、442、443、447、448、449、450、451、453、453、454、457、461、465、466、469、473、 474, 475, 477, 478, 479, 482, 483, 488, 490, 503, 508, 514, 515, 516, 526, 527, 528, 529, 536, 541, 542, 544, 545, 550, 554, 557, 558, 559, 562, 569, 575, 577, 578, 579, 589, 594, 601, 603, 604, 612, 613, 641, 644, 651, 656, 660, 662, 663, 664, 667, 671, 677, 685, 687, 691, 69 1, 694, 697, 698, 699, 701, 702, 704, 706, 707, 708, 713, 714, 715, 716, 718, 721, 723, 724, 725, 728, 731, 732, 735, 740, 742, 743, 745, 747, 748, 749, 753, 758, 760, 762, 768, 769, 770, 787, 792, 798, 804, 807, 808, 809, 812, 815, 820, 824, 825 or 831 or a combination thereof, wherein the position is relative to the position corresponding to SEQ Reference sequence of ID NO: 220, 226, 272, 328, 546, 710 or 1208.

[0022] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, or to a reference sequence corresponding to SEQ ID NO: 220, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, or to a reference sequence corresponding to SEQ ID NO: 220.

[0023] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 222-268, or to a reference sequence corresponding to an even numbered SEQ ID NO: 222-268, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, or to the reference sequence corresponding to SEQ ID NO: 220.

[0024] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation or set of mutations at amino acid position 515, 594, 559, 760, 541, 753, 402, 398, 758, 399, 575, 257 / 758, 558, 601, 762, 589, or 544, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 220.

[0025] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 226, or to a reference sequence corresponding to SEQ ID NO: 226, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 226, or to a reference sequence corresponding to SEQ ID NO: 226.

[0026] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 270-320, or to a reference sequence corresponding to SEQ ID NO: 270-320, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 226, or to the reference sequence corresponding to SEQ ID NO: 226.

[0027] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation or set of mutations at the following amino acid positions: 515 / 760, 514 / 515 / 760, 478 / 515 / 760, 660, 178, 402 / 515, 377 / 515, 515, 514 / 515, 402, 503, 239, 73, 528 / 541, 59, 515 / 575, 3 / 559, 377 / 515 / 528 / 541 / 554 / 557, 402 / 514 / 515 / 541 / 559, 514 / 515 / 541 / 559, 514 / 515 / 559, 515 / 559, 515 / 541 / 559, 377 / 515 / 559, 402 / 515 / 559 or 515 / 541 / 557, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 226.

[0028] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 272, or to a reference sequence corresponding to SEQ ID NO: 272, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 272, or to a reference sequence corresponding to SEQ ID NO: 272.

[0029] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 322-534, or a reference sequence corresponding to an even numbered SEQ ID NO: 322-534, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 272, or relative to the reference sequence corresponding to SEQ ID NO: 272.

[0030] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises a mutation or set of mutations at at least the following amino acid positions: 402 / 528 / 541 / 660, 257 / 604 / 660, 178 / 257 / 402 / 404 / 528 / 557 / 660, 402 / 604 / 660, 257 / 402 / 541 / 660, 377 / 402 / 541 / 660, 257 / 377 / 402 / 660, 483 / 660, 257 / 402 / 257 / 377 / 402 / 660, 178 / 257 / 402 / 404 / 528 / 557 / 660. 77 / 402 / 403 / 483 / 604 / 660、377、178 / 402 / 403 / 528 / 541 / 660、377 / 601 / 660、178 / 377、377 / 490 / 660、402 / 403 / 483 / 554 / 557、178 / 257 / 377 / 558 / 660、257 / 402 / 403 / 483 / 660、178 / 554 / 557 / 558 / 660、554 / 557 / 660、178 / 483 / 541 / 545 / 554 / 557 / 604 / 660、792、402 / 403 / 483 / 60 4 / 660, 178, 474, 257 / 377 / 541 / 604, 743, 396, 479, 377 / 403 / 541 / 604, 475, 178 / 257 / 403 / 528, 448, 577, 395, 769, 691, 677, 704, 377 / 541 / 575 / 604 / 660, 178 / 257 / 601 / 604, 685, 377 / 604 / 660, 749, 482, 291, 579, 178 / 541 / 550 / 604, 477, 178 / 257, 257 / 402 / 490 / 528 / 541 / 660 , 328, 740, 368, 721 / 745, 578, 687, 541 / 601 / 604, 257 / 528, 257 / 528 / 554, 768, 770, 257 / 377 / 483 / 541, 542, 257 / 402 / 403, 257 / 403 / 541, 604, 402 / 604, 257 / 316 / 541 / 575 / 604, 327, 403 / 541 / 604 / 660, 257 / 377 / 403 / 483 / 541 / 601 / 604 or 402 / 528 / 541 / 601, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 272.

[0031] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 328, or to a reference sequence corresponding to SEQ ID NO: 328, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 328, or to a reference sequence corresponding to SEQ ID NO: 328.

[0032] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 536-674, or a reference sequence corresponding to an even numbered SEQ ID NO: 536-674, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 328, or relative to the reference sequence corresponding to SEQ ID NO: 328.

[0033] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation or set of mutations at the following amino acid positions: 79, 257 / 474, 677, 562, 178 / 395 / 745, 291 / 395 / 687 / 745, 257 / 395 / 677, 303 / 338, 474 / 475, 359, 474 / 475 / 6 77 / 704, 395 / 687, 671, 233, 699, 379, 257 / 792, 257 / 395, 21, 257 / 448 / 474 / 475, 713, 641, 442, 656, 475 / 704, 742, 667, 257 / 448, 257 / 316 / 395 / 482 / 687 / 745, 804, 70 4. 178 / 257 / 395 / 482 / 579 / 687 / 745, 257 / 579, 257 / 291 / 395 / 475 / 541 / 687, 257 / 768, 812, 475, 662, 536, 748, 808, 291 / 316 / 395 / 687, 613, 257 / 291 / 316, 257 / 291 / 57 9, 257 / 316 / 541 / 745, 351, 257 / 316 / 395 / 745, 185, 257 / 316 / 687, 291 / 316 / 475 / 687 / 745, 257 / 291 / 395 / 740 / 745, 450, 473, 124, 430, 413, 66 or 365, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 328.

[0034] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 546, or to a reference sequence corresponding to SEQ ID NO: 546, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 546, or to a reference sequence corresponding to SEQ ID NO: 546.

[0035] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 676-1190, or to a reference sequence corresponding to an even numbered SEQ ID NO: 676-1190, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 546, or to the reference sequence corresponding to SEQ ID NO: 546.

[0036] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation or set of mutations at the following amino acid positions: 438, 697, 457, 716, 79 / 474 / 536 / 699, 536, 392, 708, 431, 395 / 474 / 536 / 699, 453, 427, 79 / 395 / 699, 378, 370, 706, 257 / 395 / 474 / 475 / 536 / 662, 474 / 536 / 562, 257 / 395 / 474 / 536, 79 / 257 / 395 / 474 / 475 / 536 / 562, 79 / 395 / 536 / 699, 372, 257 / 395 / 792, 390, 79 / 474 / 536 / 699 / 792, 409, 443, 448, 381, 707, 416, 474 / 475 / 662, 79 / 395 / 474 / 475 / 536 / 792, 474 / 475 / 536 / 662, 382, 257 / 474 / 536 / 562 / 66 2. 257 / 395 / 474 / 475 / 792, 79 / 474 / 662 / 699, 725, 447, 79 / 257 / 699, 701, 257 / 474 / 475 / 536 / 562 / 662, 702, 342 / 716, 233 / 562 / 713, 704, 474 / 475 / 536 / 562 / 662、454、327 / 427、257 / 316 / 474 / 536 / 792、417、425、536 / 562、714、233、303 / 613、21 / 79 / 303 / 562 / 613、79 / 562、435、257 / 536 / 699 / 792、79 / 536、 453 / 667、79 / 303 / 338 / 613 / 804 / 812、79 / 257 / 395 / 536 / 662 / 792、79 / 338 / 562、79 / 233 / 303 / 662、79 / 662 / 699、79 / 395 / 475 / 662、79 / 536 / 562 / 699、536 / 662、21 / 79 / 338 / 641 / 699、662、21 / 79 / 442 / 699、79 / 303 / 662、79 / 178 / 257 / 662 / 699、79 / 257 / 536 / 562 / 662、562 / 699、359 / 613 / 742 / 808 / 812、21 / 79 / 338 / 379 / 562 / 662 / 804 / 812、731、21 / 79 / 359 / 442 / 662、79 / 338 / 359 / 562、21 / 613 / 662 / 742、21 / 79 / 303 / 662 / 699 / 742 / 808、21 / 79 / 338 / 442 / 613 / 699、79 / 303 / 338 / 562 / 613 / 662、303 / 338 / 562 / 699 / 808 / 812、79 / 257、257 / 662 / 792、79 / 536 / 699、79 / 359 / 713 / 742 / 808、21 / 359 / 442 / 808 / 812、79 / 257 / 475 / 536 / 662、21 / 79 / 338、79 / 713、79 / 562 / 641、373、79 / 178 / 257 / 316 / 536 / 662、21 / 233 / 338 / 562 / 662、257 / 316 / 536、21 / 79 / 303 / 442 / 662、21 / 79 / 233 / 303 / 338 / 379 / 562 / 713、338 / 442 / 699、426、21 / 338 / 359 / 808 / 812、79 / 178 / 233 / 442 / 562 / 812、79 / 474 / 536 / 662 / 699、21 / 79 / 338 / 379 / 662、721、21 / 79 / 233 / 338 / 613 / 641、374、338 / 359 / 379、461、451、715、79 / 303 / 338 / 641 / 713、393、79 / 257 / 474 / 536 / 699、79、437、257 / 536 / 562 / 662、21 / 79 / 379 / 442 / 562 / 662、362、410、338 / 812、21 / 662 / 808 / 812、21 / 303 / 338、418、338 / 442、449、379 / 641、316 / 536 / 562、257 / 536 / 792、21 / 79 / 442 / 613、257 / 536 / 662 / 792、257 / 474 / 529 / 536 / 662、412、79 / 257 / 536 / 662 / 699、257 / 316 / 475 / 536 / 792、388、380 / 454、21 / 79 / 233 / 303 / 613 / 699 / 809、724、21 / 79 / 338 / 613、79 / 178 / 536 / 792、449 / 747、21 / 79 / 379 / 613 / 713、79 / 316 / 536 / 662 / 699、376、21 / 562 / 662、79 / 257 / 262 / 536 / 562 / 699、718、395 / 475 / 536 / 562 / 662 / 699、79 / 233 / 338 / 359 / 379 / 442 / 562 / 613 / 812、371、316 / 475 / 562 / 662 / 792、79 / 257 / 316 / 792、728、79 / 233 / 338、432、79 / 178 / 257 / 316 / 395 / 536 / 662 / 699、79 / 303 / 713, 233 / 379 / 442 / 562 / 613 / 662 / 713, 21 / 742, 732, 79 / 257 / 395 / 562 / 662 / 699, 21 / 233 / 338 / 379 / 442 / 562 / 613 / 662 / 812, 429, 562 / 699, 2 / 374, 723, 434, 420 or 699, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 546.

[0037] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 710, or to a reference sequence corresponding to SEQ ID NO: 710, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 710, or to a reference sequence corresponding to SEQ ID NO: 710.

[0038] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 1192-1326, or a reference sequence corresponding to an even numbered SEQ ID NO: 1192-1326, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710.

[0039] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises a mutation or set of mutations at at least amino acid position(s): 68, 78, 466, 807, 612, 147, 356, 488, 603, 271, 508, 787, 412 / 417 / 427, 251, 697, 562, 79 / 434 / 562, 820, 275, 378 / 381 / 697, 469, 412 / 417 / 697, 798, 79 / 388, 651, 825, 51, 370, 745, 815, 465, 317, 569, 341, 824, 18, 181, 735, or 336, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 710.

[0040] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 1208, or to a reference sequence corresponding to SEQ ID NO: 1208, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 1208, or to a reference sequence corresponding to SEQ ID NO: 1208.

[0041] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 1328-1378, or to a reference sequence corresponding to an even numbered SEQ ID NO: 1328-1378, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 1208, or to the reference sequence corresponding to SEQ ID NO: 1208.

[0042] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation or set of mutations at the following amino acid positions: 68 / 251 / 603 / 612 / 798, 68 / 603, 68 / 78 / 370, 370, 251 / 798, 68 / 370 / 603 / 612, 51 / 370 / 412 / 603 / 612, 412 / 603 / 798, 251 / 370 / 412 / 612, 370 / 798, 78 / 251, 251, 68 / 370 / 508 / 798, 251 / 370 / 508 / 798. or 51 / 68 / 251 / 370 / 603, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 1208.

[0043] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one mutation provided in Tables 4.1, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 9.3, 10.1, 10.2, 11.1, and 11.2, wherein the position of the mutation is relative to the reference sequence of SEQ ID NO: 2, 220, 226, 272, 328, 546, 710, or 1208 as provided in the corresponding Tables.

[0044] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation or set of mutations provided in Tables 4.1, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 9.3, 10.1, 10.2, 11.1, and 11.2, wherein the position of the mutation or set of mutations is relative to a reference sequence of SEQ ID NO: 2, 220, 226, 272, 328, 546, 710, or 1208 as provided in the corresponding Tables.

[0045] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to residues 12 to 844 of the even-numbered SEQ ID NOs among SEQ ID NOs: 4-1378, or the sequence corresponding to the even-numbered SEQ ID NOs among SEQ ID NOs: 2-1378.

[0046] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence comprising residues 12 to 844 of an even-numbered SEQ ID NO: 4-1378, or an amino acid sequence comprising an even-numbered SEQ ID NO: 2-1378.

[0047] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence comprising residues 12 to 844 of SEQ ID NO: 220, 226, 272, 328, 546, 710, or 1208, or an amino acid sequence comprising SEQ ID NO: 2, 220, 226, 272, 328, 546, 710, or 1208.

[0048] In some embodiments, the engineered DNA polymerase is characterized by at least one improved property compared to a reference DNA polymerase. In some embodiments, the improved property of the engineered DNA polymerase is selected from increased activity, increased stability, increased thermostability, increased processivity, increased fidelity, increased product yield, and increased resistance or tolerance to inhibitors, or any combination thereof, compared to a reference DNA polymerase having a sequence corresponding to SEQ ID NO: 2, 220, 226, 272, 328, 546, 710, or 1208. In some embodiments, the improved property of the engineered DNA polymerase is compared to a reference DNA polymerase having a sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 and / or a reference DNA polymerase having a sequence corresponding to SEQ ID NO: 2.

[0049] In some further embodiments, the engineered DNA polymerase is purified.In some embodiments, the engineered DNA polymerase is provided in solution, or immobilized on a substrate, for example, on a solid substrate or membrane or particle.

[0050] In another aspect, the present disclosure provides a recombinant polynucleotide comprising a polynucleotide sequence encoding an engineered DNA polymerase disclosed herein. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding at least one engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 220, 226, 272, 328, 546, 710 or 1208, or to a reference sequence corresponding to SEQ ID NO: 2, 220, 226, 272, 328, 546, 710 or 1208, wherein the amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 220, 226, 272, 328, 546, 710 or 1208, or to a reference sequence corresponding to SEQ ID NO: One or more mutations of the reference sequence of NO: 2, 220, 226, 272, 328, 546, 710 or 1208.

[0051] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 2532 of an odd-numbered SEQ ID NO: 1-1377, or a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO: 1-1377, wherein the recombinant polynucleotide encodes a DNA polymerase.

[0052] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to nucleotide residues 34 to 2532 of SEQ ID NO: 1, 219, 225, 271, 327, 545, 709, or 1207, or a reference polynucleotide sequence corresponding to SEQ ID NO: 1, 219, 225, 271, 327, 545, 709, or 1207, wherein the recombinant polynucleotide encodes a DNA polymerase.

[0053] In some embodiments, the polynucleotide sequence of the recombinant polynucleotide is codon optimized. In some embodiments, the polynucleotide sequence is codon optimized for expression in bacterial cells, fungal cells, or mammalian cells.

[0054] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 2532 of SEQ ID NO: 1, 219, 225, 271, 327, 545, 709, or 1207, or a polynucleotide sequence comprising SEQ ID NO: 1, 219, 225, 271, 327, 545, 709, or 1207.

[0055] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 2532 of an odd-numbered SEQ ID NO among SEQ ID NOs: 1-1377, or a polynucleotide sequence comprising an odd-numbered SEQ ID NO among SEQ ID NOs: 1-1377.

[0056] In some embodiments, the recombinant polynucleotide is operably linked to a regulatory sequence. In some embodiments, the regulatory sequence comprises a promoter, particularly a heterologous promoter.

[0057] In a further aspect, the present disclosure provides an expression vector comprising at least one recombinant polynucleotide encoding an engineered DNA polymerase as provided herein. In another aspect, the present disclosure also provides a host cell transformed with at least one expression vector as provided herein.

[0058] In another aspect, the disclosure provides a method for producing an engineered DNA polymerase polypeptide, the method comprising culturing a host cell as described herein under suitable culture conditions to produce at least one engineered DNA polymerase. In some embodiments, the method further comprises recovering the engineered DNA polymerase from the culture and / or host cell. In some embodiments, the method further comprises a step of purifying the engineered DNA polymerase.

[0059] In another aspect, the disclosure provides a composition comprising at least one engineered DNA polymerase disclosed herein. In some embodiments, the composition comprises at least a buffer. In some embodiments, the composition further comprises one or more DNA polymerase substrates, such as nucleotide substrates and oligonucleotide primer substrates. In another embodiment, the composition comprises a DNA template, particularly a heterologous DNA template.

[0060] In a further aspect, the present disclosure provides the use of an engineered DNA polymerase in a method for preparing a complementary DNA copy of all or part of a target DNA. In some embodiments, the present disclosure provides a method for preparing a complementary DNA copy of all or part of a target DNA, comprising contacting the target DNA with an engineered DNA polymerase as described herein in the presence of an appropriate substrate under conditions suitable for DNA polymerase-mediated production of a DNA complementary to the target DNA.

[0061] In some embodiments, an engineered DNA polymerase is used to detect a target DNA, the method comprising contacting a sample suspected of containing the target DNA with an engineered DNA polymerase of the present disclosure in the presence of an appropriate substrate under conditions suitable for DNA polymerase-mediated production of DNA complementary to the target DNA, and detecting the presence of the complementary DNA. In some embodiments, the sample is a biological sample. In some embodiments, the sample is an environmental sample. In some embodiments, detecting the complementary DNA is by amplifying the complementary DNA, for example, by polymerase chain reaction (PCR), particularly including qPCR (e.g., ), hot start PCR, touchdown PCR, asymmetric PCR, multiplex PCR, long or long-distance PCR, assembly PCR and inverse PCR; or by isothermal amplification reactions, including LAMP, whole genome amplification (WGA) and multiple displacement amplification. In some embodiments, the engineered DNA polymerase can be used with a reverse transcriptase to detect the target RNA, for example, by RT-PCR.

[0062] In a further aspect, the present disclosure also provides a kit comprising at least one engineered DNA polymerase disclosed herein. In some embodiments, the kit further comprises one or more of a buffer, a nucleotide substrate, and / or an oligonucleotide primer substrate. In some embodiments, the kit comprises a template DNA, particularly a control DNA template. DETAILED DESCRIPTION

[0063] The present disclosure provides engineered DNA polymerase polypeptides and compositions thereof, as well as polynucleotides encoding engineered DNA polymerase polypeptides. The present disclosure also provides methods for using engineered DNA polymerase polypeptides and compositions thereof for diagnosis and other purposes. In some embodiments, the engineered DNA polymerase polypeptides particularly exhibit enhanced or increased properties, particularly under conditions involving low concentrations of DNA input, high throughput analysis, and / or sequencing reaction conditions: i) thermal stability; ii) polymerization activity; iii) high replication fidelity; iv) high processability; or v) inhibitor tolerance or resistance; or any combination of i), ii), iii), iv), and / or v). In some embodiments, the engineered DNA polymerase is particularly suitable for polymerase chain reaction.

[0064] In some embodiments, the engineered DNA polymerases can be used in diagnostic and research applications using minute amounts of DNA or RNA from a sample, including cell-free DNA and cell-free RNA, circulating tumor DNA, DNA isolated from circulating tumor cells, circulating fetal DNA, synthetic DNA, DNA isolated from forensic samples, DNA isolated from virally infected cells, fine needle aspirates, or single cells isolated by FACS (fluorescence activated cell sorting), laser capture microscopy, or microfluidic devices.

[0065] Abbreviations and definitions

[0066] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Generally, the nomenclature used herein and the laboratory procedures for cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry and nucleic acid chemistry described below are those well-known and commonly used in the art. Such techniques are known and described in numerous texts and references well-known to those skilled in the art. Standard techniques or modifications thereof are used for chemical syntheses and chemical analyses.

[0067] Although exemplary methods and materials are described herein, any suitable methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. It should be understood that the present invention is not limited to the described ad hoc methods, protocols, and reagents, as these may vary depending on the context in which they are used by those skilled in the art. Accordingly, the terms defined below will be more fully described by reference to the application as a whole.

[0068] As used herein, the singular "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0069] As used herein, the term "comprising" and its cognates are used in their inclusive sense (ie, equivalent to the term "including" and its corresponding cognates).

[0070] It will also be understood that while embodiments are described using the term "comprising" and its cognates, embodiments may also be described using the language "consisting essentially of" or "consisting of.

[0071] In addition, numerical ranges are inclusive of the numbers defining the ranges. Therefore, every numerical range disclosed herein is intended to encompass every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. Every maximum (or minimum) numerical limit disclosed herein is also intended to include every lower (or higher) numerical limit, as if such lower (or higher) numerical limits were expressly written herein.

[0072] As used herein, the term "about" means an acceptable error about a particular value. In some cases, "about" means within 0.05%, 0.5%, 1.0%, or 2.0% of a given value. In some cases, "about" means within 1, 2, 3, or 4 standard deviations of a given value.

[0073] Additionally, the headings provided herein are not limitations of the various aspects or embodiments of the invention, which may be had by reference to the application as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the application as a whole.

[0074] The "EC" number refers to the enzyme nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reaction they catalyze.

[0075] "ATCC" refers to the American Type Culture Collection, whose biorepository collection includes genes and strains.

[0076] "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

[0077] "Protein," "polypeptide," and "peptide" are used interchangeably to refer to a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modifications (eg, glycosylation or phosphorylation).

[0078] " Amino acid " is mentioned in this article by its commonly known three letter symbol or the single letter symbol recommended by IUPAC-IUB Biochemical Nomenclature Commission (Biochemical Nomenclature Commission).Similarly, nucleotide may be mentioned by its commonly recognized single letter code.Amino acid abbreviation for genetic coding is conventional and as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamic acid (Glu or E), glutamine (Gln or Q), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) and valine (Val or V). When three-letter abbreviations are used, an amino acid may be in the L or D configuration about the α-carbon (Cα), unless specifically preceded by "L" or "D" or otherwise clear from the context in which the abbreviation is used. For example, when "Ala" designates alanine without specifying the configuration about the α-carbon, "D-Ala" and "L-Ala" designate D-alanine and L-alanine, respectively. When single-letter abbreviations are used, uppercase letters designate amino acids that are in the L configuration about the α-carbon, and lowercase letters designate amino acids that are in the D configuration about the α-carbon. For example, "A" designates L-alanine, and "a" designates D-alanine. When a polypeptide sequence is presented as a string of single-letter or three-letter abbreviations (or mixtures thereof), the sequence is presented in the amino (N) to carboxyl (C) direction according to general convention.

[0079] "Fusion protein" and "chimeric protein" and "chimera" refer to hybrid proteins produced by joining two or more polynucleotides that originally encoded separate proteins. In some embodiments, the fusion protein is produced by recombinant technology (e.g., molecular biology techniques known in the art).

[0080] "Polymerase" refers to a class of enzymes that polymerize nucleoside triphosphates. In some embodiments, a polymerase uses a template nucleic acid chain to synthesize a complementary nucleic acid chain. The template chain and the synthesized nucleic acid chain can independently be DNA or RNA. Polymerases known in the art include, but are not limited to, DNA polymerases (e.g., Escherichia coli (E. coli) DNA pol I, Thermus aquaticus (T. aquaticus) DNA polymerase (Taq)), DNA-dependent RNA polymerases, and reverse transcriptases. As used herein, a polymerase is a polypeptide or protein containing enough amino acids to perform the desired enzymatic function of the polymerase. In some embodiments, the polymerase does not contain all the amino acids found in the natural enzyme, but only contains those amino acids sufficient to allow the polymerase to perform the desired catalytic activity, including but not limited to 5'-3' polymerization, 5'-3' exonuclease, and 3'-5' exonuclease activity. In some embodiments, the polymerase includes a DNA polymerase classified under EC 2.7.7.7.

[0081] "DNA polymerase activity," "synthetic activity," and "polymerase activity" are used interchangeably herein and refer to the ability of an enzyme to synthesize new DNA chains by incorporating deoxyribonucleoside triphosphates or analogs thereof.

[0082] "Polynucleotide" is used herein to refer to a polymer comprising at least two nucleotides, wherein the nucleotides are deoxyribonucleotides or ribonucleotides or a mixture of deoxyribonucleotides and ribonucleotides. In some embodiments, the abbreviations for genetically encoded nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless otherwise indicated, the abbreviated nucleosides may be ribonucleosides or 2'-deoxyribonucleosides. Nucleosides may be designated as ribonucleosides or 2'-deoxyribonucleosides on an individual basis or on an aggregate basis. When a nucleic acid sequence is presented as a string of single-letter abbreviations, the sequence is presented in the 5' to 3' direction according to common practice and does not indicate a phosphate group. The term "DNA" refers to deoxyribonucleic acid. The term "RNA" refers to ribonucleic acid.

[0083] "Duplex" and "ds" refer to a double-stranded nucleic acid (e.g., DNA or RNA) molecule composed of two single-stranded polynucleotides that are complementary in their sequence (e.g., A pairs with T or U, C pairs with G), arranged in an antiparallel 5' to 3' orientation, and held together by hydrogen bonds between the nucleobases (e.g., adenine [A], guanine [G], cytosine [C], thymine [T], and uridine [U]).

[0084] "Engineered," "recombinant," "non-naturally occurring," and "variant" when used in reference to a cell, polynucleotide, or polypeptide refer to such material or material corresponding to the native or naturally occurring form of that material that has been modified in a manner that does not otherwise exist in nature, or is identical thereto, but is produced or derived from synthetic materials and / or through manipulation using recombinant techniques.

[0085] "Wild-type" and "naturally occurring" refer to forms found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and has not been intentionally modified by human manipulation.

[0086] "Coding sequence" refers to the portion of a nucleic acid (eg, a gene) that encodes the amino acid sequence of a protein.

[0087] " Percentage (%) sequence identity " refers to the comparison in polynucleotide and polypeptide, and is determined by comparing the sequences of two best comparisons on a comparison window, wherein compared with the reference sequence for the best comparison of two sequences, the part of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (that is, rooms). Percentage may be calculated by: determining the number of positions at which identical nucleic acid bases or amino acid residues occur in two sequences, to obtain the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window, and the result is multiplied by 100, to obtain the percentage of sequence identity. Alternatively, percentage may be calculated by: determining the number of positions at which identical nucleic acid bases or amino acid residues occur in two sequences or that nucleic acid bases or amino acid residues are compared with rooms, to obtain the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window, and the result is multiplied by 100, to obtain the percentage of sequence identity. Those skilled in the art will appreciate that there are many determination algorithms that can be used for comparing two sequences. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85:2444), computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection, as known in the art. Examples of algorithms suitable for determining percent sequence identity and sequence similarity include, but are not limited to, BLAST and BLAST 2.0 algorithms (see, e.g., Altschul et al., J. Mol. Biol., 1990, 215:403-410; and Altschul et al., Nucleic Acids Res., 1977, 3389-3402). Software for performing BLAST analyses is publicly available through the website of the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length "W" in the query sequence that either match or satisfy a positive-valued threshold score "T" when aligned with a word of the same length in a database sequence.T is called the neighborhood word score threshold (see Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence until the cumulative alignment score can be increased. For nucleotide sequences, the parameters "M" (reward score for a pair of matching residues; always >0) and "N" (penalty score for mismatched residues; always <0) are used to calculate the cumulative score. For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The word hit extension in each direction stops when the following occurs: the cumulative alignment score drops by a number "X" from the maximum value it reaches; the cumulative score drops to zero or below due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses the following as default values: word length (W) 11, expectation (E) 10, M=5, N=-4, and comparison of two chains. For amino acid sequences, the BLASTP program can use the following as defaults: word length (W) 3, expectation (E) 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89: 10915). Exemplary determinations of sequence alignments and % sequence identity can be made using the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI) using the default parameters provided.

[0088] A "reference sequence" refers to a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Typically, a reference sequence is at least 20 nucleotides or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length, or the full length of a nucleic acid or polypeptide. Because two polynucleotides or polypeptides may each (1) contain a sequence that is similar between the two sequences (i.e., a portion of the complete sequence), and (2) may further contain a sequence that is different between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing the sequences of the two polynucleotides or polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. In some embodiments, a "reference sequence" can be based on a primary amino acid sequence, wherein the reference sequence is a sequence that may have one or more changes in the primary sequence. For example, the phrase "the reference sequence corresponding to SEQ ID NO:2 having a leucine at the residue corresponding to X478" (or "the reference sequence corresponding to SEQ ID NO:2 having a leucine at the residue corresponding to position 478") refers to the reference sequence in which the corresponding residue at position X478 in SEQ ID NO:2 (e.g., methionine) has been changed to a leucine.

[0089] A "comparison window" refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues in which a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and wherein a portion of the sequence in the comparison window may contain 20% or fewer additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues, and optionally includes windows of 30, 40, 50, 100, or longer.

[0090] When used in the context of numbering a given amino acid or polynucleotide sequence, "corresponding to," "reference," and "relative to," refer to the numbering of the residues of a given amino acid or polynucleotide sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue numbers or residue positions of a given polymer are specified with respect to the reference sequence, rather than by the actual numerical position of the residues within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of an engineered DNA polymerase, can be aligned with a reference sequence by introducing gaps to optimize residue matching between the two sequences. In these cases, the numbering of the residues in a given amino acid or polynucleotide sequence is prepared with respect to the reference sequence to which it has been aligned, despite the presence of the gaps. In some embodiments, the sequence is tagged (e.g., with a histidine tag).

[0091] "Mutation" refers to a change in a nucleic acid sequence. In some embodiments, the mutation results in a change in the encoded polypeptide sequence (i.e., compared to the original sequence without the mutation). In some embodiments, the mutation comprises a substitution so that a different amino acid is produced. In some alternative embodiments, the mutation comprises an addition so that an amino acid is added (e.g., inserted) into the original polypeptide sequence. In some further embodiments, the mutation comprises a deletion so that an amino acid is deleted from the original polypeptide sequence. Any number of mutations may be present in a given sequence.

[0092] "Amino acid difference" and "residue difference" refer to the difference in the amino acid residue at a position in a polypeptide sequence relative to the amino acid residue at the corresponding position in the reference sequence. The position of the amino acid difference is generally referred to as "Xn" in this article, where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, "the residue difference at position X478 compared to SEQ ID NO:2" (or "the residue difference at position 478 compared to SEQ ID NO:2") refers to the difference in the amino acid residue at the polypeptide position corresponding to position 478 of SEQ ID NO:2. Thus, if the reference polypeptide of SEQ ID NO:2 has a methionine at position 478, then "the residue difference at position X478 compared to SEQ ID NO:2" refers to an amino acid substitution of any residue other than methionine at the polypeptide position corresponding to position 478 of SEQ ID NO:2. In some cases herein, the specific amino acid residue differences at positions are indicated as "XnY," where "Xn" specifies the corresponding residue and position (as described above) of a reference polypeptide, and "Y" is the single-letter identifier of the amino acid found in the engineered polypeptide (i.e., a residue different from the reference polypeptide). In some cases (e.g., in the tables of the Examples), the disclosure also provides specific amino acid differences represented by the conventional notation "AnB," where A is the single-letter identifier of the residue in the reference sequence, "n" is the numbering of the residue position in the reference sequence, and B is the single-letter identifier of the residue replacement in the sequence of the engineered polypeptide. In some embodiments, "replacement" comprises the deletion of an amino acid and can be represented by the "-" symbol. In some embodiments, the phrase "amino acid residue nB" represents an amino acid residue present at a defined amino acid position in an engineered polypeptide that may or may not be a replacement in the context of a reference polypeptide or amino acid sequence.

[0093] In some cases, the polypeptides of the present disclosure may include one or more amino acid residue differences relative to a reference sequence, which are indicated by a list of designated positions at which residue differences exist relative to the reference sequence. In some embodiments, when more than one amino acid can be used in a specific residue position of a polypeptide, the various amino acid residues that can be used are separated by " / " (e.g., X478L / X478I, X478L / I, or 478L / I). The present disclosure includes engineered polypeptide sequences comprising one or more amino acid differences, including either / or both conservative and non-conservative amino acid substitutions, as well as amino acid insertions and deletions in the sequence.

[0094] "Amino acid substitution set" and "substitution set" refer to a set of amino acid substitutions within a polypeptide sequence. In some embodiments, the substitution set comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions. In some embodiments, the substitution set refers to an amino acid substitution set present in any variant DNA polymerase polypeptide listed in any table in the examples. In these substitution sets, the various substitutions are separated by a semicolon (";"; for example, M478L; R516K; E541D) or a slash (" / "; for example, M478L / R516K / E541D or 478L / 516K / 541D). In some embodiments, the phrase "mutation set" is used.

[0095] "Conservative amino acid substitutions" refer to the replacement of a residue with a different residue having a similar side chain, and thus generally involve the replacement of an amino acid in a polypeptide with an amino acid within the same or similar defined amino acid class. By way of example and not limitation, an amino acid with an aliphatic side chain may be replaced by another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain may be replaced by another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain may be replaced by another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain may be replaced by another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain may be replaced by another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and a hydrophobic or hydrophilic amino acid may be replaced by another hydrophobic or hydrophilic amino acid, respectively.

[0096] "Non-conservative substitutions" refer to the replacement of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affect: (a) the structure of the peptide backbone in the region of the substitution (e.g., proline for glycine); (b) charge or hydrophobicity; and / or (c) the bulk of the side chain. By way of example and not limitation, exemplary non-conservative substitutions include substitutions of acidic amino acids with basic or aliphatic amino acids; substitutions of aromatic amino acids with small amino acids; and substitutions of hydrophilic amino acids with hydrophobic amino acids.

[0097] "Deletion" refers to the modification of a polypeptide by removing one or more amino acids from a reference polypeptide. A deletion can comprise the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, constituting up to 10% of the total number of amino acids in the reference enzyme, or up to 20% of the total number of amino acids, while retaining enzymatic activity and / or retaining the improved properties of the engineered polymerase. Deletions can be directed to internal portions and / or terminal portions of a polypeptide. In various embodiments, deletions can comprise contiguous segments or can be non-contiguous. As noted above, in some embodiments, a deletion is indicated by a "-" and may be present in a substitution or substitution set.

[0098] "Insertion" refers to the modification of a polypeptide by the addition of one or more amino acids from a reference polypeptide. Insertions can be located in internal portions of a polypeptide or at the carboxyl or amino termini. Insertions, as used herein, include fusion proteins as are known in the art. Insertions can be contiguous stretches of amino acids or separated by one or more amino acids in a naturally occurring polypeptide.

[0099] "Combinations" or "combination" in the context of any list of mutations (e.g., substitutions) or mutated amino acid positions refers to any combination of the listed mutations and / or amino acid positions. In some embodiments, "combinations" or "combination" refers to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more of the listed mutations and / or amino acid positions.

[0100] "Functional fragment" and "biologically active fragment" are used interchangeably herein to refer to a polypeptide having amino-terminal and / or carboxyl-terminal deletions and / or internal deletions, but in which the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is compared (e.g., a full-length engineered DNA polymerase of the invention) and retains substantially all of the activity of the full-length polypeptide.

[0101] "Isolated polypeptide" refers to a polypeptide that is substantially separated from other contaminants (e.g., proteins, lipids, and polynucleotides) that naturally accompany it. The term encompasses polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., host cells or in vitro synthesis). The recombinant DNA polymerase polypeptide may be present in a cell, in a cell culture medium, or prepared in various forms such as lysates or isolated preparations. Therefore, in some embodiments, the recombinant DNA polymerase polypeptide provided herein is an isolated polypeptide.

[0102] "Substantially pure polypeptide" or "purified polypeptide" refers to a composition wherein the polypeptide species is the predominant species present (i.e., on a molar or weight basis, it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially pure composition when the target species constitutes at least about 50% of the macromolecular species present by mole or % weight. Generally, a substantially pure DNA polymerase composition will constitute about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species present in the composition by mole or % weight. In some embodiments, the target species is purified to basic homogeneity (i.e., the contaminant species cannot be detected in the composition by conventional detection methods), wherein the composition is substantially composed of a single macromolecular species. Solvent species, small molecules (<500 daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant DNA polymerase polypeptide is a substantially pure polypeptide composition.

[0103] "Improved enzyme properties" refers to an engineered DNA polymerase polypeptide that exhibits an improvement in any enzyme property compared to a reference DNA polymerase polypeptide, e.g., a wild-type DNA polymerase polypeptide (e.g., the DNA polymerase polypeptide sequence of SEQ ID NO: 2) or another engineered DNA polymerase polypeptide. Improved properties include, but are not limited to, such properties as increased protein expression, increased thermal activity, increased thermal stability, increased stability, increased enzymatic activity, increased substrate specificity and / or affinity, increased specific activity, increased resistance to substrate and / or end-product inhibition, increased chemical stability, improved chemical selectivity, improved solvent stability, increased solubility, increased fidelity, increased processability, increased inhibitor resistance or tolerance, and altered temperature profile.

[0104] "Increased enzymatic activity" and "enhanced catalytic activity" refer to improved properties of an engineered DNA polymerase polypeptide, which can be expressed as an increase in specific activity (e.g., product / time / weight protein produced) and / or an increase in the percentage conversion of substrate to product (e.g., the percentage conversion of a starting amount of substrate to product over a specified time period using a specified amount of DNA polymerase) compared to a reference DNA polymerase (e.g., wild-type DNA polymerase and / or another engineered DNA polymerase). Exemplary methods for determining enzymatic activity are provided in the Examples. Any property associated with enzymatic activity may be affected, including classical enzyme properties Km, Vmax, or kcat, changes in which can result in increased enzymatic activity. The improvement in enzymatic activity can be from about 1.1-fold the enzymatic activity of the corresponding wild-type enzyme to as much as 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, or more the enzymatic activity of a naturally occurring DNA polymerase or another engineered DNA polymerase from which the DNA polymerase polypeptide is derived.

[0105] "Hybridization stringency" refers to the hybridization conditions, such as washing conditions, in the hybridization of nucleic acids. Generally, hybridization reactions are performed under conditions of lower stringency, followed by washes of different but higher stringency (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 2001; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, 2003). The term "moderately stringent hybridization" refers to conditions that allow target DNA to bind to complementary nucleic acids that have about 60% identity, preferably about 75% identity, about 85% identity, with the target DNA, accompanied by greater than about 90% identity with the target polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization at 42°C in 50% formamide, 5×Denhart's solution, 5×SSPE, 0.2% SDS, followed by washes at 42°C in 0.2×SSPE, 0.2% SDS. "High stringency hybridization" generally refers to conditions that are about 10°C or lower from the thermal melting temperature, Tm, as determined for a defined polynucleotide sequence under solution conditions. In some embodiments, high stringency conditions refer to conditions that allow only the hybridization of those nucleic acid sequences that form stable hybrids in 0.018 M NaCl at 65°C (i.e., if a hybrid is not stable in 0.018 M NaCl at 65°C, it is also unstable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization under conditions equivalent to 50% formamide, 5× Denhart's solution, 5× SSPE, 0.2% SDS at 42°C, followed by a wash in 0.1× SSPE and 0.1% SDS at 65°C. Another high stringency condition includes hybridization under conditions equivalent to hybridization in 5X SSC containing 0.1% (w:v) SDS at 65° C., and a wash in 0.1× SSC containing 0.1% SDS at 65° C. Other high stringency hybridization conditions, as well as moderate stringency conditions, are described in the references cited above.

[0106] "Codon optimized" refers to the change of the codons of a polynucleotide encoding a protein to those codons preferentially used in a particular organism, so that the encoded protein is more efficiently expressed in that organism. Although the genetic code is degenerate, in that most amino acids are represented by a few codons called "synonymous" or "synonymous" codons, it is well known that the codon usage by a particular organism is non-random and biased towards specific codon triplets. This codon usage bias may be higher for a given gene, genes with a common function or ancestral origin, highly expressed proteins relative to low copy number proteins, and aggregated protein coding regions of the genome of an organism. In some embodiments, the polynucleotide encoding a DNA polymerase is codon optimized for optimal production from the host organism selected for expression.

[0107] "Control sequences" herein refer to all components necessary or advantageous for the expression of the polynucleotides and / or polypeptides of the present disclosure. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter sequence, a signal peptide sequence, an initiation sequence, and a transcription terminator. In some embodiments, the control sequence includes at least a promoter and transcription and translation termination signals. In some embodiments, the control sequence is provided with a linker for the purpose of introducing specific restriction sites to facilitate connection of the control sequence to the coding region of the nucleic acid sequence encoding the polypeptide.

[0108] "Operably linked" refers to a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide and, where appropriate, the encoded polypeptide of interest.

[0109] "Promoter" or "promoter sequence" refers to a nucleic acid sequence, e.g., a coding sequence, that is recognized by a host cell for expression of a polynucleotide of interest. The promoter sequence contains transcriptional control sequences that mediate expression of the polynucleotide of interest. The promoter may be any nucleic acid sequence that shows transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and may be derived from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0110] "Suitable reaction conditions" or "suitable conditions" refer to those conditions in an enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffer, co-solvents, etc.) under which a DNA polymerase polypeptide of the present disclosure is able to convert a substrate into a desired product compound. Exemplary "suitable reaction conditions" are provided herein (see, Examples).

[0111] "Product" in the context of an enzymatic conversion process refers to a compound or molecule that results from the action of a DNA polymerase polypeptide on a substrate.

[0112] "Culturing" refers to growing a population of cells, such as microbial cells, under suitable conditions using any suitable medium (eg, liquid, gel, or solid).

[0113] A "vector" is a polynucleotide construct for introducing a polynucleotide sequence into a cell. In some embodiments, a vector is an expression vector operably linked to appropriate control sequences that are capable of expressing the polynucleotide of interest and, where appropriate, the encoded polypeptide in a suitable host. In some embodiments, an "expression vector" has a promoter sequence operably linked to a DNA sequence (e.g., a transgene) to drive expression in a host cell, and in some embodiments, further comprises a transcription termination sequence.

[0114] "Expression" includes any step involved in the production of the polypeptide, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from the cell.

[0115] "Production" refers to the production of proteins and / or other compounds by cells. It is intended that the term encompasses any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses the secretion of a polypeptide from a cell.

[0116] "Heterologous" or "recombinant" refers to the relationship between two or more nucleic acid or polypeptide sequences (eg, promoter sequence, signal peptide, termination sequence, etc.) that are derived from different sources and not associated in nature.

[0117] "Host cell" and "host strain" refer to a suitable host for an expression vector comprising a DNA as provided herein (e.g., a polynucleotide sequence encoding at least one DNA polymerase variant). In some embodiments, the host cell is a prokaryotic or eukaryotic cell transformed or transfected with a vector constructed using recombinant DNA techniques as known in the art.

[0118] By "analog" is meant a polypeptide having more than 70% sequence identity but less than 100% sequence identity (e.g., more than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to a reference polypeptide. In some embodiments, the analog includes non-naturally occurring amino acid residues, including but not limited to homoarginine, ornithine, and norvaline, as well as naturally occurring amino acids. In some embodiments, the analog also includes one or more D-amino acid residues and non-peptide bonds between two or more amino acid residues.

[0119] An "effective amount" means an amount sufficient to produce the desired result. One of ordinary skill in the art can determine the effective amount by using routine experimentation.

[0120] "Cell-free DNA" or "cfDNA" refers to DNA that circulates freely in the bloodstream and is not contained by or associated with cells. In some embodiments, cell-free DNA comprises DNA originally derived from and released from normal somatic or germline cells, cancer cells, fetal cells, microbial cells, or viruses.

[0121] "Cell-free RNA" or "cfRNA" refers to RNA that circulates freely in the bloodstream and is not contained by or associated with cells. In some embodiments, cell-free RNA comprises RNA originally derived from and released from normal somatic or germline cells, cancer cells, fetal cells, microbial cells, or viruses.

[0122] "Amplification" refers to the replication of a nucleic acid. In some embodiments, the term refers to the replication of a specific template nucleic acid.

[0123] "Polymerase chain reaction" and "PCR" refer to a method for generating multiple copies of a nucleic acid template of interest by repeated cycles of denaturation, annealing, and primer extension with a polymerase in the presence of nucleic acid primers, such as described in "PCR: Methods and Protocols," Methods in Molecular Biology, Springer Protocols (2017), and "Quantitative Real-Time PCR: Methods and Protocols," Methods in Molecular Biology, Springer Protocols (2014), which are incorporated herein by reference. The sequence of denaturation, annealing, and extension constitutes one "cycle." The steps of denaturation, primer annealing, and polymerase extension can be repeated (i.e., using multiple cycles) to amplify the nucleic acid template. In some embodiments, the annealing and extension processes occur in a single step, sometimes referred to as two-step PCR. Accordingly, as used herein, the term "PCR" includes many variations of this method, including, among others, qPCR, hot-start PCR, touchdown PCR, asymmetric PCR, multiplex PCR, long or long-distance PCR, assembly PCR, and inverse PCR.

[0124] When used in reference to methods employing DNA polymerases, "target" refers to a region of nucleic acid used to prepare complementary DNA. The "target" is separated from other nucleic acids present in methods employing DNA polymerases. In some embodiments, a "segment" is a region of nucleic acid within a target sequence.

[0125] When used in the context of a DNA polymerase, "target DNA" refers to all or a portion of the DNA that is the target for making a complementary DNA copy. The target DNA can be the entire DNA sequence or a portion thereof, such as a segment of a DNA sequence.

[0126] "Target RNA" refers to all or a portion of an RNA that is the target for making a complementary DNA copy. The target RNA can be the entire RNA sequence or a portion thereof, such as a segment of an RNA sequence.

[0127] "Sample template" refers to nucleic acid derived from a sample for the presence of a target nucleic acid being analyzed. In contrast, "background template" refers to nucleic acid other than the sample template, which may or may not be present in the sample. Background template may be inadvertently included in the sample, which may result from carryover, or may be due to the presence of nucleic acid contaminants from which the target nucleic acid is purified. For example, in some embodiments, nucleic acid from organisms other than those to be detected may be present in the test sample as background. However, it is not intended that the present invention be limited to any specific nucleic acid sample or template.

[0128] "Amplifiable nucleic acid" is used to refer to a nucleic acid that may be amplified by any amplification method, including but not limited to PCR. In most embodiments, the amplifiable nucleic acid comprises a sample template.

[0129] "PCR product," "PCR fragment," and "amplification product" refer to the resulting product obtained after two or more rounds of PCR amplification (or other amplification method, as indicated by the context), which typically includes denaturation, annealing, and extension steps. These terms encompass situations in which amplification of one or more segments of one or more target sequences is already present.

[0130] "Amplification reagents" and "PCR reagents" refer to those reagents required for amplification (e.g., deoxyribonucleotide triphosphates, buffer, etc.), in addition to primers, nucleic acid template, and amplification enzyme. Typically, the amplification reagents are placed and contained in a reaction vessel (e.g., a test tube, microwell, etc.) along with the other reaction components. It is not intended that the present invention be limited to any specific amplification reagent, as any suitable reagent may be used in the present invention.

[0131] " Primer " refers to an oligonucleotide (i.e., a sequence of nucleotides) whether naturally occurring or synthetic, recombinant or produced by amplification, which can serve as a starting point for nucleic acid synthesis when placed under conditions in which a primer extension product complementary to a nucleic acid chain is induced to synthesize (i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase, and at a suitable temperature and pH). In some embodiments, the primer is single-stranded, but in some embodiments, the primer is double-stranded. In some embodiments, the primer has enough length to initiate the synthesis of extension products in the presence of a nucleic acid polymerase. Exact primer length depends on many factors, as known to those skilled in the art.

[0132] "Probe" refers to an oligonucleotide (i.e., a sequence of nucleotides) that is naturally occurring, synthetic, recombinant, or produced by amplification, which is capable of hybridizing with another oligonucleotide of interest. The probe can be used to detect, identify, and / or separate a specific gene sequence of interest. In some embodiments, the probe is labeled with a "reporter molecule" (also referred to as a "label") that contributes to probe detection in a suitable detection system (e.g., fluorescence, radioactivity, luminescence, enzymatic, and other systems). It is not contemplated that the present invention is limited to any specific detection system or label. Primers, deoxyribonucleotides, and deoxyribonucleosides may contain labels. In fact, it is not contemplated that the labeling composition of the present invention is limited to any specific component. Illustrative labels include, but are not limited to, 32P, 35S, and fluorescent molecules (e.g., fluorescent dyes, including but not limited to green fluorescent protein).

[0133] When referring to the use of polymerase, "fidelity" is expected to refer to the accuracy of the template-guided complementary base incorporation in the synthesized DNA chain relative to the template chain. Typically, fidelity is measured based on the incorporation frequency of incorrect bases in the newly synthesized nucleic acid chain. The incorporation of incorrect bases can result in point mutations, insertions, or deletions. Fidelity can be calculated according to any method known in the art (see, e.g., Tindall and Kunkel, Biochem., 1988, 27: 6008-6013; and Barnes, Gene, 1992, 112: 29-35). Polymerases or polymerase variants can exhibit high fidelity or low fidelity. As used herein, "high fidelity" refers to that a polymerase has an accurate base incorporation frequency exceeding a predetermined value. As used herein, the term "low fidelity" refers to that a polymerase has an accurate base incorporation frequency below a predetermined value. In some embodiments, the predetermined value is the desired frequency of accurate base incorporation or the fidelity of a known polymerase (i.e., a reference polymerase).

[0134] "Altered fidelity" refers to a polymerase variant whose fidelity is different from the fidelity of the parent polymerase or reference polymerase from which the polymerase variant is derived. In some embodiments, the altered fidelity is higher than the fidelity of the parent or reference polymerase, while in some other embodiments, the altered fidelity is lower than the fidelity of the parent or reference polymerase. The altered fidelity can be determined by assaying the activities of the parent and variant polymerases and comparing them using any suitable assay known in the art.

[0135] "Processivity" refers to the ability of a nucleic acid modification enzyme, such as a DNA polymerase, to remain bound to a template or substrate and to perform multiple modification reactions. Processivity is typically measured by the number of catalytic events that occur per binding event.

[0136] "Altered processivity" refers to a polymerase or variant thereof having a processivity that is different from the processivity of a parent or reference polymerase from which the variant is derived. In some embodiments, the altered processivity is greater than the processivity of the parent or reference enzyme, while in some other embodiments, the altered processivity is less than the processivity of the parent or reference enzyme. The altered processivity can be determined by assaying the activities of the parent / reference and variant enzymes and comparing their activities using any suitable assay known in the art.

[0137] "Subject" encompasses mammals such as humans, non-human primates, livestock, companion animals, and laboratory animals (eg, rodents and lagamorphs). The term is intended to encompass females as well as males.

[0138] "Patient" means any subject being evaluated for a disease, treated for a disease, or experiencing a disease. In some embodiments, the "sample" used for reaction with DNA polymerase is obtained from a patient.

[0139] As used herein, "sample" refers to a material or substance for reacting with a nucleic acid polymerase, such as for detecting the presence of a target nucleic acid or preparing a DNA copy of the target nucleic acid for sequencing or generating a cDNA library. In some embodiments, the sample is a "biological sample," which refers to a sample of a biological tissue or fluid. Such samples are typically from humans, but include tissues isolated from non-human primates or rodents, such as mice and rats, and include sections of tissue such as biopsies and autopsy samples, frozen sections obtained for histological purposes, blood, plasma, serum, sputum, feces, tears, mucus, hair, skin, etc. A "biological sample" also refers to a cell or cell colony or a certain amount of tissue or fluid from an organism. In some embodiments, a biological sample has been taken out from an animal, but the term "biological sample" can also refer to cells or tissues analyzed in vivo, that is, without having to take out from an animal, including cell cultures. Typically, a "biological sample" contains cells from an animal or organism, but the term can also refer to non-cellular biological materials, such as the non-cellular fraction of blood, saliva, or urine. Numerous types of biological samples can be used with the enzymes, compositions and methods of the present disclosure, including but not limited to tissue biopsies, blood samples, cheek scrapings, saliva samples or nipple discharge. As used herein, "tissue biopsy" refers to the amount of tissue removed from an animal, preferably a human, for diagnostic analysis. In a cancer patient, tissue may be removed from a tumor, allowing analysis of cells within the tumor. "Tissue biopsy" can refer to any type of biopsy, such as a needle biopsy, fine needle biopsy, surgical biopsy, etc. In some embodiments, the sample can be from an environmental source, such as, by way of example and not limitation, water (e.g., oceans, rivers, garbage / sewers, etc.), soil, air, vents, or surfaces (e.g., floors, machines, counters, etc.).

[0140] Engineered DNA polymerase polypeptides

[0141] In one aspect, present disclosure provides archaeal dna polymerase, including engineered archaeal dna polymerase polypeptide variants.In some embodiments, archaeal dna polymerase and engineered archaeal dna polymerase polypeptide variants can be used for performing polymerase reaction, including preparing the complementary DNA of target DNA target / template.In some embodiments, the engineered archaeal dna polymerase variants of present disclosure can be used for effectively producing complementary DNA from whole or part of DNA template, for example, for order-checking (for example, NGS order-checking), amplification (for example, PCR) and diagnostic method, for example, for detecting target nucleic acid.These engineered archaeal dna polymerase variants can be used in solution, and for use in immobilized embodiments.In some embodiments, engineered archaeal dna polymerase can prepare and be used as non-fusion polypeptide or fusion polypeptide.

[0142] In some embodiments herein, when referring to a specific DNA polymerase variant (i.e., an engineered DNA polymerase polypeptide) by reference to a modification of a specific amino acid residue in the sequence of a wild-type DNA polymerase or a reference DNA polymerase polypeptide, it is understood that variants of another DNA polymerase modified in an equivalent position (as determined by an optional amino acid sequence alignment between the respective amino acid sequences) are encompassed herein. For example, for substitutions at designated amino acid positions numbered with reference to SEQ ID: 2, equivalent amino acid positions can be readily determined for another sequence, such as a reference sequence of SEQ ID NO: 220, 226, 272, 328, 546, 710, or 1208.

[0143] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 220, 226, 272, 328, 546, 710 or 1208, or to a reference sequence corresponding to SEQ ID NO: 2, 220, 226, 272, 328, 546, 710 or 1208, wherein the amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 220, 226, 272, 328, 546, 710 or 1208, or to a reference sequence corresponding to SEQ ID NO: One or more mutations of the reference sequence of NO: 2, 220, 226, 272, 328, 546, 710 or 1208.

[0144] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2.

[0145] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO: 2.

[0146] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, 226, 272, 328, 546, 710, or 1208, or a reference sequence corresponding to SEQ ID NO: 220, 226, 272, 328, 546, 710, or 1208, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0147] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 4-1378, or to a reference sequence corresponding to an even numbered SEQ ID NO: 4-1378, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO: 2.

[0148] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises mutations at at least the following amino acid positions: 2, 3, 21, 39, 51, 59, 66, 68, 72, 73, 78, 79, 87, 124, 147, 178, 181, 185, 199, 216, 224, 233, 239, 241, 251, 257, 262, 271, 275, 288, 291, 303, 316, 317, 327, 328, 336, 338, 341, 342, 351, 356, 359, 362, 365, 368, 370, 371, 372. 2、373、374、376、377、378、379、380、381、382、388、390、392、393、395、396、398、399、402、403、404、409、410、412、413、416、417、418、420、425、426、427、429、430、431、432、434、435、437、438、442、443、447、448、449、450、451、453、453、454、457、461、465、466、469、473、 474, 475, 477, 478, 479, 482, 483, 488, 490, 503, 508, 514, 515, 516, 526, 527, 528, 529, 536, 541, 542, 544, 545, 550, 554, 557, 558, 559, 562, 569, 575, 577, 578, 579, 589, 594, 601, 603, 604, 612, 613, 641, 644, 651, 656, 660, 662, 663, 664, 667, 671, 677, 685, 687, 691, 69 792, 798, 804, 807, 808, 809, 812, 815, 820, 824, 825 or 831 , or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0149] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least mutations 2-, 3N, 21A / G / N, 39F, 51T, 59K, 66R, 68E / G / I / K / Q / R / T, 72A, 73I, 78N / S / T / V, 79A / M / R / T, 87V, 124A, 147A / H / M / Q, 178L, 181Q / R, 185T, 199P, 216K, 224L, 233H / K / L, 239V, 241E, 251A / I / L, 257V, 262N, 271G, 275A / T, 288R, 291L, 303T, 316G, 317R, 327E / L, 328L / V, 336 E, 338V, 341H / T / Y, 342G, 351H, 356I / S, 359G / V, 362M / P / S, 365T, 368H, 370 G / S / V, 371L, 372V / Y, 373G / T, 374S / V / Y, 376F / S, 377C, 378C / F / L / S, 379G, 3 80I, 381E / K, 382C / S, 388C / M / P / V, 390S / T, 392A / C / S / T / V, 393G / R / S / W, 39 5G / H / S, 396F / L / P / R / S, 398G / Q, 399G / T, 402R / Y, 403I, 404T, 409S, 410V / W, 412M / Q / R / V, 413T, 416S, 417R / W, 418K, 420V, 425T, 426T, 427D / F / H / I / L / R / T / Y, 429F, 430G, 431G / L / R / V, 432T, 434N, 435K / P, 437N / P / S, 438F / S, 442G , 443A, 447R / S / W, 448C / I / T / V, 449A / G / L / V, 450V, 451L, 453L / S, 454D / F / G / L / W, 457N / R / W / Y, 461L / Q / S, 465Y, 466Y, 469T / V, 473N, 474A / C / G, 475A / S / V / Y, 477V, 478I / L, 479L, 482S, 483D, 488W, 490W, 503R, 508K, 514I, 515H / T , 516K, 526S, 527A, 528A / S, 529S / V, 536V, 541D / G / N / S, 542R, 544M, 545L, 55 0C / T, 554G / N, 557I, 558G / L / T, 559R, 562K, 569M / Q, 575W, 577S, 578A, 579L / Q, 589G, 594A, 601T, 603M / R, 604H, 612D, 613H / R, 641A, 644Q, 651Q, 656Q / S,660V, 662S, 663E, 664A, 667S / T / W, 671W, 677L, 685R, 687A, 691S / W, 694D, 697A / I / L / V, 698E, 699V, 701M, 702S / V, 70 4L / V, 706I / S / V, 707W, 708L, 713G, 714I, 715H / L, 716E / L, 718F / Q, 721A / M / N, 723Y, 724F / L, 725V, 728S, 731I / T, 732L / Q, 735L, 740S / V, 742K, 743H / V, 745S / T / V, 747S, 748G, 749M, 753A, 758G, 760T / W / Y, 762A, 768S, 769Q, 770E, 787M / N, 792F / Y, 798I, 804R, 807M, 808W / Y, 809K, 812R / S, 815L / R / S / T, 820E / S / T, 824M, 825E, or 831G, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0150] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the mutations H2-, H3N, P21A / G / N, Y39F, V51T, R59K, K66R, D68E / G / I / K / Q / R / T, V72A, V73I, A78N / S / T / V, K79A / M / R / T, A87V, P124A, C147A / H / M / Q, V178L, K181Q / R, P185T, R199P, R216K, V224L, R233H / K / L, L239V, R241E, R251A / I / L, A257V, D262N, A271G, R275A / T, E288R, F291L, G303T, E316G, G317R, K327E / L, E328L / V, A336E, A338V, A341H / T / Y, E342G, P351H, A356I / S, K359G / V, R362M / P / S, L365T, D368H, A370G / S / V, V371L, L37 2V / Y, A373G / T, L374S / V / Y, E376F / S, G377C, V378C / F / L / S, A379G, L380I, D 381E / K, P382C / S, L388C / M / P / V, A390S / T, L392A / C / S / T / V, L393G / R / S / W, P3 95G / H / S, A396F / L / P / R / S, T398G / Q, N399G / T, G402R / Y, V403I, A404T, G409 S, E410V / W, T412M / Q / R / V, E413T, A416S, E417R / W, R418K, L420V, L425T, F4 26T, Q427D / F / H / I / L / R / T / Y, L429F, F430G, P431G / L / R / V, R432T, S434N, E4 35K / P, L437N / P / S, L438F / S, Q442G, E443A, P447R / S / W, L448C / I / T / V, S449A / G / L / V, R450V, V451L, A453LS, H454D / F / G / L / W, A457N / R / W / Y, R461L / Q / S, P465Y, L466Y, A469T / V, E473N, L474A / C / G, E475A / S / V / Y, E477V, M478I / L, E 479L, E482S, G483D, L488W, G490W, K503R, E508K, V514I, G515H / T, R516K, A 526S, Q527A, G528A / S, A529S / V, A536V, E541D / G / N / S, L542R, L544M, Q545L,S550C / T, S554G / N, L557I, D558G / L / T, P559R, R562K, G569M / Q, F575W, Q577S, T578A, A579 L / Q, D589G, N594A, L601T, Q603M / R, R604H, E612D, E613H / R, R641A, R644Q, T651Q, W656Q / S , L660V, P662S, A663E, L664A, P667S / T / W, R671W, N677L, S685R, H687A, Q691S / W, G694D, Y 697A / I / L / V, K698E, E699V, E701M, A702S / V, I704L / V, R706I / S / V, Y707W, F708L, K713G, V7 14I, R715H / L, A716E / L, I718F / Q, T721A / M / N, E723Y, E724F / L, G725V, R728S, V731I / T, E7 32L / Q, F735L, Y740S / V, P742K, D743H / V, A745S / T / V, R747S, V748G, R749M, E753A, M758G, F 760T / W / Y, M762A, A768S, A769Q, D770E, G787M / N, L792F / Y, L798I, E804R, A807M, E808W / Y, E809K, A812R / S, K815L / R / S / T, N820E / S / T, L824M, D825E or E831G, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0151] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation at amino acid position 147, 257, 291, 395, 402, 474, 475, 478, 514, 515, 526, 527, 528, 536, 559, 604, 660, 662, 687, 745, or 760, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the mutations 147A / H / M / Q, 257V, 291L, 395G / H / S, 402R / Y, 474A / C / G, 475A / S / V / Y, 478I / L, 514I, 515H / T, 526S, 527A, 528A / S, 536V, 559R, 604H, 660V, 662S, 687A, 745S / T / V, or 760T / W / Y, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least mutations 147H, 257V, 291L, 395H / S, 402R, 474A, 475A, 478L, 514I, 515T, 526S, 527A, 528A, 536V, 559R, 604H, 660V, 662S, 687A, 745T, or 760W, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the mutations C147H, A257V, F291L, P395H / S, G402R, L474A, E475A, M478L, V514I, G515T, A526S, Q527A, G528A, A536V, P559R, R604H, L660V, P662S, H687A, A745T, or F760W, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0152] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation at amino acid position 559, wherein the position is relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a substitution 559R, wherein the position is relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a substitution P559R, wherein the position is relative to the reference sequence corresponding to SEQ ID NO: 2.

[0153] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation at amino acid position 514, 515, or 760, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation 514I, 515T, or 760W, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation V514I, G515T, or F760W, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0154] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation at amino acid position 402, 604, or 660, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation 402R, 604H, or 660V, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation G402R, R604H, or L660V, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0155] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation at amino acid position 291, 395, 687, or 745, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation 291L, 395H, 687A, or 745T, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation F291L, P395H, H687A, or A745T, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0156] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation at amino acid position 257, 395, 474, 475, 536, or 662, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation 257V, 395S, 474A, 475A, 536V, or 662S, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation A257V, H395S, L474A, E475A, A536V, or P662S, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0157] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation at amino acid position 478, 515, 526, 527, or 528, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation 478L, 515T, 526S, 527A, or 528A, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation M478L, G515T, A526S, Q527A, or V528A, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0158] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation at amino acid position 402, 478, 515, 526, 527, 528, 559, 604, 660, or 760, or a combination thereof, wherein the positions are relative to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises a mutation at least 2, 3, 4, 5, 6, 7, 8, 9, or all of amino acid positions 402, 478, 515, 526, 527, 528, 559, 604, 660, and 760. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation 402R, 478L, 515T, 526S, 527A, 528A, 559R, 604H, 660V, or 760W, or a combination thereof, wherein the positions are relative to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or all of the mutations 402R, 478L, 515T, 526S, 527A, 528A, 559R, 604H, 660V, or 760W. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the mutations G402R, M478L, G515T, A526S, Q527A, G528A, P559R, R604H, L660V, or F760W, or a combination thereof, wherein the positions are relative to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or all of the mutations G402R, M478L, G515T, A526S, Q527A, G528A, P559R, R604H, L660V, or F760W.

[0159] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation at amino acid position 478, 515, 526, 527, 528, 559, or 760, or a combination thereof, wherein the positions are relative to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises a mutation at least 2, 3, 4, 5, 6, or all of amino acid positions 478, 515, 526, 527, 528, 559, and 760. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation 478L, 515T, 526S, 527A, 528A, 559R, or 760W, or a combination thereof, wherein the positions are relative to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least 2, 3, 4, 5, 6, or all of mutations 478L, 515T, 526S, 527A, 528A, 559R, and 760W. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the mutations M478L, G515T, A526S, Q527A, G528A, P559R, or F760W, or a combination thereof, wherein the positions are relative to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least 2, 3, 4, 5, 6, or all of the mutations M478L, G515T, A526S, Q527A, G528A, P559R, and F760W.

[0160] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises a mutation at each of amino acid positions 478, 515, 526, 527, 528, 559, and 760, wherein the positions are relative to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises each of the mutations 478L, 515T, 526S, 527A, 528A, 559R, and 760W, wherein the positions are relative to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises each of the mutations M478L, G515T, A526S, Q527A, G528A, P559R, and F760W, wherein the positions are relative to SEQ ID NO: 2.

[0161] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises a mutation at each of amino acid positions 402, 478, 515, 526, 527, 528, 559, 604, 660, and 760, wherein the positions are relative to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises each of the mutations 402R, 478L, 515T, 526S, 527A, 528A, 559R, 604H, 660V, and 760W, wherein the positions are relative to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises each of the mutations G402R, M478L, G515T, A526S, 527A, 528A, 559R, 604H, 660V, and 760W, wherein the positions are relative to SEQ ID NO: 2.

[0162] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises a mutation at each of amino acid positions 147, 257, 291, 395, 402, 474, 475, 478, 515, 526, 527, 528, 536, 559, 604, 660, 662, 687, 745, and 760, wherein the positions are relative to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises each of mutations 147H, 257V, 291L, 395S, 402R, 474A, 475A, 478L, 515T, 526S, 527A, 528A, 536V, 559R, 604H, 660V, 662S, 687A, 745T, and 760W, wherein the positions are relative to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises each of the mutations C147H, A257V, F291L, P395H / S, G402R, L474A, E475A, M478L, G515T, A526S, Q527A, G528A, A536V, P559R, R604H, L660V, P662S, H687A, A745T, and F760W, wherein the positions are relative to SEQ ID NO: 2.

[0163] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation at amino acid position 147, wherein the position is relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation at amino acid position 147H, wherein the position is relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation at amino acid position C147H, wherein the position is relative to the reference sequence corresponding to SEQ ID NO: 2.

[0164] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation or set of mutations at the following amino acid positions: 541 / 554 / 557, 516 / 541 / 550 / 554 / 557, 478 / 514 / 526 / 527, 478 / 516 / 526 / 527 / 529 / 541, 288 / 478 / 514 / 516 / 541 / 557, 288 / 478 / 516 / 541 / 550 / 554 / 557, 550 / 554 / 557, 478 / 399 / 514 / 516, 399 / 516 / 526 / 528 / 529 / 554 / 557, 478 / 514 / 541 / 550 / 554 / 557. 1. 288 / 399 / 514 / 554 / 557, 478 / 514 / 554 / 557, 288 / 478 / 526 / 550 / 554, 478 / 541 / 550 / 557, 541, 288 / 514 / 526 / 541, 554 / 557, 399 / 478, 288 / 514 / 554 / 557 57, 478 / 514 / 516 / 526 / 527 / 529 / 557, 399 / 541 / 550 / 554, 516 / 529 / 541 / 554 / 557, 478 / 541 / 554 / 557, 288 / 478 / 526 / 528 / 529 / 554 / 557, 288 / 514 / 516 / 5 29 / 541、478 / 514 / 516 / 541、478 / 514 / 557、399 / 541 / 557、399 / 541、288 / 399 / 516 / 527 / 541 / 557、288 / 478 / 514 / 541、288 / 514 / 516、664 / 698、399 / 478 / 516 / 541、399 / 528 / 550、399 / 478 / 514 / 541 / 557、288 / 478 / 514 / 516、224 / 664、478 / 526 / 527 / 528、288 / 478 / 514 / 516 / 541 / 550 / 554、478 / 514 / 541 / 554、 478 / 516, 478 / 514 / 516 / 541 / 554 / 557, 288 / 399 / 478 / 516 / 541, 288 / 478 / 514 / 554 / 557, 288 / 478 / 516, 288 / 399 / 516 / 554 / 557, 550 / 554, 288 / 550 / 554 、288 / 399 / 478 / 516 / 550 / 554、399 / 514 / 541 / 550 / 554、288 / 478 / 514 / 554、199、288 / 399 / 529 / 541、399 / 478 / 554、516 / 528 / 541 / 554 / 557、39 / 199 / 698、399 / 478 / 526 / 528 / 529、288 / 399 / 514 / 541 / 550 / 554、288 / 541、288 / 478 / 514 / 550 / 554 / 557、288 / 399 / 478 / 516、526 / 529 / 554 / 557、288 / 478 / 557、288 / 478 / 514 / 550 / 554、478 / 541 / 550 / 554、516 / 526 / 528、288 / 526 / 528 / 550 / 554 / 557 、199 / 664 / 698、514 / 526 / 528 / 529 / 550 / 557、288、399 / 554 / 557、288 / 554 / 557、399 / 514 / 541、288 / 478 / 514 / 527 / 554 / 557、399 / 478 / 516 / 541 / 554、87 / 541 / 554、288 / 550 / 554 / 557、527 / 529 / 541、528 / 529 / 554、478 / 554 / 557、478 / 514 / 516 / 541 / 550 / 554 / 557、199 / 216 / 644 / 664、396 / 478 / 514 / 554 / 557 / 694、241 / 644 / 663 / 664、399 / 478 / 554 / 557、288 / 399 / 550、288 / 516 / 526 / 527 / 528 / 557、399 / 526 / 527 / 528 / 529、288 / 478 / 526、399 / 478 / 514 / 516 / 526 / 527 / 528 / 529 / or 478 / 514 / 526 / 527 / 528, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least mutations or a set of mutations 541D / 554G / 557I, 516K / 541D / 550T / 554N / 557I, 478L / 514I / 526S / 527A, 478L / 516K / 526S / 527A / 529V / 541D, 288R / 478I / 514I / 516K / 541D / 557I, 288R / 478L / 516K / 541D / 550T / 554G / 557I, 550T / 554N / 557I, 478L, 399T,<h2 style=";text-align:left;direction:ltr">514I / 516K, 399T / 516K / 526S / 528A / 529V / 554N / 557I, 478L / 516K / 541D, 478I / 541D, 288R / 399T / 514I / 554G / 557I, 478L / 514I / 554N / 557I, 288R / 478L / 526S / 550T / 554N、478L / 541D / 550T / 557I、541D、288R / 514I / 526S / 541D、5 54N / 557I、399T / 478L、288R / 514I / 554N / 557I、478L / 514I / 516K / 526S / 527 A / 529V / 557I、399T / 541D / 550T / 554N、516K / 529V / 541D / 554G / 557I、478I / 541D / 554G / 557I、288R / 478L / 526S / 528S / 529V / 554N / 557I、288R / 514I / 51 6K / 529V / 541D, 478I / 514I / 516K / 541D, 478L / 514I / 557I, 399T / 541D / 557I, 399T / 541D, 288R / 399T / 516K / 527A / 541D / 557I, 288R / 478I / 514I / 541D, 28 8R / 514I / 516K、664A / 698E、399T / 478L / 516K / 541D、399T / 528A / 550T、399T / 478I / 514I / 541D / 557I、288R / 478L / 514I / 516K、224L / 664A、478L / 526S / 5 27A / 528S、288R / 478L / 514I / 516K / 541D / 550T / 554N、478L / 514I / 541D / 554 N、478L / 516K、478L / 514I / 516K / 541D / 554N / 557I、288R / 399T / 478L / 516K / 541D, 288R / 478L / 514I / 554N / 557I, 288R / 478I / 516K, 288R / 399T / 516K / 554G / 557I, 550T / 554G, 288R / 550T / 554N, 288R / 399T / 478L / 516K / 550T / 554N 399T / 514I / 541D / 550T / 554G, 288R / 478L / 514I / 554N, 199P, 288R / 399T / 529V / 541D, 399T / 478L / 554N, 516K / 528A / 541D / 554N / 557I, 39F / 199P / 698E<h2 style=";text-align:left;direction:ltr">399T / 478L / 526S / 528S / 529V, 288R / 399T / 514I / 541D / 550T / 554N, 288R / 541D, 288R / 478I / 514I / 550T / 554N / 557I, 288R / 399T / 478L / 516K, 5 50T / 554N、526S / 529V / 554G / 557I、288R / 478I / 557I、288R / 478L / 514I / 550T / 554N、478L / 541D / 550T / 554N、516K / 526S / 528S、288R / 526S / 528 S / 550T / 554N / 557I、199P / 664A / 698E、514I / 526S / 528S / 529V / 550T / 5 57I、288R、399T / 554N / 557I、288R / 554G / 557I、399T / 514I / 541D、288R / 478L / 514I / 527A / 554N / 557I, 399T / 478I / 516K / 541D / 554N, 87V / 541D / 554N, 288R / 550T / 554N / 557I, 527A / 529V / 541D, 528S / 529V / 554N, 478 L / 554G / 557I,478L / 514I / 516K / 541D / 550T / 554G / 557I,199P / 216K / 644Q / 664A,396S / 478I / 514I / 554G / 557I / 694D,478I,241E / 644Q / 663E / 664A, 399T / 478I / 554N / 557I, 288R / 399T / 550T, 288R / 516K / 526S / 527A / 528A / 557I, 399T / 526S / 527A / 528S / 529V, 288R / 478L / 526S, 399T / 4 78L / 514I / 516K / 526S / 527A / 528S / 529V / 541D、288R / 399T / 478I / 514I / 554G / 557I、288R / 399T / 514I / 516K / 528S、478L / 554N、399T / 514I / 516 K、514I、399T / 550T / 554N、478I / 516K、288R / 478L / 514I / 528A / 541D / 5 54N、478I / 514I / 516K / 554N、478L / 514I / 541D、288R / 541D / 554N / 557I、698E or 478L / 514I / 526S / 527A / 528A, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the mutations or sets of mutations E541D / S554G / L557I, R516K / E541D / S550T / S554N / L557I, M478L / V514I / A526S / Q527A, M478L / R516K / A526S / Q527A / A529V / E541D, E288R / M478I / V514I / R516K / E541D / L557I, E288R / M478L / R516K / E541D / S550T / S554N / L557I. 554N / L557I, M478L, N399T, V514I / R516K, N399T / R516K / A526S / G528A / A529V / S554N / L557I, M478L / R516K / E541D,, M478I / E541D, E288R / N 399T / V514I / S554G / L557I, M478L / V514I / S554N / L557I, E288R / M478L / A526S / S550T / S554N, M478L / E541D / S550T / L557I, E541D, E288R / V5 14I / A526S / E541D, S554N / L557I, N399T / M478L, E288R / V514I / S554N / L557I, M478L / V514I / R516K / A526S / Q527A / A529V / L557I, N399T / E54 1D / S550T / S554N, R516K / A529V / E541D / S554G / L557I, M478I / E541D / S554G / L557I, E288R / M478L / A526S / G528S / A529V / S554N / L557I, E288 R / V514I / R516K / A529V / E541D,, M478I / V514I / R516K / E541D, M478L / V514I / L557I, N399T / E541D / L557I, N399T / E541D, E288R / N399T / R516 K / Q527A / E541D / L557I, E288R / M478I / V514I / E541D, E288R / V514I / R516K, L664A / K698E, N399T / M478L / R516K / E541D, N399T / G528A / S550T,N399T / M478I / V514I / E541D / L557I、E288R / M478L / V514I / R516K、V224L / L664A、M478L / A526S / Q527A / G528S、E288R / M478L / V514I / R516K / E541D / S550T / S554N、M478L / V514I / E541D / S554N、M478L / R516K、M478L / V514I / R516K / E541D / S554N / L557I、E288R / N399T / M478L / R516K / E541D、E288R / M478L / V514I / S554N / L557I、E288R / M478I / R516K、E288R / N399T / R516K / S554G / L557I、S550T / S554G、E288R / S550T / S554N、E288R / N399T / M478L / R516K / S550T / S554N、N399T / V514I / E541D / S550T / S554G、E288R / M478L / V514I / S554N、R199P、E288R / N399T / A529V / E541D、N399T / M478L / S554N、R516K / G528A / E541D / S554N / L557I、Y39F / R199P / K698E、N399T / M478L / A526S / G528S / A529V、E288R / N399T / V514I / E541D / S550T / S554N、E288R / E541D、E288R / M478I / V514I / S550T / S554N / L557I、E288R / N399T / M478L / R516K、S550T / S554N、A526S / A529V / S554G / L557I、E288R / M478I / L557I、E288R / M478L / V514I / S550T / S554N、M478L / E541D / S550T / S554N、R516K / A526S / G528S、E288R / A526S / G528S / S550T / S554N / L557I、R199P / L664A / K698E、V514I / A526S / G528S / A529V / S550T / L557I、E288R、N399T / S554N / L557I、E288R / S554G / L557I、N399T / V514I / E541D、E288R / M478L / V514I / Q527A / S554N / L557I、N399T / M478I / R516K / E541D / S554N, A87V / E541D / S554N, E288R / S550T / S554N / L557I, Q527A / A529V / E541D, G 528S / A529V / S554N, M478L / S554G / L557I, M478L / V514I / R516K / E541D / S550T / S554G / L557I, R199P / R216K / R 644Q / L664A, A396S / M478I / V514I / S554G / L557I / G694D, M478I, R241E / R644Q / A663E / L664A, N399T / M478I / S 554N / L557I, E288R / N399T / S550T, E288R / R516K / A526S / Q527A / G528A / L557I, N399T / A526S / Q527A / G528S / A5 29V, E288R / M478L / A526S, N399T / M478L / V514I / R516K / A526S / Q527A / G528S / A529V / E541D, E288R / N399T / M4 78I / V514I / S554G / L557I, E288R / N399T / V514I / R516K / G528S, M478L / S554N, N399T / V514I / R516K, V514I, N39 9T / S550T / S554N, M478I / R516K, E288R / M478L / V514I / G528A / E541D / S554N, M478I / V514I / R516K / S554N, M478L / V514I / E541D, E288R / E541D / S554N / L557I, K698E, or M478L / V514I / A526S / Q527A / G528A, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0165] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the following set of mutations at amino acid positions: 478 / 514 / 515 / 526 / 527 / 528, 478 / 514 / 526 / 527 / 528 / 594, 478 / 514 / 526 / 527 / 528 / 559, 478 / 514 / 526 / 527 / 528 / 760, 478 / 514 / 526 / 527 / 528 / 541, 478 / 514 / 526 / 527 / 528 / 753, 478 / 514 / 526 / 527 / 528 / 398. 478 / 514 / 526 / 527 / 528 / 558, 478 / 514 / 526 / 527 / 528 / 601, 478 / 514 / 526 / 527 / 528 / 762, 478 / 514 / 526 / 527 / 528 / 589 or 478 / 514 / 526 / 527 / 528 / 544, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the mutation set 478L / 514I / 515H / 526S / 527A / 528A, 478L / 514I / 526S / 527A / 528A / 594A, 478L / 514I / 526S / 527A / 528A / 559R, 478L / 514I / 526S / 527A / 528A / 760W, 478L / 514I / 526S / 527A / 528A / 541S, 478L / 514I / 526S / 527A / 528A / 541N, 478L / 514I / 526S / 527A / 528A / 753A, 402R / 478L / 514I / 526S / 527A / 528A, 478L / 514I / 526S / 5 27A / 528A / 760T, 398Q / 478L / 514I / 526S / 527A / 528A, 478L / 514I / 526S / 527A / 528A / 758G, 478L / 514I / 526S / 527A / 528 A / 760Y, 399G / 478L / 514I / 526S / 527A / 528A, 478L / 514I / 526S / 527A / 528A / 575W, 257V / 478L / 514I / 526S / 527A / 528A / 758G、478L / 514I / 526S / 527A / 528A / 558L、402Y / 478L / 514I / 526S / 527A / 528A、478L / 514I / 526S / 527A / 528A / 558T、47 8L / 514I / 526S / 527A / 528A / 601T, 398G / 478L / 514I / 526S / 527A / 528A, 478L / 514I / 526S / 527A / 528A / 762A, 478L / 514I / 526S / 527A / 528A / 558G, or 478L / 514I / 526S / 527A / 528A / 589G, or 478L / 514I / 526S / 527A / 528A / 544M, wherein the positions are relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the mutation or sets of mutations provided in Tables 5.1 and 5.2 relative to the reference sequence corresponding to SEQ ID NO: 2.

[0166] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the following sets of mutations at amino acid positions: 478 / 514 / 515 / 526 / 527 / 528 / 559 / 760, 478 / 515 / 526 / 527 / 528 / 559 / 760, 514 / 515 / 526 / 527 / 528 / 559 / 760, 478 / 514 / 526 / 527 / 528 / 559 / 660, 178 / 478 / 514 / 526 / 527 / 528 / 559 / 760. / 515 / 526 / 527 / 528 / 559、377 / 478 / 514 / 515 / 526 / 527 / 528 / 559、478 / 514 / 515 / 526 / 527 / 528 / 559、478 / 515 / 526 / 527 / 528 / 559、402 / 478 / 514 / 526 / 527 / 528 / 559、478 / 503 / 514 / 526 / 527 / 528 / 559、239 / 478 / 514 / 526 / 527 / 528 / 559、73 / 478 / 514 / 526 / 527 / 528 / 559、478 / 514 / 526 / 527 / 541 / 559、59 / 478 / 514 / 526 / 527 / 528 / 559、478 / 514 / 515 / 526 / 527 / 528 / 559 / 575、3 / 478 / 514 / 515 / 526 / 527 / 528、377 / 478 / 514 / 515 / 526 / 527 / 541 / 554 / 557 / 559、402 / 478 / 515 / 526 / 527 / 528 / 541、478 / 515 / 526 / 527 / 528 / 541, 478 / 515 / 526 / 527 / 528, 478 / 514 / 515 / 526 / 527 / 528, 478 / 514 / 515 / 526 / 527 / 528 / 541, 377 / 478 / 514 / 515 / 526 / 527 / 528, 402 / 478 / 514 / 515 / 526 / 527 / 528 or 478 / 514 / 515 / 526 / 527 / 528 / 541 / 557 / 559, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:2.In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the mutation set 478L / 514I / 515T / 526S / 527A / 528A / 559R / 760W, 478L / 515T / 526S / 527A / 528A / 559R / 760W, 514I / 515T / 526S / 527A / 528A / 559R / 760W, 478L / 514I / 526S / 527A / 528A / 559R / 660V, 178L / 478L / 514I / 526S / 527A / 528A / 559R, 478L / 514I / 515T / 526S / 527A / 528A / 559R A / 528A / 559R, 377C / 478L / 514I / 515T / 526S / 527A / 528A / 559R, 478L / 514 I / 515T / 526S / 527A / 528A / 559R, 478L / 515T / 526S / 527A / 528A / 559R, 402Y / 478L / 514I / 526S / 527A / 528A / 559R、478L / 503R / 514I / 526S / 527A / 528A / 559R、239V / 478L / 514I / 526S / 527A / 528A / 559R、73I / 478L / 514I / 526S / 52 7A / 528A / 559R, 478L / 514I / 526S / 527A / 541N / 559R, 59K / 478L / 514I / 526 S / 527A / 528A / 559R, 478L / 514I / 515T / 526S / 527A / 528A / 559R / 575W, 3N / 4 78L / 514I / 515T / 526S / 527A / 528A, 377C / 478L / 514I / 515H / 526S / 527A / 54 1N / 554N / 557I / 559R, 402R / 478L / 515T / 526S / 527A / 528A / 541N, 478L / 515 9A / 528A / 541N, 478L / 514I / 515T / 526S / 527A / 528A / 541N, 478L / 514I / 515T / 526S / 527A / 528A / 541N, 377C / 478L / 514I / 515T / 526S / 527A / 528A, 402Y / 478L / 514I / 515H / 526S / 527A / 528A, or 478L / 514I / 515T / 526S / 527A / 528A / 541N / 557I / 559R, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO:2.In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the set of mutations provided in Tables 6.1 and 6.2 relative to the reference sequence corresponding to SEQ ID NO:2.

[0167] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the following sets of mutations at amino acid positions: 402 / 478 / 515 / 526 / 527 / 541 / 559 / 660 / 760, 257 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760, 178 / 257 / 402 / 404 / 478 / 515 / 526 / 527 / 557 / 559 / 660 / 760, 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760, 257 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760. 60 / 760、377 / 402 / 478 / 515 / 526 / 527 / 528 / 541 / 559 / 660 / 760、257 / 377 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 660 / 760、478 / 483 / 515 / 526 / 527 / 528 / 559 / 660 / 760、257 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 760、257 / 478 / 515 / 526 / 527 / 528 / 559 / 760、257 / 377 / 478 / 515 / 526 / 527 / 528 / 559 / 760、178 / 478 / 515 / 526 / 527 / 528 / 559 / 660 / 760、257 / 377 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 760、478 / 515 / 526 / 527 / 528 / 559 / 660 / 760、178 / 257 / 377 / 402 / 403 / 478 / 48 3 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760、377 / 478 / 515 / 526 / 527 / 528 / 559 / 760、178 / 402 / 403 / 478 / 515 / 526 / 527 / 541 / 559 / 660 / 760、377 / 478 / 515 / 5 26 / 527 / 528 / 559 / 601 / 660 / 760、178 / 377 / 478 / 515 / 526 / 527 / 528 / 559 / 760、377 / 478 / 490 / 515 / 526 / 527 / 528 / 559 / 660 / 760、402 / 403 / 478 / 483 / 515 / 526 / 527 / 528 / 554 / 557 / 559 / 760、178 / 257 / 377 / 478 / 515 / 526 / 527 / 528 / 558 / 559 / 660 / 760、257 / 402 / 403 / 478 / 483 / 515 / 526 / 527 / 528 / 559 / 660 / 760、178 / 478 / 515 / 526 / 527 / 528 / 554 / 557 / 558 / 559 / 660 / 760、478 / 515 / 526 / 527 / 528 / 554 / 557 / 559 / 660 / 760、178 / 478 / 483 / 515 / 526 / 527 / 528 / 541 / 545 / 554 / 557 / 559 / 604 / 660 / 760、478 / 515 / 526 / 527 / 528 / 559 / 760 / 792、402 / 403 / 478 / 483 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760、178 / 478 / 515 / 526 / 527 / 528 / 559 / 760、474 / 478 / 515 / 526 / 527 / 528 / 559 / 760、257 / 377 / 478 / 515 / 526 / 527 / 528 / 541 / 559 / 604 / 760、478 / 515 / 526 / 527 / 528 / 559 / 743 / 760、396 / 478 / 515 / 526 / 527 / 528 / 559 / 760、478 / 479 / 515 / 526 / 527 / 528 / 559 / 760、377 / 403 / 478 / 515 / 526 / 527 / 528 / 541 / 559 / 604 / 760、475 / 478 / 515 / 526 / 527 / 528 / 559 / 760、178 / 257 / 403 / 478 / 515 / 526 / 527 / 559 / 760、448 / 478 / 515 / 526 / 527 / 528 / 559 / 760、478 / 515 / 526 / 527 / 528 / 559 / 577 / 760、395 / 478 / 515 / 526 / 527 / 528 / 559 / 760、478 / 515 / 526 / 527 / 528 / 559 / 760 / 769、478 / 515 / 526 / 527 / 528 / 559 / 691 / 760、478 / 515 / 526 / 527 / 528 / 559 / 677 / 760、478 / 515 / 526 / 527 / 528 / 559 / 704 / 760、377 / 478 / 515 / 526 / 527 / 528 / 541 / 559 / 575 / 604 / 660 / 760、178 / 257 / 478 / 515 / 526 / 527 / 528 / 559 / 601 / 604 / 760、478 / 515 / 526 / 527 / 528 / 559 / 685 / 760、377 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760、478 / 515 / 526 / 527 / 528 / 559 / 749 / 760、478 / 482 / 515 / 526 / 527 / 528 / 559 / 760、291 / 478 / 515 / 526 / 527 / 528 / 559 / 760、478 / 515 / 526 / 527 / 528 / 559 / 579 / 760、178 / 478 / 515 / 526 / 527 / 528 / 541 / 550 / 559 / 604 / 760、477 / 478 / 515 / 526 / 527 / 528 / 559 / 760、178 / 257 / 478 / 515 / 526 / 527 / 528 / 559 / 760、257 / 402 / 478 / 490 / 515 / 526 / 527 / 541 / 559 / 660 / 760、328 / 478 / 515 / 526 / 527 / 528 / 559 / 760、478 / 515 / 526 / 527 / 528 / 559 / 740 / 760、478 / 483 / 515 / 526 / 527 / 528 / 559 / 660 / 760、257 / 377 / 478 / 515 / 526 / 527 / 528 / 559 / 760、368 / 478 / 515 / 526 / 527 / 528 / 559 / 760、478 / 515 / 526 / 527 / 528 / 559 / 760 / 792、478 / 515 / 526 / 527 / 528 / 559 / 721 / 745 / 760、478 / 515 / 526 / 527 / 528 / 559 / 578 / 760、478 / 515 / 526 / 527 / 528 / 559 / 687 / 760、478 / 515 / 526 / 527 / 528 / 541 / 559 / 601 / 604 / 760、257 / 478 / 515 / 526 / 527 / 559 / 760、257 / 478 / 515 / 526 / 527 / 554 / 559 / 760、478 / 515 / 526 / 527 / 528 / 559 / 760 / 768、478 / 515 / 526 / 527 / 528 / 559 / 760 / 770、257 / 377 / 478 / 483 / 515 / 526 / 527 / 528 / 541 / 559 / 760、478 / 515 / 526 / 527 / 528 / 542 / 559 / 760、257 / 402 / 403 / 478 / 515 / 526 / 527 / 528 / 559 / 760、257 / 403 / 478 / 515 / 526 / 527 / 528 / 541 / 559 / 760、478 / 515 / 526 / 527 / 528 / 559 / 604 / 760、402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 760、257 / 316 / 478 / 515 / 526 / 527 / 528 / 541 / 559 / 575 / 604 / 760、327 / 478 / 515 / 526 / 527 / 528 / 559 / 760、or 257 / 377 / 478 / 515 / 526 / 527 / 528 / 559 / 760, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the mutation sets 402R / 478L / 515T / 526S / 527A / 541D / 559R / 660V / 760W, 257V / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W, 178L / 257V / 402R / 404T / 478L / 515T / 526S / 527A / 557I / 559R / 660V / 760W, 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W, 257V / 402Y / 478L / 515T / 526S / 527 A / 528A / 541G / 559R / 660V / 760W, 377C / 402Y / 478L / 515T / 526S / 527A / 528A / 541N / 559R / 660V / 760W, 257V / 377 C / 402Y / 478L / 515T / 526S / 527A / 528A / 559R / 660V / 760W, 478L / 483D / 515T / 526S / 527A / 528A / 559R / 660V / 76 0W, 257V / 402Y / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 257V / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 2 57V / 377C / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 178L / 478L / 515T / 526S / 527A / 528A / 559R / 660V / 760W, 257V / 377C / 402Y / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 478L / 515T / 526S / 527A / 528A / 559R / 660V / 760W,<h2 style=";text-align:left;direction:ltr">178L / 257V / 377C / 402R / 403I / 478L / 483D / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W、377C / 478L / 515T / 526S / 527A / 528A / 559R / 760W、178L / 402R / 403I / 478L / 515T / 526S / 527A / 541G / 559R / 660V / 760W、377C / 478L / 515T / 5 26S / 527A / 528A / 559R / 601T / 660V / 760W、178L / 377C / 478L / 515T / 526S / 527 A / 528A / 559R / 760W、377C / 478L / 490W / 515T / 526S / 527A / 528A / 559R / 660V / 760W、402R / 403I / 478L / 483D / 515T / 526S / 527A / 528A / 554N / 557I / 559R / 76 0W、178L / 257V / 377C / 478L / 515T / 526S / 527A / 528A / 558T / 559R / 660V / 760W、257V / 402Y / 403I / 478L / 483D / 515T / 526S / 527A / 528A / 559R / 660V / 760W、17 8L / 478L / 515T / 526S / 527A / 528A / 554N / 557I / 558T / 559R / 660V / 760W、478L / 515T / 526S / 527A / 528A / 554N / 557I / 559R / 660V / 760W、178L / 478L / 483D / 5 15T / 526S / 527A / 528A / 541N / 545L / 554N / 557I / 559R / 604H / 660V / 760W、478L / 515T / 526S / 527A / 528A / 559R / 760W / 792F、402Y / 403I / 478L / 483D / 515T / 5 26S / 527A / 528A / 559R / 604H / 660V / 760W、178L / 478L / 515T / 526S / 527A / 528A / 559R / 760W、474A / 478L / 515T / 526S / 527A / 528A / 559R / 760W、257V / 377C / 478L / 515T / 526S / 527A / 528A / 541D / 559R / 604H / 760W、478L / 515T / 526S / 527A / 528A / 559R / 743H / 760W、396L / 478L / 515T / 526S / 527A / 528A / 559R / 760W、<h2 style=";text-align:left;direction:ltr">478L / 515T / 526S / 527A / 528A / 559R / 743V / 760W, 396R / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 478L / 479L / 515T / 526S / 527A / 528A / 559R / 760W, 377 C / 403I / 478L / 515T / 526S / 527A / 528A / 541D / 559R / 604H / 760W、475A / 478L / 515T / 526S / 527A / 528A / 559R / 760W、178L / 257V / 403I / 478L / 515T / 526S / 5 27A / 559R / 760W, 448I / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 478L / 515T / 526S / 527A / 528A / 559R / 577S / 760W, 395G / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 478L / 515T / 526S / 527A / 528A / 559R / 760W / 769Q, 475V / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 448C / 478L / 515T / 526S / 527A / 528A / 55 9R / 760W, 478L / 515T / 526S / 527A / 528A / 559R / 691W / 760W, 478L / 515T / 526S / 527A / 528A / 559R / 677L / 760W, 478L / 515T / 526S / 527A / 528A / 559R / 704L / 760W, 377C / 478L / 515T / 526S / 527A / 528A / 541N / 559R / 575W / 604H / 660V / 760W, 178L / 257V / 478L / 515T / 526S / 527A / 528A / 559R / 601T / 604H / 760W, 478 L / 515T / 526S / 527A / 528A / 559R / 685R / 760W、377C / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W、478L / 515T / 526S / 527A / 528A / 559R / 749M / 7 60W, 396F / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 395H / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 475S / 478L / 515T / 526S / 527A / 528A / 559R / 760W<h2 style=";text-align:left;direction:ltr">474C / 478L / 515T / 526S / 527A / 528A / 559R / 760W、478L / 482S / 515T / 526S / 527A / 528A / 559R / 760W、291L / 478L / 515T / 526S / 527A / 528A / 559R / 760W、44 8V / 478L / 515T / 526S / 527A / 528A / 559R / 760W、478L / 515T / 526S / 527A / 528A / 559R / 579Q / 760W、178L / 478L / 515T / 526S / 527A / 528A / 541G / 550C / 559R / 604H / 760W, 477V / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 178L / 257V / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 257V / 402R / 478L / 490W / 515T / 52 6S / 527A / 541G / 559R / 660V / 760W、328L / 478L / 515T / 526S / 527A / 528A / 559 R / 760W、395S / 478L / 515T / 526S / 527A / 528A / 559R / 760W、478L / 515T / 526S / 527A / 528A / 559R / 740V / 760W, 478L / 483D / 515T / 526S / 527A / 528A / 559R / 660V / 760W, 257V / 377C / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 478L / 51 5T / 526S / 527A / 528A / 559R / 579L / 760W、368H / 478L / 515T / 526S / 527A / 528A / 559R / 760W、478L / 515T / 526S / 527A / 528A / 559R / 760W / 792Y、478L / 515T / 526S / 527A / 528A / 559R / 721A / 745T / 760W, 474A / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 478L / 515T / 526S / 527A / 528A / 559R / 743H / 760W, 478L / 51 5T / 526S / 527A / 528A / 559R / 578A / 760W、478L / 515T / 526S / 527A / 528A / 559R / 687A / 760W、478L / 515T / 526S / 527A / 528A / 541D / 559R / 601T / 604H / 760W、<h2 style=";text-align:left;direction:ltr">328V / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 396L / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 475Y / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 395 G / 478L / 515T / 526S / 527A / 528A / 559R / 760W、257V / 478L / 515T / 526S / 527A / 559R / 760W、257V / 478L / 515T / 526S / 527A / 554N / 559R / 760W、478L / 515T / 52 6S / 527A / 528A / 559R / 760W / 768S、257V / 478L / 515T / 526S / 527A / 528A / 559R / 760W、257V / 478L / 515T / 526S / 527A / 528A / 559R / 760W、396P / 478L / 515T / 5 26S / 527A / 528A / 559R / 760W、478L / 515T / 526S / 527A / 528A / 559R / 760W / 770E、257V / 377C / 478L / 483D / 515T / 526S / 527A / 528A / 541N / 559R / 760W、478L / 515T / 526S / 527A / 528A / 542R / 559R / 760W、257V / 402Y / 403I / 478L / 515T / 526S / 527A / 528A / 559R / 760W、257V / 403I / 478L / 515T / 526S / 527A / 528A / 541 N / 559R / 760W, 474G / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 478L / 515T / 526S / 527A / 528A / 559R / 604H / 760W, 402Y / 478L / 515T / 526S / 527A / 528A / 55 9R / 604H / 760W、478L / 515T / 526S / 527A / 528A / 559R / 691S / 760W、257V / 316G / 478L / 515T / 526S / 527A / 528A / 541D / 559R / 575W / 604H / 760W、327L / 478L / 5 15T / 526S / 527A / 528A / 559R / 760W、478L / 515T / 526S / 527A / 528A / 559R / 704V / 760W、403I / 478L / 515T / 526S / 527A / 528A / 541N / 559R / 604H / 660V / 760W、257V / 377C / 403I / 478L / 483D / 515T / 526S / 527A / 528A / 541D / 559R / 601T / 604H / 760W、478L / 515T / 5 26S / 527A / 528A / 559R / 740S / 760W, 478L / 515T / 526S / 527A / 528A / 559R / 760W, 257V / 478L / 515T / 526 S / 527A / 528A / 559R / 760W, 402R / 478L / 515T / 526S / 527A / 541G / 559R / 601T / 760W, 478L / 515T / 526S / 527A / 528A / 559R / 760W, or 257V / 377C / 478L / 515T / 526S / 527A / 528A / 559R / 760W, wherein the positions are relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered DNA polymerase comprises an amino acid sequence comprising at least the set of mutations provided in Tables 7.1 and 7.2 relative to the reference sequence corresponding to SEQ ID NO: 2.

[0168] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the following sets of mutations at amino acid positions: 79 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760, 257 / 402 / 474 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760, 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760, 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760, 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760. 7 / 528 / 559 / 604 / 660 / 745 / 760、291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、257 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 677 / 760、303 / 338 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760、402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760、359 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760、402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 677 / 704 / 760、395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 7 60, 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 671 / 760, 233 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760, 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 0 / 699 / 760, 379 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760, 257 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760 / 792, 257 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760、21 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 66 0 / 760、257 / 402 / 448 / 474 / E475 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760、402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 713 / 760、402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 641 / 660 / 760、402 / 442 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760、402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 656 / 660 / 760、402 / 475 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 704 / 760、402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 742 / 760、402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 667 / 760、257 / 402 / 448 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760、257 / 316 / 395 / 402 / 478 / 482 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760 / 804、402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 704 / 760、178 / 257 / 395 / 402 / 478 / 482 / 515 / 526 / 527 / 528 / 559 / 579 / 604 / 660 / 687 / 745 / 760、257 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 579 / 604 / 660 / 760、257 / 291 / 395 / 402 / 475 / 478 / 515 / 526 / 527 / 528 / 541 / 559 / 604 / 660 / 687 / 760、257 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760 / 768、402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / P667 / 760、402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760 / 812、402 / 475 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760、402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 662 / 760、402 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 760、402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 748 / 760、402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760 / 808、291 / 316 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 760、402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 613 / 660 / 760、257 / 291 / 316 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760、257 / 291 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 579 / 604 / 660 / 760、257 / 316 / 402 / 478 / 515 / 526 / 527 / 528 / 541 / 559 / 604 / 660 / 745 / 760、351 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760、257 / 316 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 745 / 760、185 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760、257 / 316 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 760、291 / 316 / 402 / 475 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、257 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 740 / 745 / 760、402 / 450 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760、402 / 473 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760、124 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760、402 / 430 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760、402 / 413 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760、66 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760 or 365 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 760, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the mutation sets 79A / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W, 257V / 402R / 474A / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W, 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W, T / 526S / 527A / 528A / 559R / 562K / 604H / 660V / 760W, 178L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 745T / 760W, 2 91L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 257V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 677L / 760W, 303T / 338V / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W, 402R / 474A / 475A / 478L / 51 5T / 526S / 527A / 528A / 559R / 604H / 660V / 760W, 79R / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W, 359G / 402R / 478L / 5 15T / 526S / 527A / 528A / 559R / 604H / 660V / 760W, 79T / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W, 402R / 474A / 475S / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 677L / 704V / 760W, 79M / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W,395S / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 760W、402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 671W / 760W、233K / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W、402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 699V / 760W、379G / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W、257V / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W / 792F、257V / 395S / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W、21A / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W、257V / 402R / L448C / 474A / 475S / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W、402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 713G / 760W、402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 641A / 660V / 760W、402R / 442G / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W、402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 656S / 660V / 760W、402R / 475S / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 704V / 760W、402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 742K / 760W、402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 667W / 760W、21N / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W、257V / 402R / L448I / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W、257V / 316G / 395H / 402R / 478L / 482S / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W / 804R、402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 704V / 760W、178L / 257V / 395S / 402R / 478L / 482S / 515T / 526S / 527A / 528A / 559R / 579L / 604H / 660V / 687A / 745T / 760W、257V / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 579L / 604H / 660V / 760W、257V / 291L / 395S / 402R / E475V / 478L / 515T / 526S / 527A / 528A / 541N / 559R / 604H / 660V / 687A / 760W、257V / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W / 768S、402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 667S / 760W、402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W / 812S、402R / 475A / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W、402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 662S / 760W、402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 760W、402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 748G / 760W、402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W / 808W、291L / 316G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 760W、402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 613H / 660V / 760W、257V / 291L / 316G / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W、257V / 291L / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 579L / 604H / 660V / 760W、257V / 316G / 402R / 478L / 515T / 526S / 527A / 528A / 541N / 559R / 604H / 660V / 745T / 760W、351H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W、257V / 316G / 395G / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 745T / 760W、185T / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W、257V / 316G / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 760W、402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W / 808Y、291L / 316G / 402R / 475V / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、257V / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 740V / 745T / 760W、402R / 450V / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W、402R / 473N / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W、21G / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W、124A / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W、402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 613R / 660V / 760W、402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 656Q / 660V / 760W、402R / 430G / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W, 402R / 413T / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W, 233L / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W, 66R / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W, 365T / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W, or 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W / 812R, wherein the positions are relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the set of mutations provided in Tables 8.1 and 8.2 relative to the reference sequence corresponding to SEQ ID NO: 2.

[0169] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the following sets of mutations at amino acid positions: 291 / 395 / 402 / 438 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760, 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 697 / 745 / 760, 291 / 395 / 402 / 457 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760, 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 716 / 745 / 760、79 / 291 / 395 / 402 / 474 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 687 / 699 / 745 / 760、291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 687 / 745 / 760、291 / 392 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 708 / 745 / 760、291 / 395 / 402 / 431 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 395 / 402 / 474 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 687 / 699 / 745 / 760、291 / 395 / 402 / 453 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、2 91 / 395 / 402 / 427 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、79 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 699 / 745 / 760、291 / 378 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 370 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 706 / 745 / 760、257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、291 / 395 / 402 / 474 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 562 / 604 / 660 / 687 / 745 / 760、257 / 291 / 395 / 402 / 474 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 687 / 745 / 760、79 / 257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 562 / 604 / 660 / 687 / 745 / 760、79 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 687 / 699 / 745 / 760、291 / 372 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、257 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760 / 792、291 / 390 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、79 / 291 / 395 / 402 / 474 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 687 / 699 / 745 / 760 / 792、291 / 395 / 402 / 409 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 395 / 402 / 443 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 395 / 402 / 448 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 381 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 707 / 745 / 760、291 / 395 / 402 / 416 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 662 / 687 / 745 / 760、79 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 687 / 745 / 760 / 792、291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、291 / 382 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、257 / 291 / 395 / 402 / 474 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 562 / 604 / 660 / 662 / 687 / 745 / 760、257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760 / 792、79 / 291 / 395 / 402 / 474 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 662 / 687 / 699 / 745 / 760、291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 725 / 745 / 760、291 / 395 / 402 / 447 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、79 / 257 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 699 / 745 / 760、291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 701 / 745 / 760、257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 562 / 604 / 660 / 662 / 687 / 745 / 760、291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 702 / 745 / 760、291 / 342 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 716 / 745 / 760、233 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 562 / 604 / 660 / 687 / 713 / 745 / 760、291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 704 / 745 / 760、291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 562 / 604 / 660 / 662 / 687 / 745 / 760、291 / 395 / 402 / 454 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 327 / 395 / 402 / 427 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、257 / 291 / 316 / 395 / 402 / 474 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 687 / 745 / 760 / 792、291 / 395 / 402 / 417 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 395 / 402 / 425 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 562 / 604 / 660 / 687 / 745 / 760、291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 714 / 745 / 760、233 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 303 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 613 / 660 / 687 / 745 / 760、21 / 79 / 291 / 303 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 562 / 604 / 613 / 660 / 687 / 745 / 760、79 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 562 / 604 / 660 / 687 / 745 / 760、291 / 395 / 402 / 435 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、257 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 687 / 699 / 745 / 760 / 792、79 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 687 / 745 / 760、291 / 395 / 402 / 453 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / P667 / 687 / 745 / 760、79 / 291 / 303 / 338 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 613 / 660 / 687 / 745 / 760 / 804 / 812、79 / 257 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760 / 792、79 / 291 / 338 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 562 / 604 / 660 / 687 / 745 / 760、79 / 233 / 291 / 303 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 662 / 687 / 745 / 760、79 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 662 / 687 / 699 / 745 / 760、79 / 291 / 395 / 402 / 475 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 662 / 687 / 745 / 760、79 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 562 / 604 / 660 / 687 / 699 / 745 / 760、291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、21 / 79 / 291 / 338 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 641 / 660 / 687 / 699 / 745 / 760、291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 662 / 687 / 745 / 760、21 / 79 / 291 / 395 / 402 / 442 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 699 / 745 / 760、79 / 291 / 303 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 662 / 687 / 745 / 760、79 / 178 / 257 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 662 / 687 / 699 / 745 / 760、79 / 257 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 562 / 604 / 660 / 662 / 687 / 745 / 760、291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 562 / 604 / 660 / 687 / 699 / 745 / 760、291 / 359 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 613 / 660 / 687 / 742 / 745 / 760 / 808 / 812、21 / 79 / 291 / 338 / 379 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 562 / 604 / 660 / 662 / 687 / 745 / 760 / 804 / 812、291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 731 / 745 / 760、21 / 79 / 291 / 359 / 395 / 402 / 442 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 662 / 687 / 745 / 760、79 / 291 / 338 / 359 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 562 / 604 / 660 / 687 / 745 / 760、21 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 613 / 660 / 662 / 687 / 742 / 745 / 760、21 / 79 / 291 / 303 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 662 / 687 / 699 / 742 / 745 / 760 / 808、21 / 79 / 291 / 338 / 395 / 402 / 442 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 613 / 660 / 687 / 699 / 745 / 760、79 / 291 / 303 / 338 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 562 / 604 / 613 / 660 / 662 / 687 / 745 / 760、291 / 303 / 338 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 562 / 604 / 660 / 687 / 699 / 745 / 760 / 808 / 812、79 / 257 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、257 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 662 / 687 / 745 / 760 / 792、79 / 291 / 359 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 713 / 742 / 745 / 760 / 808、21 / 291 / 359 / 395 / 402 / 442 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760 / 808 / 812、79 / 257 / 291 / 395 / 402 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、21 / 79 / 291 / 338 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、79 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 713 / 745 / 760、79 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 562 / 604 / 641 / 660 / 687 / 745 / 760、291 / 373 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、79 / 178 / 257 / 291 / 316 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、21 / 233 / 291 / 338 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 562 / 604 / 660 / 662 / 687 / 745 / 760、257 / 291 / 316 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 687 / 745 / 760、21 / 79 / 291 / 303 / 395 / 402 / 442 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 662 / 687 / 745 / 760、21 / 79 / 233 / 291 / 303 / 338 / 379 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 562 / 604 / 660 / 687 / 713 / 745 / 760、291 / 338 / 395 / 402 / 442 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 699 / 745 / 760、291 / 395 / 402 / 426 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、21 / 291 / 338 / 359 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760 / 808 / 812、79 / 178 / 233 / 291 / 395 / 402 / 442 / 478 / 515 / 526 / 527 / 528 / 559 / 562 / 604 / 660 / 687 / 745 / 760 / 812、79 / 291 / 395 / 402 / 474 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 699 / 745 / 760、21 / 79 / 291 / 338 / 379 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 662 / 687 / 745 / 760、291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 721 / 745 / 760、21 / 79 / 233 / 291 / 338 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 613 / 641 / 660 / 687 / 745 / 760、291 / 374 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 338 / 359 / 379 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 395 / 402 / 461 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 395 / 402 / 451 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 715 / 745 / 760、79 / 291 / 303 / 338 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 641 / 660 / 687 / 713 / 745 / 760、291 / 393 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、79 / 257 / 291 / 395 / 402 / 474 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 687 / 699 / 745 / 760、79 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 395 / 402 / 437 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、257 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 562 / 604 / 660 / 662 / 687 / 745 / 760、21 / 79 / 291 / 379 / 395 / 402 / 442 / 478 / 515 / 526 / 527 / 528 / 559 / 562 / 604 / 660 / 662 / 687 / 745 / 760、291 / 362 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 395 / 402 / 410 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 338 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760 / 812、21 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 662 / 687 / 745 / 760 / 808 / 812、21 / 291 / 303 / 338 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 395 / 402 / 418 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 338 / 395 / 402 / 442 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 395 / 402 / 449 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 379 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 641 / 660 / 687 / 745 / 760、291 / 316 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 562 / 604 / 660 / 687 / 745 / 760、257 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 687 / 745 / 760 / 792、21 / 79 / 291 / 395 / 402 / 442 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 613 / 660 / 687 / 745 / 760、257 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760 / 792、257 / 291 / 395 / 402 / 474 / 478 / 515 / 526 / 527 / 528 / 529 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、291 / 395 / 402 / 412 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、79 / 257 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 699 / 745 / 760、257 / 291 / 316 / 395 / 402 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 687 / 745 / 760 / 792、291 / 388 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 380 / 395 / 402 / 454 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、21 / 79 / 233 / 291 / 303 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 613 / 660 / 687 / 699 / 745 / 760 / 809、291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 724 / 745 / 760、21 / 79 / 291 / 338 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 613 / 660 / 687 / 745 / 760、79 / 178 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 687 / 745 / 760 / 792、291 / 395 / 402 / 449 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 747 / 760、21 / 79 / 291 / 379 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 613 / 660 / 687 / 713 / 745 / 760、79 / 291 / 316 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 699 / 745 / 760、291 / 376 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、21 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 562 / 604 / 660 / 662 / 687 / 745 / 760、79 / 257 / 262 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 562 / 604 / 660 / 687 / 699 / 745 / 760、291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 718 / 745 / 760、291 / 395 / 402 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 562 / 604 / 660 / 662 / 687 / 699 / 745 / 760、79 / 233 / 291 / 338 / 359 / 379 / 395 / 402 / 442 / 478 / 515 / 526 / 527 / 528 / 559 / 562 / 604 / 613 / 660 / 687 / 745 / 760 / 812、291 / 371 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 316 / 395 / 402 / 475 / 478 / 515 / 526 / 527 / 528 / 559 / 562 / 604 / 660 / 662 / 687 / 745 / 760 / 792、79 / 257 / 291 / 316 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760 / 792、291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 728 / 745 / 760、79 / 233 / 291 / 338 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 395 / 402 / 432 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、79 / 178 / 257 / 291 / 316 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 699 / 745 / 760、79 / 291 / 303 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 713 / 745 / 760、233 / 291 / 379 / 395 / 402 / 442 / 478 / 515 / 526 / 527 / 528 / 559 / 562 / 604 / 613 / 660 / 662 / 687 / 713 / 745 / 760、21 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 742 / 745 / 760、291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 732 / 745 / 760、79 / 257 / 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 562 / 604 / 660 / 662 / 687 / 699 / 745 / 760、21 / 233 / 291 / 338 / 379 / 395 / 402 / 442 / 478 / 515 / 526 / 527 / 528 / 559 / 562 / 604 / 613 / 660 / 662 / 687 / 745 / 760 / 812、291 / 395 / 402 / 429 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、2- / 291 / 374 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 723 / 745 / 760、291 / 395 / 402 / 434 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760、291 / 395 / 402 / 420 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760 or 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 699 / 745 / 760, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the set of mutations,

[0170] <h2 style=";text-align:left;direction:ltr">291L / 395H / 402R / 438F / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 697I / 745T / 760W、291L / 395H / 402R / A457W / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 716E / 745T / 760W、79R / 291L / 395H / 402R / 474A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 699V / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 745T / 760W、291L / 392V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 708L / 745T / 760W、291L / 395H / 402R / 431G / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 76 0W、291L / 395S / 402R / 474A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 699V / 745T / 760W、291L / 395H / 402R / A453S / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 697V / 745T / 760W、291L / 395H / 402R / 427R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、<h2 style=";text-align:left;direction:ltr">79R / 291L / 395S / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 699V / 745T / 760W、291L / 378F / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 370V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / R706S / 745T / 760W、257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、291L / 395H / 402R / 474A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 562K / 604H / 660V / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 745T / 760W、79R / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 562K / 604H / 660V / 687A / 745T / 760W、291L / 378S / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、79R / 291L / 395S / 402R / 478L / 515T / 526S / 5 27A / 528A / 536V / 559R / 604H / 660V / 687A / 699V / 745T / 760W、291L / L372V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、<h2 style=";text-align:left;direction:ltr">257V / 291L / 395S / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W / 792F、291L / 390S / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 427L / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、7 9R / 291L / 395H / 402R / 474A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 699V / 745T / 760W / 792F、291L / 395H / 402R / 409S / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 443A / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687 A / 745T / 760W、291L / 395H / 402R / 448V / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 381E / 395H / 402R / 478L / 515 T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 4 02R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 707W / 745T / 760W、291L / 381K / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 457Y / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 416S / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、<h2 style=";text-align:left;direction:ltr">291L / 395H / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 662S / 687A / 745T / 760W、79R / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 745T / 760W / 792F、291L / 395H / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、291L / 395H / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、291L / 382C / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、257V / 291L / 395H / 402R / 474A / 478L / 51 5T / 526S / 527A / 528A / 536V / 559R / 562K / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W / 792F、79R / 291L / 395H / 402R / 474A / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 662S / 687A / 699V / 7 45T / 760W, 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 725V / 745T / 760W, 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 706I / 745T / 760W, 291L / 395H / 402R / 447R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W<h2 style=";text-align:left;direction:ltr">291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 697L / 745T / 760W、79R / 257V / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 699V / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 701M / 745T / 760W、257V / 291L / 395H / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 562K / 604H / 660V / 662S / 687A / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528 A / 559R / 604H / 660V / 687A / 702V / 745T / 760W、291L / 342G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 716L / 745T / 760W、233K / 291 L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 660V / 687A / 713G / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 60 4H / 660V / 687A / 704V / 745T / 760W、291L / 395H / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 562K / 604H / 660V / 662S / 687A / 745T / 760W、2 91L / 395H / 402R / 454F / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 327E / 395H / 402R / 427T / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、257V / 291L / 316G / 395H / 402R / 474A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 745T / 760W / 792F、<h2 style=";text-align:left;direction:ltr">291L / 395H / 402R / 427D / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 370G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559 R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 417R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 425T / 4 78L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 562K / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 714I / 745T / 760W、233H / 291L / 395H / 402R / 478L / 515T / 526S / 52 7A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 392T / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 303T / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 613H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660 V / 687A / 697A / 745T / 760W、291L / 378L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 378L / 395H / 402R / 478L / 5 15T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 427H / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、<h2 style=";text-align:left;direction:ltr">21A / 79A / 291L / 303T / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 613H / 660V / 687A / 745T / 760W、291L / 370S / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 457N / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687 A / 745T / 760W、79R / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 55 9R / 562K / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 435P / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、257V / 291 L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 6 87A / 699V / 745T / 760W / 792F、79R / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / A453S / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / P667T / 687A / 745T / 760W、79A / 291L / 303T / 338V / 395H / 402R / 478L / 515T / 526S / 527 A / 528A / 559R / 604H / 613H / 660V / 687A / 745T / 760W / 804R / A812S、79R / 2 57V / 291L / 395S / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 792F、79A / 291L / A338V / 395H / 402R / 47 8L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 660V / 687A / 745T / 760W、<h2 style=";text-align:left;direction:ltr">79R / 257V / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 699V / 745T / 760W、79R / 233K / 291L / 303T / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 662S / 687A / 745T / 760W、79R / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 662S / 687A / 699V / 745T / 760W、79R / 291L / 395S / 402R / 475A / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 662S / 687A / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 704V / 745T / 760W、79R / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536 V / 559R / 562K / 604H / 660V / 687A / 699V / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745 T / 760W、21A / 79A / 291L / 338V / 395H / 402R / 478L / 515T / 526S / 527A / 528 A / 559R / 604H / R641A / 660V / 687A / 699V / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 662S / 687A / 745T / 760W、21A / 79A / 291L / 395H / 402R / 442G / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 699V / 745T / 760W、79A / 291L / 303T / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 662S / 687A / 745T / 760W、<h2 style=";text-align:left;direction:ltr">79R / 178L / 257V / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 662S / 687A / 699V / 745T / 760W、79R / 257V / 291L / 395H / 402R / 478L / 5 15T / 526S / 527A / 528A / 536V / 559R / 562K / 604H / 660V / 662S / 687A / 745T / 760 W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 660V / 6 87A / 699V / 745T / 760W、291L / 359G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 613H / 660V / 687A / 742K / 745T / 760W / 808Y / A812S、21N / 79A / 29 1L / 338V / A379G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604 H / 660V / 662S / 687A / 745T / 760W / 804R / 812S、291L / 395H / 402R / L448V / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 731I / 745T / 760W、 21N / 79R / 291L / 359G / 395H / 402R / 442G / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 662S / 687A / 745T / 760W、79A / 291L / A338V / 359V / 395H / 402R / 47 8L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 660V / 687A / 745T / 760W、21N / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 613H / 660V / 662 S / 687A / 742K / 745T / 760W、21A / 79A / 291L / 303T / 395H / 402R / 478L / 515T / 52 6S / 527A / 528A / 559R / 604H / 660V / 662S / 687A / 699V / 742K / 745T / 760W / 808Y、<h2 style=";text-align:left;direction:ltr">21A / 79A / 291L / 338V / 395H / 402R / 442G / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 613H / 660V / 687A / 699V / 745T / 760W、79A / 291L / 303T / 338V / 395H / 4 02R / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 613H / 660V / 662S / 687A / 745T / 760W、291L / 303T / 338V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 660V / 687A / 699V / 745T / 760W / 808W / 812S、79R / 257V / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、257V / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 792F、79R / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 699V / 745T / 760W、79A / 291L / 359G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 713G / 742K / 745T / 760W / 808W、21A / 291L / K359G / 395H / 402R / 442G / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W / 808W / 812S、79R / 257V / 291L / 395H / 402R / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、21N / 79A / 291L / 338V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、79A / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 713G / 745T / 760W、<h2 style=";text-align:left;direction:ltr">291L / 395H / 402R / 457R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、79A / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 641A / 660V / 687A / 745T / 760W、291L / 392C / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291 L / 373T / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、79R / 178L / 257V / 291L / 316G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、21N / 233K / 291L / 338V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 60 4H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 316G / 395H / 402R / 478L / 5 15T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 745T / 760W、21N / 7 9R / 291L / 303T / 395H / 402R / 442G / 478L / 515T / 526S / 527A / 528A / 559R / 6 04H / 660V / 662S / 687A / 745T / 760W、21N / 79R / 233K / 291L / 303T / 338V / A37 9G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 660V / 6 87A / 713G / 745T / 760W、291L / 338V / 395H / 402R / 442G / 478L / 515T / 526S / 5 27A / 528A / 559R / 604H / 660V / 687A / 699V / 745T / 760W、291L / 395H / 402R / 426T / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、<h2 style=";text-align:left;direction:ltr">21A / 291L / 338V / 359G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W / 808Y / 812S、79A / 178L / 233K / 291L / 395H / 402R / 442G / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 660V / 687A / 745T / 760W / 812S、79R / 291L / 395H / 402R / 474A / 478L / 515T / 526S / 52 7A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 699V / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 697I / 745T / 760W、291L / 390S / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、21A / 79A / 291L / 338V / 379G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 662S / 687A / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 721M / 745T / 760W、21A / 79A / 233K / 291L / 338V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 613H / 641A / 660V / 687A / 745T / 760W、2 91L / 374V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 338V / 359G / 379G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / R461L / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、<h2 style=";text-align:left;direction:ltr">291L / 395H / 402R / V451L / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 715L / 745T / 760W、79R / 291L / 303T / 338V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 641A / 660V / 687A / 713G / 745T / 760 W、291L / 393W / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 372Y / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、79R / 257V / 291L / 395H / 402R / 474A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 699V / 745T / 76 0W, 79R / 257V / 291L / 395H / 402R / 474A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 699V / 745T / 760W, 291L / 395H / 402R / 431R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 454G / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 7 60W, 79R / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 395H / 402R / 437S / 478L / 515T / 526S / 527A / 528 A / 559R / 604H / 660V / 687A / 745T / 760W、257V / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 562K / 604H / 660V / 662S / 687A / 745T / 760W、<h2 style=";text-align:left;direction:ltr">291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / V731T / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559 R / 604H / 660V / 687A / R706V / 745T / 760W、291L / 395H / 402R / 448T / 478L / 5 15T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、21A / 79A / 291 L / 379G / 395H / 402R / 442G / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 60 4H / 660V / 662S / 687A / 745T / 760W、291L / 395H / 402R / 457Y / 478L / 515T / 52 6S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 362M / 395H / 4 02R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、2 91L / 395H / 402R / 410W / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 454D / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 338V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W / 812S、21N / 79R / 291L / 338V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、21A / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 5 59R / 604H / 660V / 662S / 687A / 745T / 760W / 808Y / 812S、291L / 395H / 402R / 435K / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、<h2 style=";text-align:left;direction:ltr">21A / 291L / 303T / 338V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 378S / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 378C / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 427L / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、2 91L / 395H / 402R / 418K / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687 A / 745T / 760W、291L / 338V / 395H / 402R / 442G / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527 A / 528A / 559R / 604H / 660V / 687A / T721N / 745T / 760W、291L / 395H / 402R / 449A / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 3 95H / 402R / 457W / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 449G / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 6 60V / 687A / 745T / 760W、291L / 379G / 395H / 402R / 478L / 515T / 526S / 527A / 528 A / 559R / 604H / 641A / 660V / 687A / 745T / 760W、291L / 395H / 402R / 410V / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 431L / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、<h2 style=";text-align:left;direction:ltr">291L / 316G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 562K / 604H / 660V / 687A / 745T / 760W、257V / 291L / 395H / 402R / 478L / 515T / 526S / 527 A / 528A / 536V / 559R / 604H / 660V / 687A / 745T / 760W / 792F、21N / 79R / 291L / 39 5H / 402R / 442G / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 613H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 745T / 760W、257V / 291L / 395H / 402R / 478L / 515T / 526S / 52 7A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 792F、291L / 395H / 402R / 431V / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 7 60W, 291L / 362P / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 257V / 291L / 395H / 402R / 474A / 478L / 515T / 526S / 527A / 528A / A529S / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、291L / 382S / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 76 0W、291L / 395H / 402R / 412V / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660 V / 687A / 745T / 760W、291L / 395H / 402R / 447S / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、79R / 257V / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 699V / 745T / 760W、<h2 style=";text-align:left;direction:ltr">291L / 392T / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 461S / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 412Q / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、257V / 291L / 316G / 3 95H / 402R / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 745T / 760W / 792F、291L / 395H / 402R / L438S / 478L / 515T / 526S / 527A / 5 28A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 388C / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 380I / 39 5H / 402R / 454L / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 447R / 478L / 515T / 526S / 527A / 528A / 559R / 604 H / 660V / 687A / 745T / 760W、21N / 79R / 233K / 291L / 303T / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 613H / 660V / 687A / 699V / 745T / 760W / 8 09K, 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 724F / 745T / 760W, 291L / 395H / 402R / 461Q / 478L / 515T / 526S / 527A / 52 8A / 559R / 604H / 660V / 687A / 745T / 760W、21A / 79R / 291L / 338V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 613H / 660V / 687A / 745T / 760W、<h2 style=";text-align:left;direction:ltr">79R / 178L / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 745T / 760W / 792F、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 715H / 745T / 760W、291L / 390T / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、 291L / 395H / 402R / 449G / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 747S / 760W、291L / 395H / 402R / 449V / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 374S / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 362S / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、21A / 79R / 291L / 379G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 613H / 660V / 687A / 713G / 745T / 760W、79R / 291L / 316G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 6 99V / 745T / 760W、291L / 370S / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 417W / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 376F / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、<h2 style=";text-align:left;direction:ltr">291L / 393R / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、21N / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 660V / 662S / 687A / 745T / 760W、79R / 257V / 262N / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 562K / 6 04H / 660V / 687A / 699V / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 718Q / 745T / 760W、291L / 395S / 402R / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 562K / 604H / 660V / 662S / 687A / 699V / 745T / 760W、291L / 395H / 402R / 449L / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 388M / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、79R / 233K / 291L / 338V / 359G / 379G / 395H / 402R / 442G / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 613H / 660V / 687A / 745T / 760W / 812S, 233K / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 371L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 376S / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W<h2 style=";text-align:left;direction:ltr">291L / 316G / 395H / 402R / 475A / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 660V / 662S / 687A / 745T / 760W / 792F、291L / 395H / 402R / 478L / 515T / 52 6S / 527A / 528A / 559R / 604H / 660V / 687A / 702S / 745T / 760W、79R / 257V / 291L / 316G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 7 45T / 760W / 792F、291L / 373G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 454W / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 728S / 745T / 760W、291L / 39 5H / 402R / 447W / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、79R / 233K / 291L / 338V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / L437P / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 43 2T / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、79R / 178L / 257V / 291L / 316G / 395S / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 5 59R / 604H / 660V / 662S / 687A / 699V / 745T / 760W、79R / 291L / 303T / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 713G / 745T / 760W、<h2 style=";text-align:left;direction:ltr">291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 718F / 745T / 760W、291L / 395H / 402R / A453L / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 427F / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、233K / 29 1L / 379G / 395H / 402R / 442G / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 613H / 660V / 662S / 687A / 713G / 745T / 760W、21A / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 742K / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 68 7A / 732L / 745T / 760W、291L / 392S / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 388P / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 393S / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 7 45T / 760W, 291L / 395H / 402R / 437N / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 392A / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 393G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W<h2 style=";text-align:left;direction:ltr">291L / 395H / 402R / 412M / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、79R / 257V / 291L / 395S / 402R / 478L / 515T / 526S / 527A / 52 8A / 559R / 562K / 604H / 660V / 662S / 687A / 699V / 745T / 760W、21A / 233K / 291L / 338V / 379G / 395H / 402R / 442G / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 613H / 660V / 662S / 687A / 745T / 760W / 812S、291L / 395H / 402R / 427Y / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 732Q / 745T / 760W、291L / 395H / 402R / 429F / 478L / 515T / 526S / 527A / 528A / 559R / 6 04H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 660V / 687A / 699V / 745T / 760W、H2- / 291L / L374Y / 39 5H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687 A / 723Y / 745T / 760W、291L / 395H / 402R / 434N / 478L / 515T / 526S / 527A / 528 A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 388V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 420V / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、7A / 528A / 559R / 604H / 660V / 687A / 699V / 745T / 760W, 291L / 395H / 402R / 427I / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, or 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the set of mutations provided in Tables 9.1, 9.2, and 9.3 relative to the reference sequence corresponding to SEQ ID NO: 2.

[0171] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the following sets of mutations at amino acid positions: 68 / 257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760, 78 / 257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760, / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760 / 807、257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760 2 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、257 / 291 / 356 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、257 / 291 / 356 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、 95 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 603 / 604 / 660 / 662 / 687 / 745 / 760、257 / 271 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、257 / 291 / 395 / 402 / 474 / 475 / 478 / 508 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、<h2 style=";text-align:left;direction:ltr">257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760 / 787N、257 / 291 / 395 / 402 / 412 / 417 / 427 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、251 / 257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760 257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 697 / 745 / 760 257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 562 / 604 / 660 / 662 / 687 / 745 / 760、79 / 257 / 291 / 395 / 402 / 434N / 4 74 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 562K / 604 / 660 / 662 / 687 / 745 / 760 257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760 / 820 257 / 275 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、257 / 291 / 378 / 381 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 697 / 745 / 760 257 / 291 / 395 / 402 / 469 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760 257 / 291 / 395 / 402 / 412 / 417 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 697 / 745 / 760、257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760 / 798、79 / 257 / 291 / 388 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 651 / 660 / 662 / 687 / 745 / 760、257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760 / 825、51 / 257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、257 / 291 / 370 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760 / 815、257 / 291 / 395 / 402 / 465 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、257 / 291 / 317 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 569 / 604 / 660 / 662 / 687 / 745 / 760、257 / 291 / 341 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760 / 824、181 / 257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 5 59 / 604 / 660 / 662 / 687 / 735 / 745 / 760 or 257 / 291 / 336 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the mutation set 68R / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W, 78S / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W, 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W, 68K / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 51 5T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W, 257V / 291L / 395S / 402R / 466Y / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 807M, 257V / 291L / 395S / 4 02R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 612D / 660V / 662S / 687A / 745T / 760W、68E / 257 V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W,<h2 style=";text-align:left;direction:ltr">68T / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、147H / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 356S / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 475A / 478L / 488W / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W, 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 603R / 604H / 660V / 662S / 687A / 745T / 760W 68I / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、147Q / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 271G / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 475A / 478L / 508K / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 787N、<h2 style=";text-align:left;direction:ltr">257V / 291L / 395S / 402R / 412R / 417W / 427H / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 356I / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、78T / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、251I / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 697I / 745T / 760W、68G / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 562K / 604H / 660V / 662S / 687A / 745T / 760W、79R / 257V / 291L / 395S / 402R / 434N / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 562K / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 820T、<h2 style=";text-align:left;direction:ltr">257V / 275T / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 378F / 381E / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 697L / 745T / 760W、257V / 291L / 395S / 402R / 4 69V / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 820S、257V / 291L / 395S / 402R / 412R / 417W / 474A / 475A / 478L / 515T / 52 6S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 697L / 745T / 760W 、257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 53 6V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 798I、79R / 257V / 291L / 3 88V / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 469T / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 651Q / 660V / 662S / 687A / 745T / 760W、<h2 style=";text-align:left;direction:ltr">257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 825E、51T / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527 A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 787M、257V / 291L / 370V / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 6 04H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745V / 760W、 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 815T、257V / 291L / 395S / 402R / 474 A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745S / 760W、257V / 291L / 395S / 402R / 465Y / 474A / 475A / 478L / 515T / 526S / 52 7A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 603M / 604H / 660V / 662S / 687A / 745T / 760W、78N / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、<h2 style=";text-align:left;direction:ltr">257V / 291L / 317R / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、251A / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 815R、68T / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 569Q / 604H / 660V / 662S / 687A / 745T / 760W 147A / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W, 257V / 291L / 341T / 395S / 402 R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527 A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 824M、78V / 257V / 2 91L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、68Q / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、<h2 style=";text-align:left;direction:ltr">251L / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、181R / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 820E、181Q / 257V / 29 1L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、147M / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、 257V / 275A / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 569M / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 735L / 745T / 760W、257V / 291L / 341Y / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 336E / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 341H / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536 V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 815L or 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 815S, wherein the positions are relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the set of mutations provided in Tables 10.1 and 10.2 relative to the reference sequence corresponding to SEQ ID NO: 2.

[0172] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the following sets of mutations at amino acid positions: 68 / 147 / 251 / 257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 603 / 604 / 612 / 660 / 662 / 687 / 745 / 760 / 798, 68 / 147 / 257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 603 / 604 / 660 / 662 / 687 / 745 / 760 / 798. / 257 / 291 / 370 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、147 / 257 / 291 / 370 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、147 / 251 / 257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760 / 798、68 / 147 / 257 / 291 / 370 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 603 / 604 / 612 / 660 / 662 / 687 / 745 / 760、51 / 147 / 257 / 291 / 370 / 395 / 402 / 412 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 603 / 604 / 612 / 660 / 662 / 687 / 745 / 760、147 / 257 / 291 / 395 / 402 / 412 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 603 / 604 / 660 / 662 / 687 / 745 / 760 / 798、147 / 251 / 257 / 291 / 370 / 395 / 402 / 412 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 612 / 660 / 662 / 687 / 745 / 760、147 / 257 / 291 / 370 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760 / 798、78 / 147 / 251 / 257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、147 / 251 / 257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、68 / 147 / 257 / 291 / 370 / 395 / 402 / 474 / 475 / 478 / 508 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760 / 798、147 / 251 / 257 / 291 / 370 / 395 / 402 / 474 / 475 / 478 / 508 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、78 / 147 / 257 / 291 / 395 / 402 / 474 / 475 / 478 / 508 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、78 / 147 / 257 / 291 / 370 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 697 / 745 / 760 / 798、51 / 147 / 251 / 257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760、68 / 147 / 257 / 291 / 370 / 395 / 402 / 474 / 475 / 478 / 508 / 515 / 526 / 527 / 528 / 536 / 559 / 603 / 604 / 660 / 662 / 687 / 697 / 745 / 760、78 / 147 / 251 / 257 / 291 / 370 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 697 / 745 / 760 / 798、78 / 147 / 257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 603 / 604 / 660 / 662 / 687 / 745 / 760 / 798 / 831、147 / 257 / 291 / 370 / 395 / 402 / 412 / 474 / 475 / 478 / 508 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 612 / 660 / 662 / 687 / 745 / 760、72 / 147 / 257 / 291 / 395 / 402 / 474 / 475 / 478 / 508 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760 / 798、51 / 147 / 251 / 257 / 291 / 395 / 402 / 412 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 660 / 662 / 687 / 745 / 760 / 798、51 / 147 / 251 / 257 / 291 / 3 70 / 395 / 402 / 474 / 475 / 478 / 508 / 515 / 526 / 527 / 528 / 536 / 559 / 603 / 604 / 660 / 662 / 687 / 745 / 760 / 798、68 / 78 / 147 / 251 / 257 / 291 / 370 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 55 9 / 603 / 604 / 612 / 660 / 662 / 687 / 697 / 745 / 760 or 51 / 68 / 147 / 251 / 257 / 291 / 370 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 603 / 604 / 660 / 662 / 687 / 745 / 760, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the mutation set 68R / 147H / 251I / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 603M / 604H / 612D / 660V / 662S / 687A / 745T / 760W / 798I, 68R / 147H / 257V / 291L / 395S / 402R / 474 A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 603M / 604H / 660V / 662S / 687A / 745T / 760W、68R / 78N / 147H / 257V / 291L / 370V / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W,<h2 style=";text-align:left;direction:ltr">147H / 257V / 291L / 370V / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、147H / 251I / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 798I、68G / 147H / 257V 51T / 147H / 51T / 147H / 257V / 291L / 370V / 395S / 402R / 412R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 603M / 604H / 612D / 660V / 662S / 687A / 745T / 760 W、147H / 257V / 291L / 395S / 402R / 412R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 603M / 604H / 660V / 662S / 687A / 745T / 760W / 79 8I、147H / 251I / 257V / 291L / 370V / 395S / 402R / 412R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 612D / 660V / 662S / 687A / 7 45T / 760W, 147H / 257V / 291L / 370V / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 798I, 78N / 147H / 251I / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W<h2 style=";text-align:left;direction:ltr">147H / 251I / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、68R / 147H / 257V / 291L / 370V / 395S / 402R / 474A / 475A / 478L / 508K / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 798I、147H / 25 1I / 257V / 291L / 370V / 395S / 402R / 474A / 475A / 478L / 508K / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、78N / 147H / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 508K / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、78N / 147H / 257V / 291L / 370V / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 697L / 745T / 760W / 798I、51T / 147H / 251I / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、68G / 147H / 257V / 291 L / 370V / 395S / 402R / 474A / 475A / 478L / 508K / 515T / 526S / 527A / 528A / 536V / 559R / 603M / 604H / 660V / 662S / 687A / 697L / 745T / 760W、78N / 147H / 251I / 257V / 291L / 370V / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 697L / 745T / 760W / 798I、78N / 147H / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 603M / 604H / 660V / 662S / 687A / 745T / 760W / 798I / 831G, 147H / 257V / 291L / 370V / 395S / 402R / 412R / 474A / 475A / 478L / 508K / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 612D / 660V / 662S / 687A / 745T / 760W, 72A / 147H / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 508K / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 798I、51T / 147H / 251I / 257V / 291L / 395S / 402R / 412R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 798I, 51T / 147H / 251I / 257V / 291L / 370V / 395S / 402R / 474A / 475A / 478L / 508K / 515T / 526S / 527A / 528A / 536V / 559R / 603M / 604H / 660V / 662S / 687A / 745T / 760W / 798I, 68G / 78N / 147H / 251I / 257V / 291L / 370V / 395S / 402R / 474A / 475A / or 51T / 68T / 147H / 251I / 257V / 291L / 370V / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 603M / 604H / 612D / 660V / 662S / 687A / 697L / 745T / 760W, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO:2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the set of mutations provided in Tables 11.1 and 11.2 relative to the reference sequence corresponding to SEQ ID NO: 2.

[0173] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 4-1378, or a reference sequence corresponding to an even numbered SEQ ID NO: 2-1378, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, 226, 272, 328, 546, 710 or 1208, or relative to the reference sequence corresponding to SEQ ID NO: 220, 226, 272, 328, 546, 710 or 1208.

[0174] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, 226, 272, 328, 546, 710 or 1208, or to a reference sequence corresponding to SEQ ID NO: 220, 226, 272, 328, 546, 710 or 1208, wherein the amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, 226, 272, 328, 546, 710 or 1208, or to a reference sequence corresponding to SEQ ID NO: One or more mutations of the reference sequence of NO: 220, 226, 272, 328, 546, 710 or 1208.

[0175] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises mutations at at least the following amino acid positions: 2, 3, 21, 39, 51, 59, 66, 68, 72, 73, 78, 79, 87, 124, 147, 178, 181, 185, 199, 216, 224, 233, 239, 241, 251, 257, 262, 271, 275, 288, 291, 303, 316, 317, 327, 328, 336, 338, 341, 342, 351, 356, 359, 362, 365, 368, 370, 371, 372、373、374、376、377、378、379、380、381、382、388、390、392、393、395、396、398、399、402、403、404、409、410、412、413、416、417、418、420、425、426、427、429、430、431、432、434、435、437、438、442、443、447、448、449、450、451、453、454、457、461、465、466、469、473、 474, 475, 477, 478, 479, 482, 483, 488, 490, 503, 508, 514, 515, 516, 526, 527, 528, 529, 536, 541, 542, 544, 545, 550, 554, 557, 558, 559, 562, 569, 575, 577, 578, 579, 589, 594, 601, 603, 604, 612, 613, 641, 644, 651, 656, 660, 662, 663, 664, 667, 671, 677, 685, 687, 691, 72, 328, 546, 710, or 1208, wherein the position is relative to a reference sequence corresponding to SEQ ID NO: 220, 226, 272, 328, 546, 710, or 1208.

[0176] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least mutations or amino acid residues 2-, 3N, 21A / G / N, 39F, 51T, 59K, 66R, 68E / G / I / K / Q / R / T, 72A, 73I, 78N / S / T / V, 79A / M / R / T, 87V, 124A, 147A / H / M / Q, 178L, 181Q / R, 185T, 199P, 216K, 224L, 233H / K / L, 239V, 241E, 251A / I / L, 257V, 262N, 271G, 275A / T, 288R, 291L, 303T, 316G, 317R, 327E / L, 328 L / V, 336E, 338V, 341H / T / Y, 342G, 351H, 356I / S, 359G / V, 362M / P / S, 365T, 3 68H, 370G / S / V, 371L, 372V / Y, 373G / T, 374S / V / Y, 376F / S, 377C, 378C / F / L / S , 379G, 380I, 381E / K, 382C / S, 388C / M / P / V, 390S / T, 392A / C / S / T / V, 393G / R / S / W, 395G / H / S, 396F / L / P / R / S, 398G / Q, 399G / T, 402R / Y, 403I, 404T, 409S, 410V / W, 412M / Q / R / V, 413T, 416S, 417R / W, 418K, 420V, 425T, 426T, 427D / F / H / I / L / R / T / Y, 429F, 430G, 431G / L / R / V, 432T, 434N, 435K / P, 437N / P / S, 438 F / S, 442G, 443A, 447R / S / W, 448C / I / T / V, 449A / G / L / V, 450V, 451L, 453L / S, 454D / F / G / L / W, 457N / R / W / Y, 461L / Q / S, 465Y, 466Y, 469T / V, 473N, 474A / C / G , 475A / S / V / Y, 477V, 478I / L / M, 479L, 482S, 483D, 488W, 490W, 503R, 508K, 5 14I / V, 515H / T, 516K, 526S, 527A, 528A / G / S, 529S / V, 536V, 541D / G / N / S, 542 R, 544M, 545L, 550C / T, 554G / N, 557I, 558G / L / T, 559P / R, 562K, 569M / Q, 575 W, 577S, 578A, 579L / Q, 589G, 594A, 601T, 603M / R, 604H, 612D, 613H / R, 641A,644Q、651Q、656Q / S、660V、662S、663E、664A、667S / T / W、671W、677L、685R、687A、691S / W、694D、697A / I / L / V、698E、699V、7 01M, 702S / V, 704L / V, 706I / S / V, 707W, 708L, 713G, 714I, 715H / L, 716E / L, 718F / Q, 721A / M / N, 723Y, 724F / L, 725V, 728S, 73 1I / T, 732L / Q, 735L, 740S / V, 742K, 743H / V, 745S / T / V, 747S, 748G, 749M, 753A, 758G, 760T / W / Y, 762A, 768S, 769Q, 770E, 787M / N, 792F / Y, 798I, 804R, 807M, 808W / Y, 809K, 812R / S, 815L / R / S / T, 820E / S / T, 824M, 825E, and 831G, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 220, 226, 272, 328, 546, 710, or 1208.

[0177] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, or to a reference sequence corresponding to SEQ ID NO: 220, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, or to a reference sequence corresponding to SEQ ID NO: 220.

[0178] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 222-268, or to a reference sequence corresponding to an even numbered SEQ ID NO: 222-268, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, or to the reference sequence corresponding to SEQ ID NO: 220.

[0179] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation or set of mutations at amino acid position 515, 594, 559, 760, 541, 753, 402, 398, 758, 399, 575, 257 / 758, 558, 601, 762, 589, or 544, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 220. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least mutation or set of mutations 515H, 594A, 559R, 760W, 541S, 541N, 753A, 402R, 760T, 398Q, 758G, 760Y, 399G, 575W, 257V / 758G, 558L, 402Y, 558T, 601T, 398G, 762A, 558G, 589G, or 544M, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 220. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the mutation or set of mutations G515H, N594A, P559R, F760W, E541S, E541N, E753A, G402R, F760T, T398Q, M758G, F760Y, N399G, F575W, A257V / M758G, D558L, G402Y, D558T, L601T, T398G, M762A, D558G, D589G, or L544M, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 220.

[0180] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 226, or to a reference sequence corresponding to SEQ ID NO: 226, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 226, or to a reference sequence corresponding to SEQ ID NO: 226.

[0181] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 270-320, or to a reference sequence corresponding to SEQ ID NO: 270-320, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 226, or to the reference sequence corresponding to SEQ ID NO: 226.

[0182] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation or set of mutations at the following amino acid positions: 515 / 760, 514 / 515 / 760, 478 / 515 / 760, 660, 178, 402 / 515, 377 / 515, 515, 514 / 515, 402, 503, 239, 73, 528 / 541, 59, 515 / 575, 3 / 559, 377 / 515 / 528 / 541 / 554 / 557, 402 / 514 / 515 / 541 / 559, 514 / 515 / 541 / 559, 514 / 515 / 559, 515 / 559, 515 / 541 / 559, 377 / 515 / 559, 402 / 515 / 559 or 515 / 541 / 557, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 226. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least mutations or mutation sets 515T / 760W, 514V / 515T / 760W, 478M / 515T / 760W, 660V, 178L, 402Y / 515T, 377C / 515T, 515T, 514V / 515T, 402Y, 503R, 239V, 73I, 528G / 541N, 59K, 515T / 575W, 3N / 515T / 559P , 377C / 515H / 528G / 541N / 554N / 557I, 402R / 514V / 515T / 541N / 559P, 514V / 515T / 541G / 559P, 514V / 515T / 559P, 515T / 559P, 515T / 541N / 559P, 377C / 515T / 559P, 402Y / 515H / 559P or 515T / 541N / 557I, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 226.In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation or set of mutations G515T / F760W, I514V / G515T / F760W, L478M / G515T / F760W, L660V, V178L, G402Y / G515T, G377C / G515T, G515T, I514V / G515T, G402Y, K503R, L239V, V73I, A528G / E541N, R59K, G515T / F575W, H3N / G515T / R559P, G377C / G515H / A528G / E541N / S554N / L557I, G402R / I514V / G515T / E541N / R559P, I514V / G515T / E541G / R559P, I514V / G515T / R559P, G515T / R559P, G515T / E541N / R559P, G377C / G515T / R559P, G402Y / G515H / R559P or G515T / E541N / L557I, wherein the positions are relative to the reference sequence corresponding to SEQ ID NO: 226.

[0183] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 272, or to a reference sequence corresponding to SEQ ID NO: 272, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 272, or to a reference sequence corresponding to SEQ ID NO: 272.

[0184] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 322-534, or a reference sequence corresponding to an even numbered SEQ ID NO: 322-534, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 272, or relative to the reference sequence corresponding to SEQ ID NO: 272.

[0185] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises a mutation or set of mutations at at least the following amino acid positions: 402 / 528 / 541 / 660, 257 / 604 / 660, 178 / 257 / 402 / 404 / 528 / 557 / 660, 402 / 604 / 660, 257 / 402 / 541 / 660, 377 / 402 / 541 / 660, 257 / 377 / 402 / 660, 483 / 660, 257 / 402 / 257 / 377 / 402 / 660, 178 / 257 / 402 / 404 / 528 / 557 / 660. 77 / 402 / 403 / 483 / 604 / 660、377、178 / 402 / 403 / 528 / 541 / 660、377 / 601 / 660、178 / 377、377 / 490 / 660、402 / 403 / 483 / 554 / 557、178 / 257 / 377 / 558 / 660、257 / 402 / 403 / 483 / 660、178 / 554 / 557 / 558 / 660、554 / 557 / 660、178 / 483 / 541 / 545 / 554 / 557 / 604 / 660、792、402 / 403 / 483 / 60 4 / 660, 178, 474, 257 / 377 / 541 / 604, 743, 396, 479, 377 / 403 / 541 / 604, 475, 178 / 257 / 403 / 528, 448, 577, 395, 769, 691, 677, 704, 377 / 541 / 575 / 604 / 660, 178 / 257 / 601 / 604, 685, 377 / 604 / 660, 749, 482, 291, 579, 178 / 541 / 550 / 604, 477, 178 / 257, 257 / 402 / 490 / 528 / 541 / 660 , 328, 740, 368, 721 / 745, 578, 687, 541 / 601 / 604, 257 / 528, 257 / 528 / 554, 768, 770, 257 / 377 / 483 / 541, 542, 257 / 402 / 403, 257 / 403 / 541, 604, 402 / 604, 257 / 316 / 541 / 575 / 604, 327, 403 / 541 / 604 / 660, 257 / 377 / 403 / 483 / 541 / 601 / 604 or 402 / 528 / 541 / 601, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 272. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the mutations or mutation sets 402R / 528G / 541D / 660V, 257V / 604H / 660V,178L / 257V / 402R / 404T / 528G / 557I / 660V、402R / 604H / 660V、257V / 402Y / 541G / 660V、377C / 402Y / 541N / 660V、257V / 377C / 402Y / 660V、483D / 660V、257V / 402Y、257V、257V / 377C、178L / 660V、257V / 377C / 402Y、660V、178L / 257V / 377C / 402R / 403I / 483D / 604H / 660V、377C、178L / 402R / 403I / 528G / 541G / 660V、377C / 601T / 660V、178L / 377C、377C / 490W / 660V、402R / 403I / 483D / 554N / 557I、178L / 257V / 377C / 558T / 660V、257V / 402Y / 403I / 483D / 660V、178L / 554N / 557I / 558T / 660V、554N / 557I / 660V、178L / 483D / 541N / 545L / 554N / 557I / 604H / 660V、792F、402Y / 403I / 483D / 604H / 660V、178L、474A、257V / 377C / 541D / 604H、743H、396L、743V、396R、479L、377C / 403I / 541D / 604H、475A、178L / 257V / 403I / 528G、448I、577S、395G、769Q、475V、448C、691W、677L、704L、377C / 541N / 575W / 604H / 660V、178L / 257V / 601T / 604H、685R、377C / 604H / 660V、749M、396F、395H、475S、474C、482S、291L、448V、579Q、178L / 541G / 550C / 604H、477V、178L / 257V、257V / 402R / 490W / 528G / 541G / 660V、328L、395S、740V、483D / 660V、257V / 377C、579L、368H、792Y、721A / 745T、474A、743H、578A、687A、541D / 601T / 604H、328V、396L、475Y、395G、257V / 528G、257V / 528G / 554N、768S、257V、257V、396P、770E、257V / 377C / 483D / 541N、542R、257V / 402Y / 403I、: 257V / 403I / 541N, 474G, 604H, 402Y / 604H, 691S, 257V / 316G / 541D / 575W / 604H, 327L, 704V, 403I / 541N / 604H / 660V, 257V / 377C / 403I / 483D / 541D / 601T / 604H, 740S, 257V, 402R / 528G / 541G / 601T or 257V / 377C, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 272. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the mutations or mutation sets G402R / A528G / E541D / L660V, A257V / R604H / L660V, V178L / A257V / G402R / A404T / A528G / L557I / L660V, G402R / R604H / L660V, A257V / G402Y / E541G / L660V, G377C / G402Y / E541N / L660V, A257V / G377C / G402Y / L660V, G483D / L660V, A257V / G402Y, A257V, A257V / G377C, V178L / L660V, A257V / G377C / G402Y, L660V, V178L / A257 V / G377C / G402R / V403I / G483D / R604H / L660V, G377C, V178L / G402R / V403I / A528G / E541G / L660V, G377C / L601T / L660V, V1 78L / G377C, G377C / G490W / L660V, G402R / V403I / G483D / S554N / L557I, V178L / A257V / G377C / D558T / L660V, A257V / G402Y / V403I / G483D / L660V, V178L / S554N / L557I / D558T / L660V, S554N / L557I / L660V, V178L / G483D / E541N / Q545L / S554N / L557 I / R604H / L660V, L792F, G402Y / V403I / G483D / R604H / L660V, V178L, L474A, A257V / G377C / E541D / R604H, D743H, A396L, D7 43V, A396R, E479L, G377C / V403I / E541D / R604H, E475A, V178L / A257V / V403I / A528G, L448I, Q577S, P395G, A769Q, E475V,L448C, Q691W, N677L, I704L, G377C / E541N / F575W / R604H / L660V, V178L / A257V / L601T / R604H, S685R, G37 7C / R604H / L660V, R749M, A396F, P395H, E475S, L474C, E482S, F291L, L448V, A579Q, V178L / E541G / S550C / R 604H, E477V, V178L / A257V, A257V / G402R / G490W / A528G / E541G / L660V, E328L, P395S, Y740V, G483D / L660 V, A257V / G377C, A579L, D368H, L792Y, T721A / A745T, L474A, D743H, T578A, H687A, E541D / L601T / R604H, E3 28V, A396L, E475Y, P395G, A257V / A528G, A257V / A528G / S554N, A768S, A257V, A257V, A396P, D770E, A257V / G377C / G483D / E541N, L542R, A257V / G402Y / V403I, A257V / V403I / E541N, L474G, R604H, G402Y / R604H, Q69 1S, A257V / E316G / E541D / F575W / R604H, K327L, I704V, V403I / E541N / R604H / L660V, A257V / G377C / V403I / G483D / E541D / L601T / R604H, Y740S, A257V, G402R / A528G / E541G / L601T or A257V / G377C, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 272.

[0186] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 328, or to a reference sequence corresponding to SEQ ID NO: 328, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 328, or to a reference sequence corresponding to SEQ ID NO: 328.

[0187] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 536-674, or a reference sequence corresponding to an even numbered SEQ ID NO: 536-674, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 328, or relative to the reference sequence corresponding to SEQ ID NO: 328.

[0188] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation or set of mutations at the following amino acid positions: 79, 257 / 474, 677, 562, 178 / 395 / 745, 291 / 395 / 687 / 745, 257 / 395 / 677, 303 / 338, 474 / 475, 359, 474 / 475 / 6 77 / 704, 395 / 687, 671, 233, 699, 379, 257 / 792, 257 / 395, 21, 257 / 448 / 474 / 475, 713, 641, 442, 656, 475 / 704, 742, 667, 257 / 448, 257 / 316 / 395 / 482 / 687 / 745, 804, 70 4. 178 / 257 / 395 / 482 / 579 / 687 / 745, 257 / 579, 257 / 291 / 395 / 475 / 541 / 687, 257 / 768, 812, 475, 662, 536, 748, 808, 291 / 316 / 395 / 687, 613, 257 / 291 / 316, 257 / 291 / 57 9, 257 / 316 / 541 / 745, 351, 257 / 316 / 395 / 745, 185, 257 / 316 / 687, 291 / 316 / 475 / 687 / 745, 257 / 291 / 395 / 740 / 745, 450, 473, 124, 430, 413, 66 or 365, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 328.In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least mutations or a set of mutations 79A, 257V / 474A, 677L, 562K, 178L / 395H / 745T, 291L / 395H / 687A / 745T, 257V / 395H / 677L, 303T / 338V, 474A / 475A, 79R, 359G, 79T, 474A / 475S / 677L / 704V, 79M, 395S / 687 A. 671W, 233K, 699V, 379G, 257V / 792F, 257V / 395S, 21A, 257V / 448C / 474A / 475S, 713G, 641A, 442G, 656S, 47 5S / 704V, 742K, 667W, 21N, 257V / 448I, 257V / 316G / 395H / 482S / 687A / 745T, 804R, 704V, 178L / 257V / 395S / 4 82S / 579L / 687A / 745T, 257V / 579L, 257V / 291L / 395S / 475V / 541N / 687A, 257V / 768S, 667S, 812S, 475A, 662S , 536V, 748G, 808W, 291L / 316G / 395H / 687A, 613H, 257V / 291L / 316G, 257V / 291L / 579L, 257V / 316G / 541N / 74 5T, 351H, 257V / 316G / 395G / 745T, 185T, 257V / 316G / 687A, 808Y, 291L / 316G / 475V / 687A / 745T, 257V / 291L / 395H / 740V / 745T, 450V, 473N, 21G, 124A, 613R, 656Q, 430G, 413T, 233L, 66R, 365T or 812R, wherein the positions are relative to the reference sequence corresponding to SEQ ID NO: 328.In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the mutation or set of mutations K79A, A257V / L474A, N677L, R562K, V178L / P395H / A745T, F291L / P395H / H687A / A745T, A257V / P395H / N677L, G303T / A338V, L474A / E475A, K79R, K359G, K79T, L474A / E475S / N677L / I704V, K79M, P395S / H687A, R6 71W, R233K, E699V, A379G, A257V / L792F, A257V / P395S, P21A, A257V / L448C / L474A / E475S, K713G, R641A, Q442G, W656S, E475S / I704V, P742K, P667W, P21N, A257V / L448I, A257V / E316G / P395H / E482S / H687A / A745T, E804R, I704V, V178L / A257V / P395S / E48 2S / A579L / H687A / A745T, A257V / A579L, A257V / F291L / P395S / E475V / E541N / H687A, A257V / A768S, P667S, A812S, E475A, P662S , A536V, V748G, E808W, F291L / E316G / P395H / H687A, E613H, A257V / F291L / E316G, A257V / F291L / A579L, A257V / E316G / E541N / A7 45T, P351H, A257V / E316G / P395G / A745T, P185T, A257V / E316G / H687A, E808Y, F291L / E316G / E475V / H687A / A745T, A257V / F291L / P395H / Y740V / A745T, R450V, E473N, P21G, P124A, E613R, W656Q, F430G, E413T, R233L, K66R, L365T or A812R, wherein the positions are relative to the reference sequence corresponding to SEQ ID NO: 328.

[0189] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 546, or to a reference sequence corresponding to SEQ ID NO: 546, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 546, or to a reference sequence corresponding to SEQ ID NO: 546.

[0190] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 676-1190, or to a reference sequence corresponding to an even numbered SEQ ID NO: 676-1190, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 546, or to the reference sequence corresponding to SEQ ID NO: 546.

[0191] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation or set of mutations at the following amino acid positions: 438, 697, 457, 716, 79 / 474 / 536 / 699, 536, 392, 708, 431, 395 / 474 / 536 / 699, 453, 427, 79 / 395 / 699, 378, 370, 706, 257 / 395 / 474 / 475 / 536 / 662, 474 / 536 / 562, 257 / 395 / 474 / 536, 79 / 257 / 395 / 474 / 475 / 536 / 562, 79 / 395 / 536 / 699, 372, 257 / 395 / 792, 390, 79 / 474 / 536 / 699 / 792, 409, 443, 448, 381, 707, 416, 474 / 475 / 662, 79 / 395 / 474 / 475 / 536 / 792, 474 / 475 / 536 / 662, 382, 257 / 474 / 536 / 562 / 66 2. 257 / 395 / 474 / 475 / 792, 79 / 474 / 662 / 699, 725, 447, 79 / 257 / 699, 701, 257 / 474 / 475 / 536 / 562 / 662, 702, 342 / 716, 233 / 562 / 713, 704, 474 / 475 / 536 / 562 / 662、454、327 / 427、257 / 316 / 474 / 536 / 792、417、425、536 / 562、714、233、303 / 613、21 / 79 / 303 / 562 / 613、79 / 562、435、257 / 536 / 699 / 792、79 / 536、 453 / 667、79 / 303 / 338 / 613 / 804 / 812、79 / 257 / 395 / 536 / 662 / 792、79 / 338 / 562、79 / 233 / 303 / 662、79 / 662 / 699、79 / 395 / 475 / 662、79 / 536 / 562 / 699、536 / 662、21 / 79 / 338 / 641 / 699、662、21 / 79 / 442 / 699、79 / 303 / 662、79 / 178 / 257 / 662 / 699、79 / 257 / 536 / 562 / 662、562 / 699、359 / 613 / 742 / 808 / 812、21 / 79 / 338 / 379 / 562 / 662 / 804 / 812、731、21 / 79 / 359 / 442 / 662、79 / 338 / 359 / 562、21 / 613 / 662 / 742、21 / 79 / 303 / 662 / 699 / 742 / 808、21 / 79 / 338 / 442 / 613 / 699、79 / 303 / 338 / 562 / 613 / 662、303 / 338 / 562 / 699 / 808 / 812、79 / 257、257 / 662 / 792、79 / 536 / 699、79 / 359 / 713 / 742 / 808、21 / 359 / 442 / 808 / 812、79 / 257 / 475 / 536 / 662、21 / 79 / 338、79 / 713、79 / 562 / 641、373、79 / 178 / 257 / 316 / 536 / 662、21 / 233 / 338 / 562 / 662、257 / 316 / 536、21 / 79 / 303 / 442 / 662、21 / 79 / 233 / 303 / 338 / 379 / 562 / 713、338 / 442 / 699、426、21 / 338 / 359 / 808 / 812、79 / 178 / 233 / 442 / 562 / 812、79 / 474 / 536 / 662 / 699、21 / 79 / 338 / 379 / 662、721、21 / 79 / 233 / 338 / 613 / 641、374、338 / 359 / 379、461、451、715、79 / 303 / 338 / 641 / 713、393、79 / 257 / 474 / 536 / 699、79、437、257 / 536 / 562 / 662、21 / 79 / 379 / 442 / 562 / 662、362、410、338 / 812、21 / 662 / 808 / 812、21 / 303 / 338、418、338 / 442、449、379 / 641、316 / 536 / 562、257 / 536 / 792、21 / 79 / 442 / 613、257 / 536 / 662 / 792、257 / 474 / 529 / 536 / 662、412、79 / 257 / 536 / 662 / 699、257 / 316 / 475 / 536 / 792、388、380 / 454、21 / 79 / 233 / 303 / 613 / 699 / 809、724、21 / 79 / 338 / 613、79 / 178 / 536 / 792、449 / 747、21 / 79 / 379 / 613 / 713、79 / 316 / 536 / 662 / 699、376、21 / 562 / 662、79 / 257 / 262 / 536 / 562 / 699、718、395 / 475 / 536 / 562 / 662 / 699、79 / 233 / 338 / 359 / 379 / 442 / 562 / 613 / 812、371、316 / 475 / 562 / 662 / 792、79 / 257 / 316 / 792、728、79 / 233 / 338、432、79 / 178 / 257 / 316 / 395 / 536 / 662 / 699、: 79 / 303 / 713, 233 / 379 / 442 / 562 / 613 / 662 / 713, 21 / 742, 732, 79 / 257 / 395 / 562 / 662 / 699, 21 / 233 / 338 / 379 / 442 / 562 / 613 / 662 / 812, 429, 562 / 699, 2 / 374, 723, 434, 420 or 699, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 546. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least mutation or mutation set 438F, 697I, 457W, 716E, 79R / 474A / 536V / 699V, 536V, 392V, 708L, 431G, 395S / 474A / 536V / 699V, 453S, 697V, 427R, 79R / 395S / 699V, 378F, 370V, 706S, 257V / 395S / 474A / 475A / 536V / 662S, 474A / 536V / 562K, 257V / 395S / 474A / 536V, 79R / 257V / 395S / 474A / 475A / 536V / 562K, 378S, 79R / 395S / 536V / 699V, 372V, 257V / 395S / 792F, 390S , 427L, 79R / 474A / 536V / 699V / 792F, 409S, 443A, 448V, 381E, 707W, 381K, 457Y, 416S, 474A / 475A / 662S, 79R / 395S / 474A / 475A / 536V / 792F, 474A / 475A / 536V / 662S, 382C, 257V / 474A / 536V / 562K / 662S, 257V / 395S / 474A / 475A / 792F, 79R / 474A / 662S / 69 9V, 725V, 706I, 447R, 697L, 79R / 257V / 699V, 701M, 257V / 474A / 475A / 536V / 562K / 662S, 702V, 342G / 716L, 233K / 562K / 713G, 7 04V, 474A / 475A / 536V / 562K / 662S, 454F, 327E / 427T, 257V / 316G / 474A / 536V / 792F, 427D, 370G, 417R, 425T, 536V / 562K, 714I , 233H, 392T, 303T / 613H, 697A, 378L, 427H, 21A / 79A / 303T / 562K / 613H, 370S, 457N, 79R / 562K, 435P, 257V / 536V / 699V / 792F,<h2 style=";text-align:left;direction:ltr">79R / 536V, 453S / 667T, 79A / 303T / 338V / 613H / 804R / 812S, 79R / 257V / 395S / 536V / 662S / 792F, 79A / 338V / 562K, 79R / 257V / 699V, 79R / 233K / 303T / 662S 79R / 662S / 699V, 79R / 395S / 475A / 662S, 704V, 79R / 536V / 562K / 699V, 536V / 662S, 21A / 79A / 338V / 641A / 699V, 662S, 21A / 79A / 442G / 699V, 79A / 303T / 6 62S, 79R / 178L / 257V / 662S / 699V, 79R / 257V / 536V / 562K / 662S, 562K / 699V, 359G / 613H / 742K / 808Y / 812S, 21N / 79A / 338V / 379G / 562K / 662S / 804R / 812 S、448V、731I、21N / 79R / 359G / 442G / 662S、79A / 338V / 359V / 562K、21N / 613H / 662S / 742K、21A / 79A / 303T / 662S / 699V / 742K / 808Y、21A / 79A / 338V / 442G / 613H / 699V, 79A / 303T / 338V / 562K / 613H / 662S, 303T / 338V / 562K / 699V / 808W / 812S, 79R / 257V, 257V / 662S / 792F, 79R / 536V / 699V, 79A / 359G / 713G / 74 2K / 808W, 21A / 359G / 442G / 808W / 812S, 79R / 257V / 475A / 536V / 662S, 21N / 79A / 338V, 79A / 713G, 457R, 79A / 562K / 641A, 392C, 373T, 79R / 178L / 257V / 316 G / 536V / 662S、21N / 233K / 338V / 562K / 662S、257V / 316G / 536V、21N / 79R / 30 3T / 442G / 662S、21N / 79R / 233K / 303T / 338V / 379G / 562K / 713G、338V / 442G / 6 99V, 426T, 21A / 338V / 359G / 808Y / 812S, 79A / 178L / 233K / 442G / 562K / 812S, 79R / 474A / 536V / 662S / 699V, 697I, 390S, 21A / 79A / 338V / 379G / 662S, 721M,<h2 style=";text-align:left;direction:ltr">21A / 79A / 233K / 338V / 613H / 641A、374V、338V / 359G / 379G、461L、451L、715L、79R / 303T / 338V / 641A / 713G、393W、372Y、79R / 257V / 474A / 536V / 699V、43 1R, 454G, 79R, 437S, 257V / 536V / 562K / 662S, 731T, 706V, 448T, 21A / 79A / 379G / 442G / 562K / 662S, 457Y, 362M, 410W, 454D, 338V / 812S, 21N / 79R / 338V, 2 1A / 662S / 808Y / 812S、435K、21A / 303T / 338V、378S、378C、427L、418K、338V / 442G、721N、449A、457W、449G、379G / 641A、410V、431L、316G / 536V / 562K、2 57V / 536V / 792F, 21N / 79R / 442G / 613H, 536V, 257V / 536V / 662S / 792F, 431V, 362P, 257V / 474A / 529S / 536V / 662S, 382S, 412V, 447S, 79R / 257V / 536V / 662 S / 699V、392T、461S、412Q、257V / 316G / 475A / 536V / 792F、438S、388C、380I / 454L、447R、21N / 79R / 233K / 303T / 613H / 699V / 809K、724F、461Q、21A / 79R / 338V / 613H, 79R / 178L / 536V / 792F, 715H, 390T, 449G / 747S, 449V, 374S, 362S, 21A / 79R / 379G / 613H / 713G, 79R / 316G / 536V / 662S / 699V, 370S, 417W, 376 F、393R、21N / 562K / 662S、79R / 257V / 262N / 536V / 562K / 699V、718Q、395S / 4 75A / 536V / 562K / 662S / 699V、449L、388M、79R / 233K / 338V / 359G / 379G / 442G / 562K / 613H / 812S、233K、371L、376S、316G / 475A / 562K / 662S / 792F、702S、79R / 257V / 316G / 792F、373G、454W、728S、447W、79R / 233K / 338V、437P、432T、79R / 178L / 257V / 316G / 395S / 536V / 662S / 699V, 79R / 303T / 713G, 718F, 453L, 427F, 233K / 379G / 442G / 562K / 613H / 662S / 713G, 21A / 742K, 732L, 392S, 388P, 393S, 437N, 392A, 393G, 412M, 79 R / 257V / 395S / 562K / 662S / 699V, 21A / 233K / 338V / 379G / 442G / 562K / 613H / 662S / 812S, 427Y, 732Q, 429F, 562K / 699V, 2- / 374Y, 723Y, 434N, 388V, 420V, 699V, 427I or 724L, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 546. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the mutation or set of mutations L438F, Y697I, A457W, A716E, K79R / L474A / A536V / E699V, A536V, L392V, F708L, P431G, H395S / L474A / A536V / E699V, A453S, Y697V, Q427R, K79R / H395S / E69 9V, V378F, A370V, R706S, A257V / H395S / L474A / E475A / A536V / P662S, L474A / A536V / R562K, A257V / H 395S / L474A / A536V, K79R / A257V / H395S / L474A / E475A / A536V / R562K, V378S, K79R / H395S / A536V / E6 99V, L372V, A257V / H395S / L792F, A390S, Q427L, K79R / L474A / A536V / E699V / L792F, G409S, E443A, L 448V, D381E, Y707W, D381K, A457Y, A416S, L474A / E475A / P662S, K79R / H395S / L474A / E475A / A536V / L 792F, L474A / E475A / A536V / P662S, P382C, A257V / L474A / A536V / R562K / P662S, A257V / H395S / L474A / E475A / L792F, K79R / L474A / P662S / E699V, G725V, R706I, P447R, Y697L, K79R / A257V / E699V, E701M,<h2 style=";text-align:left;direction:ltr">A257V / L474A / E475A / A536V / R562K / P662S,A702V,E342G / A716L,R233K / R562K / K713G,I704V,L474A / E475A / A536V / R562K / P662S,H454F,K327E / Q427T、A257V / E316G / L474A / A536V / L792F、Q427D、A370G、E417R、L425T、 A536V / R562K、V714I、R233H、L392T、G303T / E613H、Y697A、V378L、Q427H、P 21A / K79A / G303T / R562K / E613H, A370S, A457N, K79R / R562K, E435P, A257V / A536V / E699V / L792F, K79R / A536V, A453S / P667T, K79A / G303T / A338V / E 613H / E804R / A812S, K79R / A257V / H395S / A536V / P662S / L792F, K79A / A338V / R562K, K79R / A257V / E699V, K79R / R233K / G303T / P662S, K79R / P662S / E 699V、K79R / H395S / E475A / P662S、I704V、K79R / A536V / R562K / E699V、A53 6V / P662S、P21A / K79A / A338V / R641A / E699V、P662S、P21A / K79A / Q442G / E 699V, K79A / G303T / P662S, K79R / V178L / A257V / P662S / E699V, K79R / A257V / A536V, R562K / P662S, R562K / E699V, K359G / E613H / P742K / E808Y / A812S P21N / K79A / A338V / A379G / R562K / P662S / E804R / A812S,L448V,V731I,P21N / K79R / K359G / Q442G / P662S,K79A / A338V / K359V / R562K,P21N / E613H / P662S / P742K, P21A / K79A / G303T / P662S / E699V / P742K / E808Y, P21A / K79A / A338V / Q442G / E613H / E699V, K79A / G303T / A338V / R562K / E613H / P662S<h2 style=";text-align:left;direction:ltr">G303T / A338V / R562K / E699V / E808W / A812S、K79R / A257V、A257V / P662S / L79 2F、K79R / A536V / E699V、K79A / K359G / K713G / P742K / E808W、P21A / K359G / Q4 42G / E808W / A812S、K79R / A257V / E475A / A536V / P662S、P21N / K79A / A338V、K79A / K713G、A457R、K79A / R562K / R641A、L392C、A373T、K79R / V178L / A257V / E316G / A536V / P662S,P21N / R233K / A338V / R562K / P662S,A257V / E316G / A536V,P21N / K79R / G303T / Q442G / P662S,P21N / K79R / R233K / G303T / A338V / A37 9G / R562K / K713G、A338V / Q442G / E699V、F426T、P21A / A338V / K359G / E808Y / A812S、K79A / V178L / R233K / Q442G / R562K / A812S、K79R / L474A / A536V / P662S / E699V,Y697I,A390S,P21A / K79A / A338V / A379G / P662S,T721M,P21A / K79A / R233K / A338V / E613H / R641A,L374V,A338V / K359G / A379G,R461L,V451L,R 715L, K79R / G303T / A338V / R641A / K713G, L393W, L372Y, K79R / A257V / L474A / A536V / E699V, P431R, H454G, K79R, L437S, A257V / A536V / R562K / P662S, V73 1T、R706V、L448T、P21A / K79A / A379G / Q442G / R562K / P662S、A457Y、R362M、E 410W、H454D、A338V / A812S、P21N / K79R / A338V、P21A / P662S / E808Y / A812S、 E435K、P21A / G303T / A338V、V378S、V378C、Q427L、R418K、A338V / Q442G、T72 1N、S449A、A457W、S449G、A379G / R641A、E410V、P431L、E316G / A536V / R562K、<h2 style=";text-align:left;direction:ltr">A257V / A536V / L792F、P21N / K79R / Q442G / E613H、A536V、A257V / A536V / P6 62S / L792F、P431V、R362P、A257V / L474A / A529S / A536V / P662S、P382S、T4 12V, P447S, K79R / A257V / A536V / P662S / E699V, L392T, R461S, T412Q, A257V / E316G / E475A / A536V / L792F, L438S, L388C, L380I / H454L, P447R, P21N / K79R / R233K / G303T / E613H / E699V / E809K,E724F,R461Q,P21A / K79R / A338V / E613H,K79R / V178L / A536V / L792F,R715H,A390T,S449G / R747S,S44 9V、L374S、R362S、P21A / K79R / A379G / E613H / K713G、K79R / E316G / A536V / P662S / E699V、A370S、E417W、E376F、L393R、P21N / R562K / P662S、K79R / A25 7V / D262N / A536V / R562K / E699V、I718Q、H395S / E475A / A536V / R562K / P66 2S / E699V、S449L、L388M、K79R / R233K / A338V / K359G / A379G / Q442G / R562 K / E613H / A812S、R233K、V371L、E376S、E316G / E475A / R562K / P662S / L792 F、A702S、K79R / A257V / E316G / L792F、A373G、H454W、R728S、P447W、K79R / R 233K / A338V, L437P, R432T, K79R / V178L, A257V, E316G, H395S, A536V, P662S, E699V, K79R, G303T, K713G, I718F, A453L, Q427F, R233K / A379G / Q44 2G / R562K / E613H / P662S / K713G,P21A / P742K,E732L,L392S,L388P,L393S,L437N,L392A,L393G,T412M,K79R / A257V / H395S / R562K / P662S / E699VP21A / R233K / A338V / A379G / Q442G / R562K / E613H / P662S / A812S, Q427Y, E732Q, L429F, R562K / E699V, H2- / L374Y, E723Y, S434N, L388V, L420V, E699V, Q427I or E724L, wherein the positions are relative to the reference sequence corresponding to SEQ ID NO: 546.

[0192] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 710, or to a reference sequence corresponding to SEQ ID NO: 710, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 710, or to a reference sequence corresponding to SEQ ID NO: 710.

[0193] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 1192-1326, or a reference sequence corresponding to an even numbered SEQ ID NO: 1192-1326, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710.

[0194] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises a mutation or set of mutations at at least amino acid position(s): 68, 78, 466, 807, 612, 147, 356, 488, 603, 271, 508, 787, 412 / 417 / 427, 251, 697, 562, 79 / 434 / 562, 820, 275, 378 / 381 / 697, 469, 412 / 417 / 697, 798, 79 / 388, 651, 825, 51, 370, 745, 815, 465, 317, 569, 341, 824, 18, 181, 735, or 336, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 710. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least mutations or a combination of mutations 68R, 78S, 68K, 466Y, 807M, 612D, 68E, 68T, 147H, 356S, 488W, 603R, 68I, 147Q, 271G, 508K, 787N, 412R / 417W / 427H, 356I, 78T, 251I, 697I, 68G, 562K, 79R / 434N / 562K, 820T, 275T, 378F / 381E / 697L, 469V, 820S, 412 R / 417W / 697L, 798I, 79R / 388V, 469T, 651Q, 825E, 51T, 787M, 370V, 745V, 815T, 745S, 465Y, 603M, 78N, 317R, 251A, 815R, 68T, 569Q, 147A, 341T, 824M, 78V, 68Q, 251L, 181R, 820E, 181Q, 147M, 275A, 569M, 735L, 341Y, 336E, 341H, 815L or 815S, wherein the positions are relative to the reference sequence corresponding to SEQ ID NO: 710.In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the mutation or set of mutations D68R, A78S, D68K, L466Y, A807M, E612D, D68E, D68T, C147H, A356S, L488W, Q603R, D68I, C147Q, A271G, E508K, G787N, T412R / E417W / Q427H, A356I, A78T, R251I, Y697I, D68G, R562K, K79R / S434N / R562K, N820T, R275T, V378F / D381E / Y697L, A469V, N820S, T412R / E417W / Y697L, L798I, K79R / L388V, A469T, T651Q, D825E, V51T, G787M, A370V, T745V, K815T, T745S, P465Y, Q603M, A78N, G317R, R251A, K815R, D68T, G569Q, C147A, A341T, L824M, A78V, D68Q, R251L, K181R, N820E, K181Q, C147M, R275A, G569M, F735L, A341Y, A336E, A341H, K815L or K815S, wherein the positions are relative to the corresponding sequence of SEQ Reference sequence of ID NO:710.

[0195] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 1208, or to a reference sequence corresponding to SEQ ID NO: 1208, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 1208, or to a reference sequence corresponding to SEQ ID NO: 1208.

[0196] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 1328-1378, or to a reference sequence corresponding to an even numbered SEQ ID NO: 1328-1378, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 1208, or to the reference sequence corresponding to SEQ ID NO: 1208.

[0197] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation or set of mutations at the following amino acid positions: 68 / 251 / 603 / 612 / 798, 68 / 603, 68 / 78 / 370, 370, 251 / 798, 68 / 370 / 603 / 612, 51 / 370 / 412 / 603 / 612, 412 / 603 / 798, 251 / 370 / 412 / 612, 370 / 798, 78 / 251, 251, 68 / 370 / 508 / 798, 251 / 370 / 508 / 798. or 51 / 68 / 251 / 370 / 603, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 1208. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least the mutation or set of mutations 68R / 251I / 603M / 612D / 798I, 68R / 603M, 68R / 78N / 370V, 370V, 251I / 798I, 68G / 370V / 603M / 612D, 51T / 370V / 412R / 603M / 612D, 412R / 603M / 798I, 251I / 370V / 412R / 612D, 370V / 798I, 78N / 251I, 251I, 68R / 370V / 508K / 798I, 251I / 370V / 508K, 78N / 508K, / 370V / 697L / 798I, 51T / 251I, 68G / 370V / 508K / 603M / 697L, 78N / 251I / 370V / 697L / 798I, 78N / 603M / 798I / 831G, 370V / 412R / 508K / 612D, 72A / 508K / 798I, 51T / 251I / 412R / 798I, 51T / 251I / 370V / 508K / 603M / 798I, 68G / 78N / 251I / 370V / 603M / 612D / 697L or 51T / 68T / 251I / 370V / 603M, wherein the positions are relative to the position corresponding to SEQ Reference sequence of ID NO:1208.In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one mutation or set of mutations D68R / R251I / Q603M / E612D / L798I, D68R / Q603M, D68R / A78N / A370V, A370V, R251I / L798I, D68G / A370V / Q603M / E612D, V51T / A370V / T 412R / Q603M / E612D, T412R / Q603M / L798I, R251I / A370V / T412R / E612D, A370V / L798I, A7 8N / R251I, R251I, D68R / A370V / E508K / L798I, R251I / A370V / E508K, A78N / E508K, A78N / A 370V / Y697L / L798I, V51T / R251I, D68G / A370V / E508K / Q603M / Y697L, A78N / R251I / A370V / Y697L / L798I, A78N / Q603M / L798I / E831G, A370V / T412R / E508K / E612D, V72A / E508K / L7 98I, V51T / R251I / T412R / L798I, V51T / R251I / A370V / E508K / Q603M / L798I, D68G / A78N / R251I / A370V / Q603M / E612D / Y697L or V51T / D68T / R251I / A370V / Q603M, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 1208.

[0198] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one mutation provided in Tables 4.1, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 9.3, 10.1, 10.2, 11.1, and 11.2 relative to the reference sequence of SEQ ID NO: 2, 220, 226, 272, 328, 546, 710, or 1208.

[0199] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least a mutation or set of mutations provided in Tables 4.1, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 9.3, 10.1, 10.2, 11.1, and 11.2 relative to a reference sequence corresponding to SEQ ID NO: 2, 220, 226, 272, 328, 546, 710, or 1208.

[0200] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence comprising a mutation or set of mutations as provided in Tables 4.1, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 9.3, 10.1, 10.2, 11.1, and 11.2.

[0201] In some embodiments, the engineered DNA polymerase comprises a sequence comprising residues 12 to 844 of an engineered DNA polymerase shown in Tables 4.1, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 9.3, 10.1, 10.2, 11.1, and 11.2, or a sequence comprising residues 12 to 844 of an engineered DNA polymerase shown in Tables 4.1, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 9.3, 10.1, 10.2, 11.1, and 11.2. .1, 7.2, 8.1, 8.2, 9.1, 9.2, 9.3, 10.1, 10.2, 11.1 and 11.2 have amino acid sequences that have at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

[0202] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to residues 12 to 844 of the even-numbered SEQ ID NOs among SEQ ID NOs: 4-1378, or the sequence corresponding to the even-numbered SEQ ID NOs among SEQ ID NOs: 2-1378.

[0203] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence comprising residues 12 to 844 of an even-numbered SEQ ID NO in SEQ ID NO: 4-1378, or an amino acid sequence comprising an even-numbered SEQ ID NO in SEQ ID NO: 2-1378. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions, insertions and / or deletions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally comprises 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally comprises 1, 2, 3, 4 or 5 substitutions. In some embodiments, the substitutions comprise non-conservative substitutions and / or conservative substitutions. In some embodiments, the substitutions comprise conservative substitutions. In some embodiments, the substitutions comprise non-conservative substitutions. In some embodiments, guidance regarding non-conservative and conservative substitutions is provided by the variants disclosed herein.

[0204] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence comprising residues 12 to 844 of an even-numbered SEQ ID NO: 4-220, 222-268, 270-320, 322-538, 540-674, 676-1190, 1192-1326, or 1328-1378, or an amino acid sequence comprising an even-numbered SEQ ID NO: 2-220, 222-268, 270-320, 322-538, 540-674, 676-1190, 1192-1326, and 1328-1378. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally comprises 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally comprises 1, 2, 3, 4 or 5 substitutions. In some embodiments, the substitution comprises a non-conservative substitution or a conservative substitution. In some embodiments, the substitution comprises a conservative substitution. In some embodiments, the substitution comprises a non-conservative substitution.

[0205] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence comprising residues 12 to 844 of SEQ ID NO: 220, 226, 272, 328, 546, 710, or 1208, or an amino acid sequence comprising SEQ ID NO: 2, 220, 226, 272, 328, 546, 710, or 1208. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally comprises 1, 2, 3, 4, or 5 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally comprises 1, 2, 3, 4, or 5 substitutions. In some embodiments, the engineered DNA polymerase comprises 1, 2, 3, or 4 substitutions in the amino acid sequence.

[0206] In some embodiments, the engineered DNA polymerase is provided as a fusion protein. In some embodiments, the engineered DNA polymerase as described herein can be fused to various peptide sequences, such as, for example, and not limited to, polypeptide tags that can be used for detection and / or purification. In some embodiments, the fusion protein of the engineered DNA polymerase comprises glycine-histidine or a histidine tag (His-tag). In some embodiments, the fusion protein of the engineered DNA polymerase comprises an epitope tag, such as c-myc, FLAG, V5, or hemagglutinin (HA). In some embodiments, the fusion protein of the engineered DNA polymerase comprises GST, SUMO, Strep, MBP, or a GFP tag. In some embodiments, fusion is for the amino (N-) end of the engineered DNA polymerase polypeptide. In some embodiments, fusion is for the carboxyl (C-) end of the engineered DNA polymerase polypeptide.

[0207] In some embodiments, the engineered DNA polymerases of the present disclosure have DNA polymerase activity.In some embodiments, the engineered DNA polymerases have at least one improved or enhanced property compared to a reference DNA polymerase.

[0208] In some embodiments, the engineered DNA polymerase has increased activity compared to a reference DNA polymerase. In some embodiments, the engineered DNA polymerase has at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, or 20-fold or more increase in activity compared to a reference DNA polymerase.

[0209] In some embodiments, the engineered DNA polymerase has increased stability compared to a reference DNA polymerase. In some embodiments, the engineered DNA polymerase has increased thermostability compared to a reference DNA polymerase.

[0210] In some embodiments, the engineered DNA polymerase has increased processivity compared to a reference DNA polymerase. In some embodiments, the engineered DNA polymerase has increased fidelity compared to a reference DNA polymerase.

[0211] In some embodiments, compared with a reference DNA polymerase, the engineered DNA polymerase has an increased input DNA template sensitivity. In some embodiments, compared with a reference DNA polymerase, for example, in a PCR reaction, the engineered DNA polymerase has an increased product yield. In some embodiments, compared with a reference DNA polymerase, the engineered DNA polymerase has an increased product yield, with about 25, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450 or about 500 copies of the input target DNA (e.g., as provided in the Examples).

[0212] In some embodiments, the engineered DNA polymerase has increased resistance or tolerance to an inhibitor, such as guanidine isothiocyanate (GITC), compared to a reference DNA polymerase.

[0213] In some embodiments, the reference DNA polymerase comprises a sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or a sequence corresponding to SEQ ID NO: 2, 220, 226, 272, 328, 546, 710, or 1208. In some embodiments, the reference DNA polymerase has an amino acid sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or an amino acid sequence corresponding to SEQ ID NO: 2.

[0214] In some embodiments, the engineered DNA polymerase has one or more improved properties compared to a reference DNA polymerase selected from: i) increased activity, ii) increased stability, iii) increased thermostability, iv) increased processivity, v) increased fidelity, vi) increased sensitivity to the input DNA template, vii) increased product yield, and viii) increased resistance or tolerance to inhibitors, or any combination of i), ii), iii), vi), v), vi), vii), and viii). In some embodiments, the reference DNA polymerase has an amino acid sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or an amino acid sequence corresponding to SEQ ID NO: 2, 220, 226, 272, 328, 546, 710, or 1208. In some embodiments, the reference DNA polymerase has an amino acid sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or an amino acid sequence corresponding to SEQ ID NO: 2. In some embodiments, specific improvements in DNA polymerase properties are provided in the Examples.

[0215] In some embodiments, the engineered DNA polymerase polypeptides described herein are isolated compositions. In some embodiments, the engineered DNA polymerase polypeptides are purified, as further discussed herein.

[0216] In some embodiments, present disclosure further provides functional fragments or bioactive fragments of engineered DNA polymerase polypeptide as herein described.Therefore, for each and every embodiment of engineered DNA polymerase in this article, functional fragments or bioactive fragments of engineered DNA polymerase are provided in conjunction with.In some embodiments, functional fragments or bioactive fragments of engineered DNA polymerase comprise at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% of the activity of its DNA polymerase polypeptide (that is, parent DNA polymerase).In some embodiments, functional fragments or bioactive fragments comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the parent sequence of DNA polymerase. In some embodiments, the functional fragment is truncated to less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, less than 50 amino acids, less than 55 amino acids, less than 60 amino acids, less than 65 amino acids, or less than 70 amino acids.

[0217] In some embodiments, the functional fragments or biologically active fragments of the engineered DNA polymerase polypeptides described herein include at least one mutation or set of mutations in the amino acid sequence of the engineered DNA polymerase described herein. Accordingly, in some embodiments, the functional fragments or biologically active fragments of the engineered DNA polymerase exhibit enhanced or improved properties associated with the mutation or set of mutations in the parent DNA polymerase.

[0218] Polynucleotides encoding engineered polypeptides, expression vectors and host cells

[0219] In another aspect, the present disclosure provides recombinant polynucleotides encoding the engineered DNA polymerases described herein. In some embodiments, the recombinant polynucleotides are operably linked to one or more heterologous regulatory sequences that control gene expression to produce a recombinant polynucleotide construct capable of expressing the DNA polymerase. In some embodiments, an expression construct containing at least one heterologous polynucleotide encoding the engineered DNA polymerase polypeptide is introduced into an appropriate host cell to express the corresponding DNA polymerase polypeptide.

[0220] As will be apparent to those skilled in the art, the availability of protein sequences and the knowledge of the codons corresponding to the various amino acids provide a description of all polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, in which the same amino acid is encoded by alternative or synonymous codons, allows the preparation of extremely large numbers of nucleic acids, all of which encode the engineered DNA polymerases of the present disclosure. Therefore, the present disclosure provides methods and compositions for producing each and every possible polynucleotide variation that can be prepared by selecting combinations based on possible codon selections, the polynucleotides encoding the engineered DNA polymerase polypeptides described herein, and all such polynucleotide variations are considered to be specifically disclosed for any engineered DNA polymerase polypeptide described herein, including the amino acid sequences presented in the Examples (e.g., Tables 4.1, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 9.3, 10.1, 10.2, 11.1, and 11.2) and the sequence listing.

[0221] In some embodiments, codons are preferably optimized to be utilized for protein production by a selected host cell. In some embodiments, preferred codons in bacteria are used for expression in bacteria. In some embodiments, preferred codons in fungal cells are used for expression in fungi. In some embodiments, preferred codons in mammalian cells are used for expression in mammalian cells. In some embodiments, the codon-optimized polynucleotide encoding the engineered DNA polymerase polypeptides described herein contains preferred codons at about 40%, 50%, 60%, 70%, 80%, 90% or greater than 90% of the codon positions in the full-length coding region.

[0222] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 220, 226, 272, 328, 546, 710 or 1208, or to a reference sequence corresponding to SEQ ID NO: 2, 220, 226, 272, 328, 546, 710 or 1208, wherein the amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 220, 226, 272, 328, 546, 710 or 1208, or to a reference sequence corresponding to SEQ ID NO: One or more mutations of the reference sequence of NO: 2, 220, 226, 272, 328, 546, 710 or 1208.

[0223] As described above, in some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2.

[0224] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, 226, 272, 328, 546, 710, or 1208, or a reference sequence corresponding to SEQ ID NO: 220, 226, 272, 328, 546, 710, or 1208, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0225] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 4-1378, or a sequence corresponding to an even numbered SEQ ID NO: 2-1378, wherein the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0226] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least mutations at the following amino acid positions: 2, 3, 21, 39, 51, 59, 66, 68, 72, 73, 78, 79, 87, 124, 147, 178, 181, 185, 199, 216, 224, 233, 239, 241, 251, 257, 262, 271, 275, 288, 291, 303, 316, 317, 327, 328, 336, 338, 341, 342, 351, 356. , 359, 362, 365, 368, 370, 371, 372, 373, 374, 376, 377, 378, 379, 380, 381, 382, 388, 390, 392, 393, 395, 396, 398, 399, 402, 403, 404, 409, 410, 412, 413, 416, 417, 418, 420, 425, 426, 427, 429, 430, 431, 432, 434, 435, 437, 438, 442, 443, 447, 448, 449, 450, 451, 453, 454, 457, 461 ,465,466,469,473,474,475,477,478,479,482,483,488,490,503,508,514,515,516,526,527,528,529,536,541,542,544,545,550,554,557,558,559,562,569,575,577,578,579,589,594,601,603,604,612,613,641,644,651,656,660,662,663,664,667,671,677,685 , 687, 691, 694, 697, 698, 699, 701, 702, 704, 706, 707, 708, 713, 714, 715, 716, 718, 721, 723, 724, 725, 728, 731, 732, 735, 740, 742, 743, 745, 747, 748, 749, 753, 758, 760, 762, 768, 769, 770, 787, 792, 798, 804, 807, 808, 809, 812, 815, 820, 824, 825 or 831, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0227] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a mutation at amino acid position 147, 257, 291, 395, 402, 474, 475, 478, 514, 515, 526, 527, 528, 536, 559, 604, 660, 662, 687, 745, or 760, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0228] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a mutation at amino acid position 478, 515, 526, 527, or 528, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0229] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a mutation at amino acid position 402, 478, 515, 526, 527, 528, 559, 604, 660, or 760, or a combination thereof, wherein the positions are relative to SEQ ID NO: 2.

[0230] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a mutation at amino acid position 478, 515, 526, 527, 528, 559, or 760, or a combination thereof, wherein the positions are relative to SEQ ID NO: 2.

[0231] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a mutation or set of mutations at the following amino acid positions: 541 / 554 / 557, 516 / 541 / 550 / 554 / 557, 478 / 514 / 526 / 527, 478 / 516 / 526 / 527 / 529 / 541, 288 / 478 / 514 / 516 / 541 / 557, 288 / 478 / 516 / 541 / 550 / 554 / 557, 550 / 554 / 557, 478 / 514 / 526 / 527, 478 / 516 / 526 / 527 / 529 / 541. 8 / 529 / 554 / 557, 478 / 516 / 541, 478 / 541, 288 / 399 / 514 / 554 / 557, 478 / 514 / 554 / 557, 288 / 478 / 526 / 550 / 554, 478 / 541 / 550 / 557, 541, 288 / 514 / 526 / 5 41, 554 / 557, 399 / 478, 288 / 514 / 554 / 557, 478 / 514 / 516 / 526 / 527 / 529 / 557, 399 / 541 / 550 / 554, 516 / 529 / 541 / 554 / 557, 478 / 541 / 554 / 557, 288 / 478 / 5 26 / 528 / 529 / 554 / 557、288 / 514 / 516 / 529 / 541、478 / 514 / 516 / 541、478 / 514 / 557、399 / 541 / 557、399 / 541、288 / 399 / 516 / 527 / 541 / 557、288 / 478 / 514 / 541、288 / 514 / 516、664 / 698、399 / 478 / 516 / 541、399 / 528 / 550、399 / 478 / 514 / 541 / 557、288 / 478 / 514 / 516、224 / 664、478 / 526 / 527 / 528、288 / 478 / 514 / 516 / 541 / 550 / 554、478 / 514 / 541 / 554、478 / 516、478 / 514 / 516 / 541 / 554 / 557、288 / 399 / 478 / 516 / 541、288 / 478 / 514 / 554 / 557、288 / 478 / 516、288 / 399 / 516 / 554 / 557、550 / 554、288 / 550 / 554、288 / 399 / 478 / 516 / 550 / 554、399 / 514 / 541 / 550 / 554、288 / 478 / 514 / 554、199、288 / 399 / 529 / 541、399 / 478 / 554、516 / 528 / 541 / 554 / 557、39 / 199 / 698、399 / 478 / 526 / 528 / 529、288 / 399 / 514 / 541 / 550 / 554、288 / 541、288 / 478 / 514 / 550 / 554 / 557、288 / 399 / 478 / 516、526 / 529 / 554 / 557、288 / 478 / 557、288 / 478 / 514 / 550 / 554、478 / 541 / 550 / 554、516 / 526 / 528、 288 / 526 / 528 / 550 / 554 / 557, 199 / 664 / 698, 514 / 526 / 528 / 529 / 550 / 557, 288, 399 / 554 / 557, 288 / 554 / 557, 399 / 514 / 541, 288 / 478 / 514 / 527 / 554 / 557, 399 / 478 / 516 / 541 / 554, 288 / 550 / 554 / 557, 527 / 529 / 541, 528 / 529 / 554, 478 / 5 54 / 557、478 / 514 / 516 / 541 / 550 / 554 / 557、199 / 216 / 644 / 664、396 / 478 / 514 / 554 / 557 / 694、241 / 644 / 663 / 664、399 / 478 / 554 / 557、288 / 399 / 550、288 / 516 / 526 / 527 / 528 / 557、399 / 526 / 527 / 528 / 529、288 / 478 / 526、399 / 478 / 514 / 516 / 526 / 527 / or 478 / 514 / 526 / 527 / 528, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0232] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a set of mutations at the following amino acid positions: 478 / 514 / 515 / 526 / 527 / 528, 478 / 514 / 526 / 527 / 528 / 594, 478 / 514 / 526 / 527 / 528 / 559, 478 / 514 / 526 / 527 / 528 / 760, 478 / 514 / 526 / 527 / 528 / 541, 478 / 514 / 526 / 527 / 528 / 753, 478 / 514 / 526 / 527 / 528 / 764. 478 / 514 / 526 / 527 / 528 / 589 or 478 / 514 / 526 / 527 / 528 / 544, wherein the positions are relative to the position corresponding to SEQ Reference sequence of ID NO:2.

[0233] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a mutation or set of mutations provided in Tables 6.1 and 6.2, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0234] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a mutation or set of mutations provided in Tables 7.1 and 7.2, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0235] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a mutation or set of mutations provided in Tables 8.1 and 8.2, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0236] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a mutation or set of mutations provided in Tables 9.1, 9.2, and 9.3, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0237] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a mutation or set of mutations provided in Tables 10.1 and 10.2, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0238] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a mutation or set of mutations provided in Tables 11.1 and 11.2, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0239] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, 226, 272, 328, 546, 710 or 1208, or to a reference sequence corresponding to SEQ ID NO: 220, 226, 272, 328, 546, 710 or 1208, wherein the amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, 226, 272, 328, 546, 710 or 1208, or to a reference sequence corresponding to SEQ ID NO: One or more mutations of the reference sequence of NO: 220, 226, 272, 328, 546, 710 or 1208.

[0240] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 4-1378, or a reference sequence corresponding to an even numbered SEQ ID NO: 2-1378, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, 226, 272, 328, 546, 710, or 1208, or relative to the reference sequence corresponding to SEQ ID NO: 220, 226, 272, 328, 546, 710, or 1208.

[0241] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least mutations at the following amino acid positions: 2, 3, 21, 39, 51, 59, 66, 68, 72, 73, 78, 79, 87, 124, 147, 178, 181, 185, 199, 216, 224, 233, 239, 241, 251, 257, 262, 271, 275, 288, 291, 303, 316, 317, 327, 328, 336, 338, 341, 342, 351, 359, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 401, 403, 404, 405, 406, 407, 411, 412, 413, 414, 420, 421. 6, 359, 362, 365, 368, 370, 371, 372, 373, 374, 376, 377, 378, 379, 380, 381, 382, 388, 390, 392, 393, 395, 396, 398, 399, 402, 403, 404, 409, 410, 412, 413, 416, 417, 418, 420, 425, 426, 427, 429, 430, 431, 432, 434, 435, 437, 438, 442, 443, 447, 448, 449, 450, 451, 453, 454, 457, 46 1, 465, 466, 469, 473, 474, 475, 477, 478, 479, 482, 483, 488, 490, 503, 508, 514, 515, 516, 526, 527, 528, 529, 536, 541, 542, 544, 545, 550, 554, 557, 558, 559, 562, 569, 575, 577, 578, 579, 589, 594, 601, 603, 604, 612, 613, 641, 644, 651, 656, 660, 662, 663, 664, 667, 671, 677, 678 85, 687, 691, 694, 697, 698, 699, 701, 702, 704, 706, 707, 708, 713, 714, 715, 716, 718, 721, 723, 724, 725, 728, 731, 732, 735, 740, 742, 743, 745, 747, 748, 749, 753, 758, 760, 762, 768, 769, 770, 787, 792, 798, 804, 807, 808, 809, 812, 815, 820, 824, 825 or 831, or a combination thereof, wherein the position is relative to SEQ Reference sequence of ID NO: 220, 226, 272, 328, 546, 710 or 1208.

[0242] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, or to a reference sequence corresponding to SEQ ID NO: 220, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, or to a reference sequence corresponding to SEQ ID NO: 220.

[0243] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 222-268, or to a reference sequence corresponding to an even numbered SEQ ID NO: 222-268, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, or to the reference sequence corresponding to SEQ ID NO: 220.

[0244] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a mutation or set of mutations at amino acid position 515, 594, 559, 760, 541, 753, 402, 398, 758, 399, 575, 257 / 758, 558, 601, 762, 589, or 544, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 220.

[0245] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 226, or to a reference sequence corresponding to SEQ ID NO: 226, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 226, or to a reference sequence corresponding to SEQ ID NO: 226.

[0246] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 270-320, or to a reference sequence corresponding to SEQ ID NO: 270-320, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 226, or to the reference sequence corresponding to SEQ ID NO: 226.

[0247] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a mutation or set of mutations at the following amino acid positions: 515 / 760, 514 / 515 / 760, 478 / 515 / 760, 660, 178, 402 / 515, 377 / 515, 515, 514 / 515, 402, 503, 239, 73, 528 / 541, 59, 515 / 575, 3 / 515 / 559, 377 / 515 / 528 / 541 / 554 / 557, 402 / 514 / 515 / 541 / 559, 514 / 515 / 541 / 559, 514 / 515 / 559, 515 / 559, 515 / 541 / 559, 377 / 515 / 559, 402 / 515 / 559 or 515 / 541 / 557, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 226.

[0248] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 272, or to a reference sequence corresponding to SEQ ID NO: 272, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 272, or to a reference sequence corresponding to SEQ ID NO: 272.

[0249] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 322-534, or a reference sequence corresponding to an even numbered SEQ ID NO: 322-534, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 272, or relative to the reference sequence corresponding to SEQ ID NO: 272.

[0250] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a mutation or set of mutations at the following amino acid positions: 402 / 528 / 541 / 660, 257 / 604 / 660, 178 / 257 / 402 / 404 / 528 / 557 / 660, 402 / 604 / 660, 257 / 402 / 541 / 660, 377 / 402 / 541 / 660, 257 / 377 / 402 / 660, 483 / 660, 257 / 402 / 257 / 377 / 402 / 660, 178 / 257 / 402 / 404 / 528 / 557 / 660, 257 / 377 / 402, 660, 178 / 257 / 377 / 402 / 403 / 483 / 604 / 660, 377, 178 / 402 / 403 / 528 / 541 / 660, 377 / 601 / 660, 178 / 377, 377 / 490 / 660, 402 / 403 / 483 / 554 / 557, 178 / 257 / 377 / 558 / 660, 257 / 402 / 403 / 483 / 660, 178 / 554 / 557 / 558 / 660, 554 / 557 / 660, 178 / 483 / 541 / 545 / 554 / 557 / 604 / 660, 92, 402 / 403 / 483 / 604 / 660, 178, 474, 257 / 377 / 541 / 604, 743, 396, 479, 377 / 403 / 541 / 604, 475, 178 / 257 / 403 / 528, 448, 577, 395, 769, 691, 677, 704, 377 / 541 / 575 / 604 / 660, 178 / 257 / 601 / 604, 685, 377 / 604 / 660, 749, 482, 291, 579, 178 / 541 / 550 / 604, 477, 178 / 257, 257 / 402 / 490 / 52 402 / 528 / 541 / 601, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 272.

[0251] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 328, or to a reference sequence corresponding to SEQ ID NO: 328, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 328, or to a reference sequence corresponding to SEQ ID NO: 328.

[0252] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 536-674, or a reference sequence corresponding to an even numbered SEQ ID NO: 536-674, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 328, or relative to the reference sequence corresponding to SEQ ID NO: 328.

[0253] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a mutation or set of mutations at the following amino acid positions: 475 / 704, 79, 257 / 474, 677, 562, 178 / 395 / 745, 291 / 395 / 687 / 745, 257 / 395 / 67 7, 303 / 338, 474 / 475, 359, 474 / 475 / 677 / 704, 395 / 687, 671, 233, 699, 379, 257 / 792, 257 / 395, 21, 257 / 448 / 474 / 475, 713, 641, 442, 656, 475 / 704, 742, 667, 257 / 448, 257 / 316 / 39 5 / 482 / 687 / 745、804、704、178 / 257 / 395 / 482 / 579 / 687 / 745、257 / 579、257 / 291 / 395 / 475 / 541 / 687、257 / 768、812、475、662、536、748、808、291 / 316 / 395 / 687、613、257 / 291 / 316、 257 / 291 / 579, 257 / 316 / 541 / 745, 351, 257 / 316 / 395 / 745, 185, 257 / 316 / 687, 291 / 316 / 475 / 687 / 745, 257 / 291 / 395 / 740 / 745, 450, 473, 124, 430, 413, 66 or 365, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 328.

[0254] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 546, or to a reference sequence corresponding to SEQ ID NO: 546, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 546, or to a reference sequence corresponding to SEQ ID NO: 546.

[0255] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 676-1190, or to a reference sequence corresponding to an even numbered SEQ ID NO: 676-1190, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 546, or to the reference sequence corresponding to SEQ ID NO: 546.

[0256] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising a mutation or set of mutations at at least the following amino acid positions: 438, 697, 457, 716, 79 / 474 / 536 / 699, 536, 392, 708, 431, 395 / 474 / 536 / 699, 453, 427, 79 / 395 / 699, 378, 370, 706, 257 / 395 / 474 / 475 / 536 / 662, 474 / 536 / 562, 257 / 395 / 474 / 536 / 699, 453, 427, 79 / 395 / 699, 378, 370, 706, 5 / 536 / 562、79 / 395 / 536 / 699、372、257 / 395 / 792、390、79 / 474 / 536 / 699 / 792、409、443、448、381、707、416、474 / 475 / 662、79 / 395 / 474 / 475 / 536 / 792、474 / 475 / 536 / 662、382、257 / 474 / 536 / 562 / 662、257 / 395 / 474 / 475 / 792、79 / 474 / 662 / 699、725、447、79 / 257 / 699、701、257 / 474 / 475 / 536 / 562 / 662 、702、342 / 716、233 / 562 / 713、704、474 / 475 / 536 / 562 / 662、454、327 / 427、257 / 316 / 474 / 536 / 792、417、425、536 / 562、714、233、303 / 613、21 / 79 / 303 / 562 / 613、79 / 562、435、257 / 536 / 699 / 792、79 / 536、453 / 667、79 / 303 / 338 / 613 / 804 / 812、79 / 257 / 395 / 536 / 662 / 792、79 / 338 / 562、79 / 233 / 303 / 662 、79 / 662 / 699、79 / 395 / 475 / 662、79 / 536 / 562 / 699、536 / 662、21 / 79 / 338 / 641 / 699、662、21 / 79 / 442 / 699、79 / 303 / 662、79 / 178 / 257 / 662 / 699、79 / 257 / 536 / 562 / 662、562 / 699、359 / 613 / 742 / 808 / 812、21 / 79 / 338 / 379 / 562 / 662 / 804 / 812、731、21 / 79 / 359 / 442 / 662、79 / 338 / 359 / 562、21 / 613 / 662 / 742、21 / 79 / 303 / 662 / 699 / 742 / 808、21 / 79 / 338 / 442 / 613 / 699、79 / 303 / 338 / 562 / 613 / 662、303 / 338 / 562 / 699 / 808 / 812、79 / 257、257 / 662 / 792、79 / 536 / 699、79 / 359 / 713 / 742 / 808、21 / 359 / 442 / 808 / 812、79 / 257 / 475 / 536 / 662、21 / 79 / 338、79 / 713、79 / 562 / 641、373、79 / 178 / 257 / 316 / 536 / 662、21 / 233 / 338 / 562 / 662、257 / 316 / 536、21 / 79 / 303 / 442 / 662、21 / 79 / 233 / 303 / 338 / 379 / 562 / 713、338 / 442 / 699、426、21 / 338 / 359 / 808 / 812、79 / 178 / 233 / 442 / 562 / 812、79 / 474 / 536 / 662 / 699、21 / 79 / 338 / 379 / 662、721、21 / 79 / 233 / 338 / 613 / 641、374、338 / 359 / 379、461、451、715、79 / 303 / 338 / 641 / 713、393、79 / 257 / 474 / 536 / 699、79、437、257 / 536 / 562 / 662、21 / 79 / 379 / 442 / 562 / 662、362、410、338 / 812、21 / 662 / 808 / 812、21 / 303 / 338、418、338 / 442、449、379 / 641、316 / 536 / 562、257 / 536 / 792、21 / 79 / 442 / 613、257 / 536 / 662 / 792、257 / 474 / 529 / 536 / 662、412、79 / 257 / 536 / 662 / 699、257 / 316 / 475 / 536 / 792、388、380 / 454、21 / 79 / 233 / 303 / 613 / 699 / 809、724、21 / 79 / 338 / 613、79 / 178 / 536 / 792、449 / 747、21 / 79 / 379 / 613 / 713、79 / 316 / 536 / 662 / 699、376、21 / 562 / 662、79 / 257 / 262 / 536 / 562 / 699、718、395 / 475 / 536 / 562 / 662 / 699、79 / 233 / 338 / 359 / 379 / 442 / 562 / 613 / 812、371、316 / 475 / 562 / 662 / 792、79 / 257 / 316 / 792、728、79 / 233 / 338, 432, 79 / 178 / 257 / 316 / 395 / 536 / 662 / 699, 79 / 303 / 713, 233 / 379 / 442 / 562 / 613 / 662 / 713, 21 / 742, 732, 79 / 257 / 395 / 562 / 662 / 699, 21 / 233 / 338 / 379 / 442 / 562 / 613 / 662 / 812, 429, 562 / 699, 2 / 374, 723, 434, 420 or 699, wherein the positions are relative to the reference sequence corresponding to SEQ ID NO: 546.

[0257] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 710, or to a reference sequence corresponding to SEQ ID NO: 710, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 710, or to a reference sequence corresponding to SEQ ID NO: 710.

[0258] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 1192-1326, or a reference sequence corresponding to an even numbered SEQ ID NO: 1192-1326, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710.

[0259] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a mutation or set of mutations at amino acid position(s): 68, 78, 466, 807, 612, 147, 356, 488, 603, 271, 508, 787, 412 / 417 / 427, 251, 697, 562, 79 / 434 / 562, 820, 275, 378 / 381 / 697, 469, 412 / 417 / 697, 798, 79 / 388, 651, 825, 51, 370, 745, 815, 465, 317, 569, 341, 824, 18, 181, 735, or 336, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 710.

[0260] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 1208, or to a reference sequence corresponding to SEQ ID NO: 1208, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 1208, or to a reference sequence corresponding to SEQ ID NO: 1208.

[0261] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 1328-1378, or to a reference sequence corresponding to an even numbered SEQ ID NO: 1328-1378, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 1208, or to the reference sequence corresponding to SEQ ID NO: 1208.

[0262] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a mutation or set of mutations at the following amino acid positions: 68 / 251 / 603 / 612 / 798, 68 / 603, 68 / 78 / 370, 370, 251 / 798, 68 / 370 / 603 / 612, 51 / 370 / 412 / 603 / 612, 412 / 603 / 798, 251 / 370 / 412 / 612, 370 / 798, 78 / 251, 251, 68 / 370 / 508 / 798. or 51 / 68 / 251 / 370 / 603, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 1208.

[0263] In some embodiments, for each of the aforementioned embodiments of the recombinant polynucleotide, the specific amino acid mutations described herein for each mutation or set of mutations can be used in the encoded engineered DNA polymerase polypeptide.

[0264] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least one mutation provided in Tables 4.1, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 9.3, 10.1, 10.2, 11.1, and 11.2, wherein the amino acid position is relative to a reference sequence of SEQ ID NO: 2, 220, 226, 272, 328, 546, 710, or 1208.

[0265] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a mutation or set of mutations provided in Tables 4.1, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 9.3, 10.1, 10.2, 11.1, and 11.2, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 2, 220, 226, 272, 328, 546, 710, or 1208.

[0266] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence comprising a mutation or set of mutations as provided in Tables 4.1, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 9.3, 10.1, 10.2, 11.1, and 11.2.

[0267] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to: comprising Tables 4.1, 5.1, 5.2, 6.1, 6.2, 7 .1, 7.2, 8.1, 8.2, 9.1, 9.2, 9.3, 10.1, 10.2, 11.1 and 11.2, or comprises the sequence of residues 12 to 844 of the engineered DNA polymerase shown in Tables 4.1, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 9.3, 10.1, 10.2, 11.1 and 11.2.

[0268] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the sequence corresponding to residues 12 to 844 of the even numbered SEQ ID NOs among SEQ ID NOs: 4-1378, or the sequence corresponding to the even numbered SEQ ID NOs among SEQ ID NOs: 2-1378.

[0269] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising residues 12 to 844 of an even-numbered SEQ ID NO in SEQ ID NOs: 4-1378 or an amino acid sequence comprising an even-numbered SEQ ID NO in SEQ ID NOs: 2-1378. In some embodiments, the amino acid sequence of the encoded engineered DNA polymerase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions in the amino acid sequence. In some embodiments, the encoded engineered DNA polymerase polypeptide optionally comprises 1, 2, 3, 4, or up to 5 substitutions in the amino acid sequence. In some embodiments, the engineered DNA polymerase polypeptide optionally comprises 1, 2, 3, or 4 substitutions in the amino acid sequence. In some embodiments, the substitution comprises a non-conservative substitution or a conservative substitution. In some embodiments, the substitution comprises a conservative substitution. In some embodiments, the substitution comprises a non-conservative substitution.

[0270] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising residues 12 to 844 of SEQ ID NO: 220, 226, 272, 328, 546, 710, or 1208, or an amino acid sequence comprising SEQ ID NO: 2, 220, 226, 272, 328, 546, 710, or 1208. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions in the amino acid sequence. In some embodiments, the encoded DNA polymerase optionally comprises 1, 2, 3, 4, up to 5 substitutions in the amino acid sequence. In some embodiments, the encoded DNA polymerase optionally comprises 1, 2, 3, or 4 substitutions in the amino acid sequence.

[0271] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 2532 of an odd-numbered SEQ ID NO: 1-1377, or a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO: 1-1377, wherein the recombinant polynucleotide encodes a DNA polymerase.

[0272] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence that has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 2532 of SEQ ID NO: 1, 219, 225, 271, 327, 545, 709, or 1207, or a reference polynucleotide sequence corresponding to SEQ ID NO: 1, 219, 225, 271, 327, 545, 709, or 1207, wherein the recombinant polynucleotide encodes a DNA polymerase.

[0273] In some embodiments, the recombinant polynucleotide encoding the engineered DNA polymerase comprises a polynucleotide sequence comprising nucleotide residues 34 to 2532 of an odd-numbered SEQ ID NO among SEQ ID NOs: 1-1377, or a polynucleotide sequence comprising an odd-numbered SEQ ID NO among SEQ ID NOs: 1-1377.

[0274] In some embodiments, the recombinant polynucleotide encoding the engineered DNA polymerase comprises a polynucleotide sequence comprising nucleotide residues 34 to 2532 of SEQ ID NO: 1, 219, 225, 271, 327, 545, 709, or 1207, or a polynucleotide sequence comprising SEQ ID NO: 1, 219, 225, 271, 327, 545, 709, or 1207.

[0275] In some embodiments, the disclosure provides recombinant polynucleotides that can hybridize under high stringency conditions to a reference polynucleotide encoding an engineered DNA polymerase polypeptide described herein, e.g., the recombinant polynucleotides provided in Tables 4.1, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 9.3, 10.1, 10.2, 11.1, and 11.2, or their reverse complements. In some embodiments, the recombinant polynucleotides hybridize under high stringency conditions to a reference polynucleotide sequence encoding an engineered DNA polymerase described herein. In some embodiments, the recombinant polynucleotide hybridizes under high stringency conditions to a reference polynucleotide corresponding to nucleotide residues 34 to 2532 of SEQ ID NO: 1, 219, 225, 271, 327, 545, 709, or 1207, or a sequence corresponding to SEQ ID NO: 1, 219, 225, 271, 327, 545, 709, or 1207, or a reverse complement thereof. In some embodiments, the recombinant polynucleotide hybridizes under high stringency conditions to a reference polynucleotide corresponding to nucleotide residues 34 to 2532 of an odd-numbered SEQ ID NO of SEQ ID NO: 1-1377, or a reference polynucleotide comprising a sequence corresponding to an odd-numbered SEQ ID NO of SEQ ID NO: 1-1377, or a reverse complement thereof.

[0276] In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes a DNA polymerase comprising an amino acid sequence having one or more residue differences compared to SEQ ID NO: 2, 220, 226, 272, 328, 546, 710 or 1208 at a residue position selected from any of the positions shown in Tables 4.1, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 9.3, 10.1, 10.2, 11.1 and 11.2. In some embodiments, a polynucleotide that hybridizes under high stringency conditions comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 34 to 2532 of SEQ ID NO: 1, 219, 225, 271, 327, 545, 709 or 1207, or a reference sequence corresponding to SEQ ID NO: 1, 219, 225, 271, 327, 545, 709 or 1207. In some additional embodiments, the polynucleotides that hybridize under high stringency conditions comprise at least one polynucleotide reference sequence corresponding to residues 34 to 2532 of the polynucleotide sequences provided in Tables 4.1, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 9.3, 10.1, 10.2, 11.1, and 11.2, or Tables 4.1, 5.1, 5.2, 6.1, 6.2 , 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 9.3, 10.1, 10.2, 11.1 and 11.2 have sequences having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

[0277] In some embodiments, the recombinant polynucleotide of any DNA polymerase of encoding this paper operates in various ways to promote the expression of DNA polymerase polypeptide.In some embodiments, the recombinant polynucleotide of encoding DNA polymerase comprises an expression vector, wherein there are one or more control sequences to regulate the expression of DNA polymerase polynucleotide and / or coded polypeptide.Depend on the expression vector utilized, the operation of the isolated polynucleotide before it is inserted into the vector may be expectation or necessity.Utilizing recombinant DNA method to be used for the technology of modifying polynucleotide and nucleotide sequence is well known in the art.In some embodiments, regulating and controlling sequence especially comprises promotor, leader sequence, polyadenylation sequence, propeptide sequence, signal peptide sequence and transcription terminator.

[0278] In some embodiments, suitable promoters are selected based on the choice of host cells. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure include, but are not limited to, promoters derived from the following: the Escherichia coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis α-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens α-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic β-lactamase genes (see, e.g., Villa-Kamaroff et al., Proc. Natl. Acad. Sci. USA 2005). Acad. Sci. USA, 1978, 75:3727-3731), and the tac promoter (see, for example, DeBoer et al., Proc. Natl Acad. Sci. USA, 1983, 80:21-25). Exemplary promoters for filamentous fungal host cells include, but are not limited to, promoters from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline proteinase, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 97 / 11665). 96 / 00787), and the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral α-amylase and Aspergillus oryzae triosephosphate isomerase), as well as mutants, truncations, and hybrid promoters thereof. Exemplary yeast cell promoters can be from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.Other useful promoters for yeast host cells are known in the art (see, eg, Romanos et al., Yeast, 1992, 8:423-488).

[0279] In some embodiments, the control sequence is a suitable transcription termination sequence (i.e., a sequence recognized by the host cell to terminate transcription). In some embodiments, the termination sequence is operably linked to the 3' end of the nucleic acid sequence encoding the DNA polymerase polypeptide. Any suitable terminator that is functional in the selected host cell can be used in the present invention. For bacterial expression, the transcription terminator can be a Rho-dependent terminator that relies on the Rho transcription factor, or a Rho-independent or intrinsic terminator that does not require a transcription factor. Exemplary transcription terminators for filamentous fungal host cells can be derived from genes for: Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger α-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be derived from genes for: Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae 3-phosphate glyceraldehyde dehydrogenase. Other available terminators for yeast host cells are known in the art (see, e.g., Romanos et al., supra).

[0280] In some embodiments, the control sequence is a suitable leader sequence (i.e., the non-translational region of the mRNA important for translation by the host cell). In some embodiments, the leader sequence is operably linked to the 5' end of the nucleotide sequence encoding the DNA polymerase polypeptide. Any suitable leader sequence with function in the selected host cell can be used in the present invention. Exemplary leaders for filamentous fungal host cells derive from genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells derive from genes for: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor, and Saccharomyces cerevisiae alcohol dehydrogenase / 3-phosphoglyceraldehyde dehydrogenase (ADH2 / GAP).

[0281] In some embodiments, the control sequence is a polyadenylation sequence (i.e., operably linked to the 3' end of the nucleic acid sequence and, when transcribed, recognized by the host cell as a sequence that signals the addition of polyadenylic acid residues to the transcribed mRNA). Any suitable polyadenylation sequence that functions in the selected host cell can be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, genes for: Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger α-glucosidase. Available polyadenylation sequences for yeast host cells are known (see, e.g., Guo and Sherman, Mol. Cell. Biol., 1995, 15: 5983-5990).

[0282] In some embodiments, the control sequence is also a signal peptide (i.e., a coding region that encodes an amino acid sequence that is linked to the amino terminus of a polypeptide and directs the encoded polypeptide to enter the secretory pathway of the cell). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region that is naturally linked in the translation reading frame to the segment of the coding region that encodes the secretory polypeptide. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence. Any suitable signal peptide coding region that directs the expressed polypeptide to enter the secretory pathway of the selected host cell can be used for expression of the engineered polypeptide. Signal peptide coding regions that are effective for bacterial host cells are signal peptide coding regions, including but not limited to those obtained from genes for: Bacillus NClB 11837 maltogenic amylase, Bacillus stearothermophilus α-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are known in the art (see, e.g., Simonen and Palva, Microbiol. Rev., 1993, 57: 109-137). In some embodiments, effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to, those derived from genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Available signal peptides for yeast host cells include, but are not limited to, those derived from genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase.

[0283] In some embodiments, the control sequence is a propeptide coding region that encodes an amino acid sequence placed at the amino terminus of a polypeptide. The resulting polypeptide is referred to as a "proenzyme," "propolypeptide," or "zymogen." The propolypeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be derived from any suitable source, including but not limited to genes for: Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha factor, Rhizomucor miehei aspartic protease, and Myceliophthora thermophila lactase (see, e.g., WO 95 / 33836). When both the signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is placed near the amino terminus of the polypeptide, and the signal peptide region is placed near the amino terminus of the propeptide region.

[0284] In some embodiments, regulatory sequences are also utilized. These sequences promote the regulation of polypeptide expression relative to host cell growth. Examples of regulatory systems are those that turn gene expression on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to, the lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, but are not limited to, the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include, but are not limited to, the TAKA α-amylase promoter, the Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter.

[0285] In yet another aspect, present disclosure provides recombinant expression vectors, which comprise the recombinant polynucleotides of the engineered DNA polymerase polypeptides of coding, and one or more expression control regions, such as promoters and terminators, replication origins, etc., depending on the host type to be introduced therein. In some embodiments, various nucleic acids as herein described and regulatory sequences are linked together (i.e., operably connected) to produce recombinant expression vectors, which comprise one or more convenient restriction sites to allow insertion or replacement of the nucleotide sequence encoding the DNA polymerase polypeptide at such sites. Alternatively, in some embodiments, nucleotide sequence of the present invention is expressed by inserting the nucleic acid sequence or the nucleic acid construct comprising the sequence into the appropriate vector for expression. In some embodiments relating to expression vector production, the coding sequence is positioned in the vector so that the coding sequence is operably connected with the appropriate control sequence for expression.

[0286] In the embodiments herein, the recombinant expression vector may be any suitable vector (e.g., a plasmid or virus) that can be readily subjected to recombinant DNA procedures and results in the expression of a polynucleotide encoding a DNA polymerase. The choice of vector generally depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

[0287] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector existing as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome). The vector may contain any means for ensuring self-replication. In some alternative embodiments, the vector is a vector that, when introduced into the host cell, is integrated into the genome and replicates together with the chromosome into which it has been integrated. In addition, in some embodiments, a single vector or plasmid is utilized, or two or more vectors or plasmids, and / or transposons, containing together the total DNA to be introduced into the genome of the host cell are used.

[0288] In some embodiments, the expression vector contains one or more selectable markers that allow easy selection of transformed cells. A "selectable marker" is a gene whose product provides biocide or viral resistance, resistance to heavy metals, prototrophy for auxotrophs, and the like. Examples of bacterial selectable markers include, but are not limited to, the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance, such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase; e.g., from Aspergillus nidulans or Aspergillus oryzae), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase; e.g., from S. hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase; e.g., from Aspergillus nidulans or Aspergillus oryzae), sC (sulfate adenylyltransferase), and trpC (anthranilate synthase), and equivalents thereof.

[0289] In another aspect, the disclosure provides a host cell comprising at least one recombinant polynucleotide encoding at least one engineered DNA polymerase polypeptide of the present invention, the recombinant polynucleotide being operably linked to one or more control sequences for expressing the engineered DNA polymerase in the host cell. Host cells suitable for expressing the polypeptide encoded by the expression vector of the present invention are well known in the art and include, but are not limited to, bacterial cells, such as Escherichia coli, Vibrio fluvialis, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells, such as Drosophila S2 cells and Spodoptera Sf9 cells; animal cells, such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Exemplary host cells also include various E. coli strains (eg, W3110 (ΔfhuA) and BL21).

[0290] Accordingly, in another aspect, the disclosure provides a method for producing an engineered DNA polymerase polypeptide, wherein the method comprises cultivating a host cell capable of expressing a polynucleotide encoding an engineered DNA polymerase polypeptide under conditions suitable for expressing or producing the encoded polypeptide. In some embodiments, the method further comprises a step of, for example, isolating the DNA polymerase polypeptide from the culture medium and / or the host cell. In some embodiments, the method further comprises purifying the DNA polypeptide polypeptide, as described herein. In some embodiments, the host cell produces more than one engineered DNA polymerase polypeptide.

[0291] Suitable culture media and growth conditions for host cells are well known in the art. Any suitable method for introducing a polynucleotide for expressing a DNA polymerase polypeptide into a cell is contemplated for use in the present invention. Suitable techniques include, but are not limited to, electroporation, gene gun particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0292] The recombinant polypeptides herein (e.g., DNA polymerase variants) can be produced using any suitable method known in the art. For example, there are a wide variety of mutagenesis techniques well known to those skilled in the art. In addition, mutagenesis kits are also available from many commercial molecular biology suppliers. The method can be used to prepare specific substitutions (site-directed mutagenesis) at limited amino acid locations, specific or random mutations (region-specific) in the restricted regions of a gene, or random mutagenesis (e.g., saturation mutagenesis) throughout a gene. Numerous suitable methods are well known to those skilled in the art, to generate enzyme variants, including but not limited to site-directed mutagenesis, cassette mutagenesis, gene synthesis, error-prone PCR, reorganization, and chemical saturation mutagenesis using PCR for single-stranded or double-stranded DNA, or any other suitable method known in the art. Non-limiting examples of methods for DNA and protein engineering are provided in the following patents: U.S. Patent No. 6,117,679; U.S. Patent No. 6,420,175; U.S. Patent No. 6,376,246; U.S. Patent No. 6,586,182; U.S. Patent No. 7,747,391; U.S. Patent No. 7,747,393; U.S. Patent No. 7,783,428; and U.S. Patent No. 8,383,346. After variants are generated, they can be screened for any desired property (e.g., high or increased activity, or low or decreased activity, increased thermal activity, increased stability, increased processability, increased fidelity, increased inhibitor resistance or tolerance, and / or pH stability, etc.).

[0293] In some embodiments, the engineered DNA polymerase polypeptides having properties disclosed herein can be obtained by subjecting the polynucleotides encoding naturally occurring or engineered DNA polymerase polypeptides to any suitable mutagenesis and / or directed evolution methods known in the art, e.g., as described herein. Exemplary directed evolution techniques are mutagenesis and / or DNA shuffling (see, e.g., Stemmer, Proc. Natl. Acad. Sci. USA, 1994, 91: 10747-10751; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767 and U.S. Patent No. 6,537,746). Other directed evolution procedures that can be used include the staggered extension process (StEP), in vitro recombination (see, e.g., Zhao et al., Nat. Biotechnol., 1998, 16:258-261), mutagenic PCR (see, e.g., Caldwell et al., PCR Methods Appl., 1994, 3:S136-S140), and cassette mutagenesis (see, e.g., Black et al., Proc. Natl. Acad. Sci. USA, 1996, 93:3525-3529), among others.

[0294] Mutagenesis and directed evolution methods can be readily applied to polynucleotides encoding DNA polymerases to generate libraries of variants that can be expressed, screened, and assayed. Any suitable mutagenesis and directed evolution methods can be used in the present invention and are known in the art (see, e.g., U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,265,201, 6,277,638, 6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344, 6,3 09,883、6,319,713、6,319,714、6,323,030、6,326,204、6,335,160、6,335,198、6,344,356、6,352,859、6,355,484、6,358,740、6,358,742、6,365,377、6,365,408、6,368,861、6,372,497、6,337,186、6,376,246、6,379,964、6,387,702、6,391,552、6,391,640、6,395,547、6,406,855、6 ,406,910、6,413,745、6,413,774、6,420,175、6,423,542、6,426,224、6,436,675、6,444,468、6,455,253、6,479,652、6,482,647、6,483,011、6,484,105、6,489,146、6,500,617、6,500,639、6,506,602、6,506,603、6,518,065、6,519,065、6,521,453、6,528,311、6,537,746、6,573,09 8、6,576,467、6,579,678、6,586,182、6,602,986、6,605,430、6,613,514、6,653,072、6,686,515、6,703,240、6,716,631、6,825,001、6,902,922、6,917,882、6,946,296、6,961,664、6,995,017、7,024,312、7,058,515、7,105,297、7,148,054、7,220,566、7,288,375、7,384,387、7,421,347、7,430,477、7,462,469、7,534,564、7,620,500、7,620,502、7,629,170、7,702,464、7,747,391、7,747,393、7,751,986、7,776,598、7,783,428、7,795,030、7,853,410、7,868,138、7,783,428、7,873,477、7,873,499、7,904,249、7,957,912、7,981,614、8,014,961、8,029,988、8,048,674、 8,058,001, 8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681, 8,383,346, 8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871, 9,593,326, 9,665,694, 9,684,771, and all relevant PCT and non-U.S. counterparts; Ling et al., Anal. Biochem., 1997, 254(2):157-78; Dale et al., Meth. Mol. Biol., 1996, 57:369-74; Smith, Ann. Rev. Genet., 1985, 19:423-462; Botstein et al., Science, 1985, 229:1193-1201; Carter, Biochem. J., 1986, 237:1-7; Kramer et al., Cell, 1984, 38:879-887; Wells et al., Gene, 1985, 34:315-323; Minshull et al., Curr. Op. Chem. Biol., 1999, 3:284-290; Christians et al., Nat. Biotechnol., 1999, 17:259-264; Crameri et al., Nature, 1998, 391:288-291; Crameri, et al., Nat. Biotechnol., 1997, 15:436-438; Zhang et al., Proc. Nat. Acad. Sci. USA, 1997, 94:4504-4509; Crameri et al., Nat. Biotechnol., 1996, 14:315-319; Stemmer, Nature, 1994, 370:389-391; Stemmer, Proc. Nat. Acad. Sci. USA, 1994,91:10747-10751; EP 3 049973; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767; WO 2009 / 152336; and WO 2015 / 048573, all o...

Claims

1. An engineered DNA polymerase or a functional fragment thereof, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 220, 226, 272, 328, 546, 710 or 1208, or to a reference sequence corresponding to SEQ ID NO: 2, 220, 226, 272, 328, 546, 710 or 1208, wherein the amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 220, 226, 272, 328, 546, 710 or 1208, or to a reference sequence corresponding to SEQ ID NO: One or more mutations of the reference sequence of NO: 2, 220, 226, 272, 328, 546, 710 or 1208.

2. The engineered DNA polymerase of claim 1 , comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO:

2.

3. The engineered DNA polymerase of claim 1 , comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, 226, 272, 328, 546, 710 or 1208, or a reference sequence corresponding to SEQ ID NO: 220, 226, 272, 328, 546, 710 or 1208, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

4. The engineered DNA polymerase of claim 1 , comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 4-1378, or a reference sequence corresponding to an even numbered SEQ ID NO: 4-1378, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

5. The engineered DNA polymerase of any one of claims 1 to 4, wherein the amino acid sequence of the engineered DNA polymerase comprises mutations at at least the following amino acid positions: 2, 3, 21, 39, 51, 59, 66, 68, 72, 73, 78, 79, 87, 124, 147, 178, 181, 185, 199, 216, 224, 233, 239, 241, 251, 257, 262, 271, 275, 288, 291, 303, 316, 317, 327, 328, 336, 338, 341, 342, 351, 356, 359, 362 , 365, 368, 370, 371, 372, 373, 374, 376, 377, 378, 379, 380, 381, 382, 388, 390, 392, 393, 395, 396, 398, 399, 402, 403, 404, 409, 410, 412, 413, 416, 417, 418, 420, 425, 426, 427, 429, 430, 431, 432, 434, 435, 437, 438, 442, 443, 447, 448, 449, 450, 451, 453, 453, 454, 457, 461, 465 , 466, 469, 473, 474, 475, 477, 478, 479, 482, 483, 488, 490, 503, 508, 514, 515, 516, 526, 527, 528, 529, 536, 541, 542, 544, 545, 550, 554, 557, 558, 559, 562, 569, 575, 577, 578, 579, 589, 594, 601, 603, 604, 612, 613, 641, 644, 651, 656, 660, 662, 663, 664, 667, 671, 677, 685, 687 , 691, 691, 694, 697, 698, 699, 701, 702, 704, 706, 707, 708, 713, 714, 715, 716, 718, 721, 723, 724, 725, 728, 731, 732, 735, 740, 742, 743, 745, 747, 748, 749, 753, 758, 760, 762, 768, 769, 770, 787, 792, 798, 804, 807, 808, 809, 812, 815, 820, 824, 825 or 831, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO:

2.

6. The engineered DNA polymerase of any one of claims 1 to 5, wherein the amino acid sequence of the engineered DNA polymerase comprises at least mutations 2-, 3N, 21A / G / N, 39F, 51T, 59K, 66R, 68E / G / I / K / Q / R / T, 72A, 73I, 78N / S / T / V, 79A / M / R / T, 87V, 124A, 147A / H / M / Q, 178L, 181Q / R, 185T, 199P, 216K, 224L, 233H / K / L, 239V, 241E, 251A / I / L, 257V, 262N, 271G, 275A / T, 288R, 291L, 303T, 16G, 317R, 327E / L, 328L / V, 336E, 338V, 341H / T / Y, 342G, 351H, 356I / S, 359 G / V, 362M / P / S, 365T, 368H, 370G / S / V, 371L, 372V / Y, 373G / T, 374S / V / Y, 37 6F / S, 377C, 378C / F / L / S, 379G, 380I, 381E / K, 382C / S, 388C / M / P / V, 390S / T , 392A / C / S / T / V, 393G / R / S / W, 395G / H / S, 396F / L / P / R / S, 398G / Q, 399G / T, 40 2R / Y, 403I, 404T, 409S, 410V / W, 412M / Q / R / V, 413T, 416S, 417R / W, 418K, 42 0V, 425T, 426T, 427D / F / H / I / L / R / T / Y, 429F, 430G, 431G / L / R / V, 432T, 434N , 435K / P, 437N / P / S, 438F / S, 442G, 443A, 447R / S / W, 448C / I / T / V, 449A / G / L / V, 450V, 451L, 453L / S, 454D / F / G / L / W, 457N / R / W / Y, 461L / Q / S, 465Y, 466Y, 469T / V, 473N, 474A / C / G, 475A / S / V / Y, 477V, 478I / L, 479L, 482S, 483D, 488 W, 490W, 503R, 508K, 514I, 515H / T, 516K, 526S, 527A, 528A / S, 529S / V, 536V, 541D / G / N / S, 542R, 544M, 545L, 550C / T, 554G / N, 557I, 558G / L / T, 559R, 562 K, 569M / Q, 575W, 577S, 578A, 579L / Q, 589G, 594A, 601T, 603M / R, 604H, 612D,613H / R, 641A, 644Q, 651Q, 656Q / S, 660V, 662S, 663E, 664A, 667S / T / W, 671W, 677L, 685R, 687A, 691S / W, 694D, 697A / I / L / V, 69 8E, 699V, 701M, 702S / V, 704L / V, 706I / S / V, 707W, 708L, 713G, 714I, 715H / L, 716E / L, 718F / Q, 721A / M / N, 723Y, 724F / L, 725V, 7 28S, 731I / T, 732L / Q, 735L, 740S / V, 742K, 743H / V, 745S / T / V, 747S, 748G, 749M, 753A, 758G, 760T / W / Y, 762A, 768S, 769Q, 770E, 787M / N, 792F / Y, 798I, 804R, 807M, 808W / Y, 809K, 812R / S, 815L / R / S / T, 820E / S / T, 824M, 825E, or 831G, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO:

2.

7. The engineered DNA polymerase of any one of claims 1-5, wherein the amino acid sequence of the engineered DNA polymerase comprises at least a mutation at amino acid position 478, 515, 526, 527 or 528, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO:

2.

8. The engineered DNA polymerase of any one of claims 1-5 and 7, wherein the amino acid sequence of the engineered DNA polymerase comprises at least the mutations 478L, 515T, 526S, 527A or 528A or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO:

2.

9. The engineered DNA polymerase of any one of claims 1-5, wherein the amino acid sequence of the engineered DNA polymerase comprises at least a mutation at amino acid position 147, 257, 291, 395, 402, 474, 475, 478, 514, 515, 526, 527, 528, 536, 559, 604, 660, 662, 687, 745, or 760, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO:

2.

10. The engineered DNA polymerase of any one of claims 1-5 and 9, wherein the amino acid sequence of the engineered DNA polymerase comprises at least mutations 147H, 257V, 291L, 395H / S, 402R, 474A, 475A, 478L, 514I, 515T, 526S, 527A, 528A, 536V, 559R, 604H, 660V, 662S, 687A, 745T or 760W, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO:

2.

11. The engineered DNA polymerase according to any one of claims 2 to 5, wherein the amino acid sequence of the engineered DNA polymerase comprises at least a mutation or set of mutations at the following amino acid positions: 541 / 554 / 557, 516 / 541 / 550 / 554 / 557, 478 / 514 / 526 / 527, 478 / 516 / 526 / 527 / 529 / 541, 288 / 478 / 514 / 516 / 541 / 557, 288 / 478 / 516 / 541 / 550 / 554 / 557, 550 / 554 / 557, 478 / 514 / 516, 399 / 516 / 526 / 528 / 529 / 554 / 557、478 / 516 / 541、478 / 541、288 / 399 / 514 / 554 / 557、478 / 514 / 554 / 557、288 / 478 / 526 / 550 / 554、478 / 541 / 550 / 557、541、288 / 514 / 526 / 541、55 4 / 557, 399 / 478, 288 / 514 / 554 / 557, 478 / 514 / 516 / 526 / 527 / 529 / 557, 399 / 541 / 550 / 554, 516 / 529 / 541 / 554 / 557, 478 / 541 / 554 / 557, 288 / 478 / 526 / 5 28 / 529 / 554 / 557, 288 / 514 / 516 / 529 / 541, 478 / 514 / 516 / 541, 478 / 514 / 557, 399 / 541 / 557, 399 / 541, 288 / 399 / 516 / 527 / 541 / 557, 288 / 478 / 514 / 541 、288 / 514 / 516、664 / 698、399 / 478 / 516 / 541、399 / 528 / 550、399 / 478 / 514 / 541 / 557、288 / 478 / 514 / 516、224 / 664、478 / 526 / 527 / 528、288 / 478 / 514 / 51 6 / 541 / 550 / 554、478 / 514 / 541 / 554、478 / 516、478 / 514 / 516 / 541 / 554 / 557、288 / 399 / 478 / 516 / 541、288 / 478 / 514 / 554 / 557、288 / 478 / 516、288 / 399 / 5 16 / 554 / 557, 550 / 554, 288 / 550 / 554, 288 / 399 / 478 / 516 / 550 / 554, 399 / 514 / 541 / 550 / 554, 288 / 478 / 514 / 554, 199, 288 / 399 / 529 / 541, 399 / 478 / 554,516 / 528 / 541 / 554 / 557、39 / 199 / 698、399 / 478 / 526 / 528 / 529、288 / 399 / 514 / 541 / 550 / 554、288 / 541、288 / 478 / 514 / 550 / 554 / 557、288 / 399 / 478 / 516、526 / 529 / 554 / 557、288 / 478 / 557、288 / 478 / 514 / 550 / 554、478 / 541 / 550 / 554、516 / 526 / 528、 288 / 526 / 528 / 550 / 554 / 557, 199 / 664 / 698, 514 / 526 / 528 / 529 / 550 / 557, 288, 399 / 554 / 557, 288 / 554 / 557, 399 / 514 / 541, 288 / 478 / 514 / 527 / 554 / 557, 399 / 478 / 516 / 541 / 554, 288 / 550 / 554 / 557, 527 / 529 / 541, 528 / 529 / 554, 478 / 5 54 / 557、478 / 514 / 516 / 541 / 550 / 554 / 557、199 / 216 / 644 / 664、396 / 478 / 514 / 554 / 557 / 694、241 / 644 / 663 / 664、399 / 478 / 554 / 557、288 / 399 / 550、288 / 516 / 526 / 527 / 528 / 557、399 / 526 / 527 / 528 / 529、288 / 478 / 526、399 / 478 / 514 / 516 / 526 / 527 / or 478 / 514 / 526 / 527 / 528, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO:

2.

12. The engineered DNA polymerase of claim 1 , comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, 226, 272, 328, 546, 710 or 1208, or a reference sequence corresponding to SEQ ID NO: 220, 226, 272, 328, 546, 710 or 1208, wherein the amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, 226, 272, 328, 546, 710 or 1208, or a reference sequence corresponding to SEQ ID NO: One or more mutations of the reference sequence of NO: 220, 226, 272, 328, 546, 710 or 1208.

13. The engineered DNA polymerase of claim 1 , comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 4-1378, or a reference sequence corresponding to an even numbered SEQ ID NO: 2-1378, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, 226, 272, 328, 546, 710, or relative to the reference sequence corresponding to SEQ ID NO: 220, 226, 272, 328, 546, 710.

14. The engineered DNA polymerase of claim 12 or 13, wherein the amino acid sequence of the engineered DNA polymerase comprises mutations at at least the following amino acid positions: 2, 3, 21, 39, 51, 59, 66, 68, 72, 73, 78, 79, 87, 124, 147, 178, 181, 185, 199, 216, 224, 233, 239, 241, 251, 257, 262, 271, 275, 288, 291, 303, 316, 317, 327, 328, 336, 338, 341, 342, 351, 356, 359, 36 2、365、368、370、371、372、373、374、376、377、378、379、380、381、382、388、390、392、393、395、396、398、399、402、403、404、409、410、412、413、416、417、418、420、425、426、427、429、430、431、432、434、435、437、438、442、443、447、448、449、450、451、453、454、457、461、465、 466, 469, 473, 474, 475, 477, 478, 479, 482, 483, 488, 490, 503, 508, 514, 515, 516, 526, 527, 528, 529, 536, 541, 542, 544, 545, 550, 554, 557, 558, 559, 562, 569, 575, 577, 578, 579, 589, 594, 601, 603, 604, 612, 613, 641, 644, 651, 656, 660, 662, 663, 664, 667, 671, 677, 685, 686 7, 691, 694, 697, 698, 699, 701, 702, 704, 706, 707, 708, 713, 714, 715, 716, 718, 721, 723, 724, 725, 728, 731, 732, 735, 740, 742, 743, 745, 747, 748, 749, 753, 758, 760, 762, 768, 769, 770, 787, 792, 798, 804, 807, 808, 809, 812, 815, 820, 824, 825 or 831 or a combination thereof, wherein the position is relative to the position corresponding to SEQ Reference sequence of ID NO: 220, 226, 272, 328, 546, 710 or 1208.

15. The engineered DNA polymerase of any one of claims 12-14, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one mutation or amino acid residue 2-, 3N, 21A / G / N, 39F, 51T, 59K, 66R, 68E / G / I / K / Q / R / T, 72A, 73I, 78N / S / T / V, 79A / M / R / T, 87V, 124A, 147A / H / M / Q, 178L, 181Q / R, 185T, 199P, 216K, 224L, 233H / K / L, 239V, 241E, 251A / I / L, 257V, 262N, 271G, 275A / T, 288R, 29 1L, 303T, 316G, 317R, 327E / L, 328L / V, 336E, 338V, 341H / T / Y, 342G, 351H, 3 56I / S, 359G / V, 362M / P / S, 365T, 368H, 370G / S / V, 371L, 372V / Y, 373G / T, 37 4S / V / Y, 376F / S, 377C, 378C / F / L / S, 379G, 380I, 381E / K, 382C / S, 388C / M / P / V, 390S / T, 392A / C / S / T / V, 393G / R / S / W, 395G / H / S, 396F / L / P / R / S, 398G / Q, 399G / T, 402R / Y, 403I, 404T, 409S, 410V / W, 412M / Q / R / V, 413T, 416S, 417R / W, 418K, 420V, 425T, 426T, 427D / F / H / I / L / R / T / Y, 429F, 430G, 431G / L / R / V, 432T, 434N, 435K / P, 437N / P / S, 438F / S, 442G, 443A, 447R / S / W, 448C / I / T / V , 449A / G / L / V, 450V, 451L, 453L / S, 454D / F / G / L / W, 457N / R / W / Y, 461L / Q / S, 4 65Y, 466Y, 469T / V, 473N, 474A / C / G, 475A / S / V / Y, 477V, 478I / L / M, 479L, 48 2S, 483D, 488W, 490W, 503R, 508K, 514I / V, 515H / T, 516K, 526S, 527A, 528A / G / S, 529S / V, 536V, 541D / G / N / S, 542R, 544M, 545L, 550C / T, 554G / N, 557I, 55 8G / L / T, 559P / R, 562K, 569M / Q, 575W, 577S, 578A, 579L / Q, 589G, 594A, 601T,603M / R, 604H, 612D, 613H / R, 641A, 644Q, 651Q, 656Q / S, 660V, 662S, 663E, 664A, 667S / T / W, 671W, 677L, 685R, 687A, 691S / W, 694D, 6 97A / I / L / V, 698E, 699V, 701M, 702S / V, 704L / V, 706I / S / V, 707W, 708L, 713G, 714I, 715H / L, 716E / L, 718F / Q, 721A / M / N, 723Y, 724F / 824M, 825E, or 831G, or a combination thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 220, 226, 272, 328, 546, 710, or 1208.

16. The engineered DNA polymerase of claim 12, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, or to a reference sequence corresponding to SEQ ID NO: 220, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, or to a reference sequence corresponding to SEQ ID NO:

220.

17. The engineered DNA polymerase of claim 13, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 222-268, or a reference sequence corresponding to an even numbered SEQ ID NO: 222-268, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 220, or relative to the reference sequence corresponding to SEQ ID NO:

220.

18. The engineered DNA polymerase of claim 16 or 17, wherein the amino acid sequence of the engineered DNA polymerase comprises at least a mutation or set of mutations at amino acid position 515, 594, 559, 760, 541, 753, 402, 398, 758, 399, 575, 257 / 758, 558, 601, 762, 589, or 544, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO:

220.

19. The engineered DNA polymerase of claim 12, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 226, or to a reference sequence corresponding to SEQ ID NO: 226, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 226, or to a reference sequence corresponding to SEQ ID NO:

226.

20. The engineered DNA polymerase of claim 13, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 270-320, or a reference sequence corresponding to SEQ ID NO: 270-320, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 226, or relative to the reference sequence corresponding to SEQ ID NO:

226.

21. The engineered DNA polymerase of claim 19 or 20, wherein the amino acid sequence of the engineered DNA polymerase comprises a mutation or set of mutations at the following amino acid positions: 515 / 760, 514 / 515 / 760, 478 / 515 / 760, 660, 178, 402 / 515, 377 / 515, 515, 514 / 515, 402, 503, 239, 73, 528 / 541, 59 , 515 / 575, 3 / 515 / 559, 377 / 515 / 528 / 541 / 554 / 557, 402 / 514 / 515 / 541 / 559, 514 / 515 / 541 / 559, 514 / 515 / 559, 515 / 559, 515 / 541 / 559, 377 / 515 / 559, 402 / 515 / 559 or 515 / 541 / 557, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO:

226.

22. The engineered DNA polymerase of claim 12, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 272, or a reference sequence corresponding to SEQ ID NO: 272, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 272, or a reference sequence corresponding to SEQ ID NO:

272.

23. The engineered DNA polymerase of claim 13, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 322-534, or a reference sequence corresponding to an even numbered SEQ ID NO: 322-534, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 272, or relative to the reference sequence corresponding to SEQ ID NO:

272.

24. The engineered DNA polymerase of claim 22 or 23, wherein the amino acid sequence of the engineered DNA polymerase comprises at least a mutation or set of mutations at the following amino acid positions: 402 / 528 / 541 / 660, 257 / 604 / 660, 178 / 257 / 402 / 404 / 528 / 557 / 660, 402 / 604 / 660, 257 / 402 / 541 / 660, 377 / 402 / 541 / 660, 257 / 377 / 402 / 660, 483 / 660, 257 / 402 / 528 / 541 / 660, 257 / 377 / 402 / 660, 178 / 257 / 402 / 404 / 528 / 557 / 660, 402 / 604 / 660 / 402, 660, 178 / 257 / 377 / 402 / 403 / 483 / 604 / 660, 377, 178 / 402 / 403 / 528 / 541 / 660, 377 / 601 / 660, 178 / 377, 377 / 490 / 660, 402 / 403 / 483 / 554 / 557, 178 / 257 / 377 / 558 / 660, 257 / 402 / 403 / 483 / 660, 178 / 554 / 557 / 558 / 660, 554 / 557 / 660, 178 / 483 / 541 / 545 / 554 / 557 / 604 / 660, 792, 40 2 / 403 / 483 / 604 / 660, 178, 474, 257 / 377 / 541 / 604, 743, 396, 479, 377 / 403 / 541 / 604, 475, 178 / 257 / 403 / 528, 448, 577, 395, 769, 691, 677, 704, 377 / 541 / 575 / 604 / 660, 178 / 257 / 601 / 604, 685, 377 / 604 / 660, 749, 482, 291, 579, 178 / 541 / 550 / 604, 477, 178 / 257, 257 / 402 / 490 / 528 / 5 or 402 / 528 / 541 / 601, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO:

272.

25. The engineered DNA polymerase of claim 12, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 328, or a reference sequence corresponding to SEQ ID NO: 328, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 328, or a reference sequence corresponding to SEQ ID NO:

328.

26. The engineered DNA polymerase of claim 13, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 536-674, or a reference sequence corresponding to an even numbered SEQ ID NO: 536-674, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 328, or relative to the reference sequence corresponding to SEQ ID NO:

328.

27. The engineered DNA polymerase of claim 25 or 26, wherein the amino acid sequence of the engineered DNA polymerase comprises at least a mutation or set of mutations at the following amino acid positions: 79, 257 / 474, 677, 562, 178 / 395 / 745, 291 / 395 / 687 / 745, 257 / 395 / 677, 303 / 338, 474 / 475, 359, 474 / 475 / 677 / 704, 395 / 687, 671, 233, 699, 379, 257 / 792, 257 / 395, 21, 257 / 448 / 474 / 475, 713, 641, 442, 656, 475 / 704, 742, 667, 257 / 448, 257 / 316 / 395 / 482 / 68 7 / 745, 804, 704, 178 / 257 / 395 / 482 / 579 / 687 / 745, 257 / 579, 257 / 291 / 395 / 475 / 541 / 687, 257 / 768, 812, 475, 662, 536, 748, 808, 291 / 316 / 395 / 687, 613, 257 / 291 / 316, 257 / : 291 / 579, 257 / 316 / 541 / 745, 351, 257 / 316 / 395 / 745, 185, 257 / 316 / 687, 291 / 316 / 475 / 687 / 745, 257 / 291 / 395 / 740 / 745, 450, 473, 124, 430, 413, 66 or 365, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO:

328.

28. The engineered DNA polymerase of claim 12, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 546, or a reference sequence corresponding to SEQ ID NO: 546, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 546, or a reference sequence corresponding to SEQ ID NO:

546.

29. The engineered DNA polymerase of claim 13, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 676-1190, or a reference sequence corresponding to an even numbered SEQ ID NO: 676-1190, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 546, or relative to the reference sequence corresponding to SEQ ID NO:

546.

30. The engineered DNA polymerase of claim 28 or 29, wherein the amino acid sequence of the engineered DNA polymerase comprises a mutation or set of mutations at at least the following amino acid positions: 438, 697, 457, 716, 79 / 474 / 536 / 699, 536, 392, 708, 431, 395 / 474 / 536 / 699, 453, 427, 79 / 395 / 699, 378, 370, 706, 257 / 395 / 474 / 475 / 536 / 662, 474 / 536 / 562, 257 / 395 / 474 / 536 / 562, 79 / 257 / 395 / 474 / 475 / 536 / 562, 395 / 536 / 699, 372, 257 / 395 / 792, 390, 79 / 474 / 536 / 699 / 792, 409, 443, 448, 381, 707, 416, 474 / 475 / 662, 79 / 395 / 474 / 475 / 536 / 792, 474 / 475 / 536 / 66 2, 382, 257 / 474 / 536 / 562 / 662, 257 / 395 / 474 / 475 / 792, 79 / 474 / 662 / 699, 725, 447, 79 / 257 / 699, 701, 257 / 474 / 475 / 536 / 562 / 662, 702, 342 / 716, 233 / 5 62 / 713、704、474 / 475 / 536 / 562 / 662、454、327 / 427、257 / 316 / 474 / 536 / 792、417、425、536 / 562、714、233、303 / 613、21 / 79 / 303 / 562 / 613、79 / 562、435、 257 / 536 / 699 / 792、79 / 536、453 / 667、79 / 303 / 338 / 613 / 804 / 812、79 / 257 / 395 / 536 / 662 / 792、79 / 338 / 562、79 / 233 / 303 / 662、79 / 662 / 699、79 / 395 / 475 / 662、79 / 536 / 562 / 699、536 / 662、21 / 79 / 338 / 641 / 699、662、21 / 79 / 442 / 699、79 / 303 / 662、79 / 178 / 257 / 662 / 699、79 / 257 / 536 / 562 / 662、562 / 699、359 / 613 / 742 / 808 / 812、21 / 79 / 338 / 379 / 562 / 662 / 804 / 812、731、21 / 79 / 359 / 442 / 662、79 / 338 / 359 / 562、21 / 613 / 662 / 742、21 / 79 / 303 / 662 / 699 / 742 / 808、21 / 79 / 338 / 442 / 613 / 699、79 / 303 / 338 / 562 / 613 / 662、303 / 338 / 562 / 699 / 808 / 812、79 / 257、257 / 662 / 792、79 / 536 / 699、79 / 359 / 713 / 742 / 808、21 / 359 / 442 / 808 / 812、79 / 257 / 475 / 536 / 662、21 / 79 / 338、79 / 713、79 / 562 / 641、373、79 / 178 / 257 / 316 / 536 / 662、21 / 233 / 338 / 562 / 662、257 / 316 / 536、21 / 79 / 303 / 442 / 662、21 / 79 / 233 / 303 / 338 / 379 / 562 / 713、338 / 442 / 699、426、21 / 338 / 359 / 808 / 812、79 / 178 / 233 / 442 / 562 / 812、79 / 474 / 536 / 662 / 699、21 / 79 / 338 / 379 / 662、721、21 / 79 / 233 / 338 / 613 / 641、374、338 / 359 / 379、461、451、715、79 / 303 / 338 / 641 / 713、393、79 / 257 / 474 / 536 / 699、79、437、257 / 536 / 562 / 662、21 / 79 / 379 / 442 / 562 / 662、362、410、338 / 812、21 / 662 / 808 / 812、21 / 303 / 338、418、338 / 442、449、379 / 641、316 / 536 / 562、257 / 536 / 792、21 / 79 / 442 / 613、257 / 536 / 662 / 792、257 / 474 / 529 / 536 / 662、412、79 / 257 / 536 / 662 / 699、257 / 316 / 475 / 536 / 792、388、380 / 454、21 / 79 / 233 / 303 / 613 / 699 / 809、724、21 / 79 / 338 / 613、79 / 178 / 536 / 792、449 / 747、21 / 79 / 379 / 613 / 713、79 / 316 / 536 / 662 / 699、376、21 / 562 / 662、79 / 257 / 262 / 536 / 562 / 699、718、395 / 475 / 536 / 562 / 662 / 699、79 / 233 / 338 / 359 / 379 / 442 / 562 / 613 / 812、371、316 / 475 / 562 / 662 / 792、79 / 257 / 316 / 792、728、79 / 233 / 338、432、79 / 178 / 257 / 316 / 395 / 536 / 662 / 699, 79 / 303 / 713, 233 / 379 / 442 / 562 / 613 / 662 / 713, 21 / 742, 732, 79 / 257 / 395 / 562 / 662 / 699, 21 / 233 / 338 / 379 / 442 / 562 / 613 / 662 / 812, 429, 562 / 699, 2 / 374, 723, 434, 420 or 699, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO:

546.

31. The engineered DNA polymerase of claim 12, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 710, or a reference sequence corresponding to SEQ ID NO: 710, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 710, or a reference sequence corresponding to SEQ ID NO:

710.

32. The engineered DNA polymerase of claim 13, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 1192-1326, or a reference sequence corresponding to an even numbered SEQ ID NO: 1192-1326, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO:

710.

33. The engineered DNA polymerase of claim 31 or 32, wherein the amino acid sequence of the engineered DNA polymerase comprises a mutation or set of mutations at at least amino acid position(s): 68, 78, 466, 807, 612, 147, 356, 488, 603, 271, 508, 787, 412 / 417 / 427, 251, 697, 562, 79 / 434 / 562, 820, 275, 378 / 381 / 697, 469, 412 / 417 / 697, 798, 79 / 388, 651, 825, 51, 370, 745, 815, 465, 317, 569, 341, 824, 18, 181, 735, or 336, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO:

710.

34. The engineered DNA polymerase of claim 12, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 1208, or a reference sequence corresponding to SEQ ID NO: 1208, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 1208, or a reference sequence corresponding to SEQ ID NO: 1208.

35. The engineered DNA polymerase of claim 13, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 1328-1378, or a reference sequence corresponding to an even numbered SEQ ID NO: 1328-1378, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 1208, or relative to the reference sequence corresponding to SEQ ID NO: 1208.

36. The engineered DNA polymerase of claim 34 or 35, wherein the amino acid sequence of the engineered DNA polymerase comprises at least a mutation or set of mutations at the following amino acid positions: 603 / 612 / 798, 68 / 603, 68 / 78 / 370, 370, 251 / 798, 68 / 370 / 603 / 612, 51 / 370 / 412 / 603 / 612, 412 / 603 / 798, 251 / 370 / 412 / 612, 370 / 798, 78 / 251, 251, 68 / 370 / 508 / 798, 251 / 370 / 603 / 612, or 51 / 68 / 251 / 370 / 603, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 1208.

37. The engineered DNA polymerase of claim 1, wherein the amino acid sequence comprises at least one mutation or set of mutations provided in Tables 4.1, 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, 8.2, 9.1, 9.2, 9.3, 10.1, 10.2, 11.1, and 11.2, wherein the mutation or set of mutations is relative to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 220, 226, 272, 328, 546, 710, or 1208, or a reference sequence corresponding to SEQ ID NO: 2, 220, 226, 272, 328, 546, 710, or 1208.

38. The engineered DNA polymerase of claim 1 , comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO: 4-1378, or a sequence corresponding to an even numbered SEQ ID NO: 2-1378.

39. The engineered DNA polymerase of claim 1, wherein the DNA polymerase comprises an amino acid sequence comprising residues 12 to 844 of an even-numbered SEQ ID NO among SEQ ID NOs: 4-1378, or an amino acid sequence comprising an even-numbered SEQ ID NO among SEQ ID NOs: 2-1378, wherein the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions in the amino acid sequence.

40. The engineered DNA polymerase of claim 39, wherein the amino acid sequence has 1, 2, 3, 4, or up to 5 substitutions.

41. The engineered DNA polymerase of claim 39 or 40, wherein the substitution comprises a conservative substitution.

42. The engineered DNA polymerase of claim 1, wherein the engineered DNA polymerase comprises an amino acid sequence comprising residues 12 to 844 of SEQ ID NO: 220, 226, 272, 328, 546, 710, or 1208, or comprises SEQ ID NO: 2, 220, 226, 272, 328, 546, 710, or 1208.

43. The engineered DNA polymerase of any one of claims 1-42, wherein the engineered DNA polymerase further comprises a fusion polypeptide.

44. The engineered DNA polymerase of any one of claims 1-43, wherein the engineered DNA polymerase has DNA polymerase activity and at least one improved property compared to a reference DNA polymerase having a sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 220, 226, 272, 328, 546, 710, or 1208, or a sequence corresponding to SEQ ID NO: 2, 220, 226, 272, 328, 546, 710, or 1208.

45. The engineered DNA polymerase of claim 44, wherein the improved property is selected from i) increased activity, ii) increased stability, iii) increased thermostability, iv) increased processivity, v) increased fidelity, vi) increased input DNA template sensitivity, vii) increased product yield, and viii) increased resistance or tolerance to inhibitors, or any combination of i), ii), iii), vi), v), vi), vii), and viii).

46. The engineered DNA polymerase of claim 44 or 45, wherein the reference DNA polymerase has a sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or a sequence corresponding to SEQ ID NO:

2.

47. The engineered DNA polymerase of any one of claims 1-46, wherein the engineered DNA polymerase is purified.

48. A recombinant polynucleotide comprising a polynucleotide sequence encoding the engineered DNA polymerase according to any one of claims 1-46.

49. The recombinant polynucleotide of claim 48, comprising a polynucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 2532 of an odd-numbered SEQ ID NO: 1-1377, or a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO: 1-1377, wherein the recombinant polynucleotide encodes a DNA polymerase.

50. The recombinant polynucleotide of claim 48 or 49, comprising a polynucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 2532 of SEQ ID NO: 1, 219, 225, 271, 327, 545, 709 or 1207, or a reference polynucleotide sequence corresponding to SEQ ID NO: 1, 219, 225, 271, 327, 545, 709 or 1207, wherein the recombinant polynucleotide encodes a DNA polymerase.

51. The recombinant polynucleotide sequence of any one of claims 48-50, wherein the polynucleotide sequence is codon optimized.

52. The recombinant polynucleotide sequence of claim 48, wherein the polynucleotide sequence comprises nucleotide residues 34 to 2532 of SEQ ID NO: 1, 219, 225, 271, 327, 545, 709, or 1207, or comprises SEQ ID NO: 1, 219, 225, 271, 327, 545, 709, or 1207.

53. The recombinant polynucleotide sequence of claim 48, wherein the polynucleotide sequence comprises residues 34 to 2532 of an odd-numbered SEQ ID NO among SEQ ID NOs: 1-1377, or comprises an odd-numbered SEQ ID NO. among SEQ ID NOs: 1-1377.

54. An expression vector comprising at least one recombinant polynucleotide according to any one of claims 48-53.

55. The expression vector of claim 54, wherein the recombinant polynucleotide is operably linked to a control sequence.

56. The expression vector of claim 55, wherein the control sequence comprises a promoter.

57. A host cell comprising the expression vector according to any one of claims 54-56.

58. The host cell of claim 57, which comprises a bacterial cell, a fungal cell, or a mammalian cell.

59. A method for producing an engineered DNA polymerase polypeptide in a host cell, comprising culturing the host cell of claim 57 or 58 under suitable culture conditions such that at least one engineered DNA polymerase is produced.

60. The method of claim 59, further comprising recovering the at least one engineered DNA polymerase from the culture and / or host cell.

61. The method of claim 59 or 60, further comprising the step of purifying the at least one engineered DNA polymerase.

62. A composition comprising a DNA polymerase having a sequence comprising residues 12 to 844 of SEQ ID NO: 2, or a sequence comprising SEQ ID NO: 2, or at least one engineered DNA polymerase according to any one of claims 1-47.

63. The composition of claim 62, comprising at least a buffer.

64. The composition of claim 62 or 63, further comprising one or more nucleotide substrates.

65. The composition of any one of claims 62-64, further comprising one or more oligonucleotide primer substrates.

66. The composition of any one of claims 62-65, further comprising a heterologous template DNA substrate.

67. A method for preparing a complementary DNA to a target DNA, comprising contacting the target DNA with a DNA polymerase comprising an amino acid sequence comprising residues 12 to 844 of SEQ ID NO: 2, or an amino acid sequence comprising SEQ ID NO: 2, or an engineered DNA polymerase according to any one of claims 1 to 47, in the presence of a substrate, under conditions suitable for producing a complementary DNA to all or a portion of the target DNA.

68. A method for detecting the presence of a target DNA, comprising contacting a sample suspected of containing the target DNA with a DNA polymerase comprising an amino acid sequence comprising residues 12 to 844 of SEQ ID NO: 2, or an amino acid sequence comprising SEQ ID NO: 2, or an engineered DNA polymerase according to any one of claims 1 to 47, in the presence of a substrate, under conditions suitable for DNA polymerase-mediated production of a DNA complementary to all or a portion of the target DNA, and detecting the presence of the complementary DNA.

69. The method of claim 68, wherein the complementary DNA is detected by amplifying the complementary DNA.

70. The method of claim 69, wherein the amplification is by polymerase chain reaction (PCR) or isothermal amplification.

71. The method of claim 70, wherein the isothermal amplification is by loop-mediated isothermal amplification (LAMP).

72. A method for amplifying a target DNA, comprising contacting the target DNA with a DNA polymerase comprising an amino acid sequence comprising residues 12 to 844 of SEQ ID NO: 2, or an amino acid sequence comprising SEQ ID NO: 2, or the engineered DNA polymerase according to any one of claims 1 to 47, in the presence of a substrate under conditions suitable for amplifying the target DNA.

73. The method of claim 72, wherein the conditions are for a polymerase chain reaction.

74. The method of claim 72, wherein the conditions are for LAMP.

75. A method for sequencing a target DNA, comprising contacting the target DNA with a DNA polymerase comprising an amino acid sequence comprising residues 12 to 844 of SEQ ID NO: 2, or an amino acid sequence comprising SEQ ID NO: 2, or an engineered DNA polymerase according to any one of claims 1 to 47, in the presence of a substrate suitable for sequencing, under conditions suitable for DNA polymerase-mediated extension of a complementary DNA of the target DNA, and determining the sequence of the target DNA.

76. A kit comprising at least a DNA polymerase comprising an amino acid sequence comprising residues 12 to 844 of SEQ ID NO: 2, or comprising an amino acid sequence of SEQ ID NO: 2, or at least one engineered DNA polymerase according to any one of claims 1-47.

77. The kit of claim 76, further comprising one or more of a buffer, a nucleotide substrate, and an oligonucleotide primer substrate.

78. The kit of claim 76 or 77, further comprising template DNA.

79. The kit of claim 78, wherein the template DNA is a control template DNA.

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