DNA polymerase variants

Abstract

The present disclosure relates to engineered DNA polymerase polypeptides and compositions thereof, as well as 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

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 383,053, filed November 9, 2022, which is incorporated herein by reference.

[0002] Reference to a sequence listing, table or computer program The Sequence Listing submitted concurrently herewith under file name CX9-227WO1_ST26.xml, created on November 8, 2023, with a file size of 4,056,133 bytes, is incorporated herein by reference.

[0003] The present disclosure provides engineered DNA polymerase polypeptides and compositions thereof, as well as polynucleotides encoding the engineered DNA polymerase polypeptides. The present disclosure also provides methods for the use of recombinant DNA polymerases or compositions thereof for diagnostics, molecular biology tools, and other purposes. [Background technology]

[0004] DNA polymerases are enzymes that synthesize DNA from deoxyribonucleotides. These enzymes are essential for DNA replication. There are various types of DNA polymerases that exhibit different properties and are found in different types of organisms. Polymerases obtained from thermophilic organisms have found widespread use in various in vitro methods, including, but not limited to, polymerase chain reaction (PCR), nucleic acid sequencing, and other diagnostic, molecular biology, and forensic applications. Although there are many commercially available thermostable DNA polymerases, such as Taq and Pfu DNA polymerases, there remains a need in the art for thermostable enzymes with improved properties, such as enhanced sensitivity, processivity, and / or fidelity. Summary of the Invention [Problem to be solved by the invention]

[0005] (Abstract) The present disclosure provides engineered DNA polymerase polypeptides and compositions thereof, as well as polynucleotides encoding the 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. [Means for solving the problem]

[0006] In one aspect, the disclosure provides a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 1088%, 109%, 1109, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1211, 1220, 1226, 1272, 1280, 1250, 1260, 1272, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, 1490, 1500, 1510, 1520, 1530, 1540, 1550, 1560, 1570, 1580, 1590, 1600, 1610, 1620, 1630, 1640, 1650, 1660, 1670, 1680, 1690, 1700, 17 Provided are engineered DNA polymerases or functional fragments thereof, comprising an amino acid sequence with 99% or greater sequence identity, 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, 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.

[0007] 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 12-844 of SEQ ID NO:2 or a reference sequence corresponding to SEQ ID NO:2, wherein the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 of SEQ ID NO:2 or a reference sequence corresponding to SEQ ID NO:2.

[0008] 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 12-844 of SEQ ID NO:2 or a reference sequence corresponding to SEQ ID NO:2, wherein the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 of SEQ ID NO:2 or a reference sequence corresponding to SEQ ID NO:2.

[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 12-844 of SEQ ID NO: 4-1378 or a reference sequence corresponding to an even-numbered SEQ ID NO: 4-1378, and the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 of SEQ ID NO: 2 or a reference sequence corresponding to SEQ ID NO: 2.

[0010] In some embodiments, the amino acid sequence of the engineered DNA polymerase is at 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, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 430, 431, 432, 433, 434, 6, 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, 70 704, 707, 808, 809, 812, 815, 820, 824, 825, or 831, or a combination thereof, wherein the positions are relative to the reference sequence corresponding to SEQ ID NO:2.

[0011] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one mutation at amino acid position 478, 515, 526, 527, or 528, or a combination thereof, wherein the positions are relative to the reference sequence corresponding to SEQ ID NO:2.

[0012] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one 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.

[0013] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one 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.

[0014] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one mutation at amino acid 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 the reference sequence corresponding to SEQ ID NO:2.

[0015] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises amino acid position(s) 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 / 516 / 541, 478 / 541, 2 88 / 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, 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 / 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 / 51 6 / 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, 4 78 / 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 / 6 98, 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 / 541, 288 / 399 / 478 / 514 / 554 / 557, At least one mutation or set of mutations in 288 / 399 / 514 / 516 / 528, 478 / 554, 399 / 514 / 516, 514, 399 / 550 / 554, 288 / 478 / 514 / 528 / 541 / 554, 478 / 514 / 516 / 554, 478 / 514 / 541, 288 / 541 / 554 / 557, 698, or 478 / 514 / 526 / 527 / 528, where the positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

[0016] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one mutation or set of mutations at amino acid position(s) of 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, where the positions are relative to the reference sequence of SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase 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, where the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

[0017] 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 12-844 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 a reference sequence corresponding to residues 12-844 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.

[0018] 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 12-844 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 a reference sequence corresponding to residues 12-844 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.

[0019] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises 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, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 441, 442, 442, 443, 444, 445, 4 78, 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, 49 0, 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: 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 12-844 of SEQ ID NO:220 or a reference sequence corresponding to SEQ ID NO:220, wherein the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 of SEQ ID NO:220 or a reference sequence corresponding to SEQ ID NO:220.

[0021] 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 12-844 of SEQ ID NO:220 or a reference sequence corresponding to an even-numbered SEQ ID NO:220, wherein the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 of SEQ ID NO:220.

[0022] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one mutation or set of mutations at amino acid position(s) 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.

[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 12-844 of SEQ ID NO:226 or a reference sequence corresponding to SEQ ID NO:226, wherein the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 of SEQ ID NO:226 or a reference sequence corresponding to SEQ ID NO:226.

[0024] 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 12-844 of SEQ ID NO:226 or a reference sequence corresponding to SEQ ID NO:226, wherein the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 of SEQ ID NO:226 or a reference sequence corresponding to SEQ ID NO:226.

[0025] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises amino acid position(s) 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 / 5 at least one mutation or set of mutations at 41 / 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, where the positions are relative 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 12-844 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 a reference sequence corresponding to residues 12-844 of SEQ ID NO:272 or a reference sequence corresponding to SEQ ID NO:272.

[0027] 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 12-844 of SEQ ID NO: 322-534 or a reference sequence corresponding to even-numbered SEQ ID NO: 322-534, and the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 of SEQ ID NO: 272 or a reference sequence corresponding to SEQ ID NO: 272.

[0028] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises amino acid position(s) 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, 257 / 377, 178 / 660, 257 / 377 / 402, 660, 178 / 257 / 377 / 402 / 403 / 4 83 / 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 / 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 / 541 / 660, 328, 740, 368, 721 / 745, 578, 687, 5 and at least one mutation or set of mutations in 41 / 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, where the positions are relative 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 12-844 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 a reference sequence corresponding to residues 12-844 of SEQ ID NO: 328 or a reference sequence corresponding to SEQ ID NO: 328.

[0030] 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 12-844 of 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 a reference sequence corresponding to residues 12-844 of SEQ ID NO: 328 or a reference sequence corresponding to SEQ ID NO: 328.

[0031] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises amino acid position(s) 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 / 68 7, 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, 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 / 3 at least one mutation or set of mutations at 95 / 745, 185, 257 / 316 / 687, 291 / 316 / 475 / 687 / 745, 257 / 291 / 395 / 740 / 745, 450, 473, 124, 430, 413, 66 or 365, where the positions are relative to the 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 12-844 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 a reference sequence corresponding to residues 12-844 of SEQ ID NO: 546 or a reference sequence corresponding to SEQ ID NO: 546.

[0033] 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 12-844 of 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 a reference sequence corresponding to residues 12-844 of SEQ ID NO: 546 or a reference sequence corresponding to SEQ ID NO: 546.

[0034] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises amino acid position(s) 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 / 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 / 56 2 / 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, 4 53 / 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、At least one mutation or set of mutations at 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, where the positions are relative to the 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 12-844 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 a reference sequence corresponding to residues 12-844 of SEQ ID NO: 710 or a reference sequence corresponding to SEQ ID NO: 710.

[0036] 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 12-844 of SEQ ID NO: 1192-1326 or a reference sequence corresponding to even-numbered SEQ ID NO: 1192-1326, wherein the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 of SEQ ID NO: 710 or a reference sequence corresponding to SEQ ID NO: 710.

[0037] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one 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.

[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 12-844 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 a reference sequence corresponding to residues 12-844 of SEQ ID NO: 1208 or a reference sequence corresponding to SEQ ID NO: 1208.

[0039] 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 12-844 of 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 a reference sequence corresponding to residues 12-844 of SEQ ID NO: 1208 or a reference sequence corresponding to SEQ ID NO: 1208.

[0040] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises amino acid position(s) 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, 78 / 508, 78 / 370 / 697 / 798, 5 and at least one mutation or set of mutations in: 1 / 251, 68 / 370 / 508 / 603 / 697, 78 / 251 / 370 / 697 / 798, 78 / 603 / 798 / 831, 370 / 412 / 508 / 612, 72 / 508 / 798, 51 / 251 / 412 / 798, 51 / 251 / 370 / 508 / 603 / 798, 68 / 78 / 251 / 370 / 603 / 612 / 697 or 51 / 68 / 251 / 370 / 603, wherein the positions are relative to the reference sequence corresponding to SEQ ID NO: 1208.

[0041] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one mutation 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, 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 table.

[0042] In some embodiments, the amino acid sequence of the engineered DNA polymerase 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 position(s) of the mutation or set of mutations are relative to the reference sequence of SEQ ID NO: 2, 220, 226, 272, 328, 546, 710 or 1208 as provided in the corresponding table.

[0043] 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 sequence corresponding to residues 12-844 of an even-numbered SEQ ID NO:4-1378 or a sequence corresponding to an even-numbered SEQ ID NO:2-1378.

[0044] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence comprising residues 12-844 of an even-numbered SEQ ID NO: 4-1378 or an amino acid sequence comprising an even-numbered SEQ ID NO: 2-1378.

[0045] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence comprising residues 12-844 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.

[0046] 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 residues 12-844 of SEQ ID NO:2 and / or a reference DNA polymerase having a sequence corresponding to SEQ ID NO:2.

[0047] In some further embodiments, the engineered DNA polymerase is purified, hi some embodiments, the engineered DNA polymerase is provided in solution or immobilized on a substrate such as a solid substrate or membrane or particle.

[0048] In another aspect, the disclosure provides a recombinant polynucleotide comprising a polynucleotide sequence encoding an engineered DNA polymerase disclosed herein. In some embodiments, the recombinant polynucleotide has a sequence identity that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 1088%, 109%, 1109, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1211, 1220, 1226, 1272, 1230, 1231, 1240, 1241, 1250, 1251, 1260, 1261, 1272, 1280, 1251, 1262, 1272, 1281, 1282, 1290, 1300, 1311, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400, 1411, 1420, 1430, 1440, 1450, 1460, 1470, 1480, 1481, 1490, 1500, 1511, 1520, 1530, 1540, 1550, The invention also includes a polynucleotide sequence encoding at least one engineered DNA polymerase comprising an amino acid sequence with 99% or greater sequence identity, wherein the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 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.

[0049] 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 34-2532 of an odd-numbered SEQ ID NO: 1-1377, or a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO: 1-1377, and the recombinant polynucleotide encodes a DNA polymerase.

[0050] In some embodiments, the recombinant polynucleotide comprises 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 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 SEQ ID NO: 1, 219, 225, 271, 327, 545, 709, or 1207, and the recombinant polynucleotide encodes a DNA polymerase.

[0051] 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 a bacterial cell, a fungal cell, or a mammalian cell.

[0052] 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.

[0053] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 2532 of an odd-numbered SEQ ID NO: 1-1377 or a polynucleotide sequence comprising an odd-numbered SEQ ID NO: 1-1377.

[0054] 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.

[0055] In a further aspect, the present disclosure provides an expression vector comprising at least one recombinant polynucleotide provided herein encoding an engineered DNA polymerase. In another aspect, the present disclosure also provides a host cell transformed with at least one expression vector provided herein.

[0056] In another aspect, the disclosure provides methods for producing an engineered DNA polymerase polypeptide, the methods comprising culturing a host cell described herein under suitable culture conditions such that at least one engineered DNA polymerase is produced. In some embodiments, the methods further comprise recovering the engineered DNA polymerase from the culture and / or the host cell. In some embodiments, the methods further comprise purifying the engineered DNA polymerase.

[0057] In another aspect, the present 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.

[0058] In a further aspect, the present disclosure provides for the use of an engineered DNA polymerase in a method for preparing all or part of a complementary DNA copy of a target DNA. In some embodiments, the present disclosure provides a method for preparing all or part of a complementary DNA copy of a target DNA, the method comprising contacting the target DNA with an engineered DNA polymerase described herein in the presence of a suitable substrate under conditions suitable for DNA polymerase-mediated production of DNA complementary to the target DNA.

[0059] In some embodiments, the engineered DNA polymerase is used to detect target DNA, the method comprising contacting a sample suspected of containing target DNA with an engineered DNA polymerase of the present disclosure in the presence of a suitable substrate under conditions suitable for DNA polymerase-mediated production of all or part 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 involves amplifying the complementary DNA, for example, by polymerase chain reaction (PCR), including qPCR (e.g., TaqMan®), hot-start PCR, touchdown PCR, asymmetric PCR, multiplex PCR, long- or long-range PCR, assembly PCR, and inverse PCR, among others, or by isothermal amplification reactions, including LAMP, whole genome amplification (WGA), and multiple displacement amplification, among others. In some embodiments, the engineered DNA polymerase may be used with a reverse transcriptase to detect target RNA, such as by RT-PCR.

[0060] In a further aspect, the present disclosure also provides kits comprising at least one engineered DNA polymerase disclosed herein. In some embodiments, the kits further comprise one or more of a buffer, a nucleotide substrate, and / or an oligonucleotide primer substrate. In some embodiments, the kits comprise a template DNA, particularly a control DNA template. DETAILED DESCRIPTION OF THE INVENTION

[0061] The present disclosure provides engineered DNA polymerase polypeptides and compositions thereof, as well as polynucleotides encoding the engineered DNA polymerase polypeptides. The disclosure also provides methods of using the engineered DNA polymerase polypeptides and compositions thereof for diagnostic and other purposes. In some embodiments, the engineered DNA polymerase polypeptides exhibit enhanced or increased i) thermostability, ii) polymerization activity, iii) high replication fidelity, iv) high processivity, or v) inhibitor tolerance or resistance, or any combination of i), ii), iii), iv), and / or v), particularly under conditions including low DNA input, high-throughput analysis and / or sequencing reaction conditions. In some embodiments, the engineered DNA polymerases are particularly applicable to polymerase chain reaction.

[0062] In some embodiments, the engineered DNA polymerases find use in diagnostic and research applications using minute amounts of DNA or RNA from samples 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.

[0063] Abbreviations and Definitions Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures of cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry, and nucleic acid chemistry described below are those well known and commonly employed in the art. Such techniques are known and described in numerous texts and references well known to those of ordinary skill in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analyses.

[0064] Although any suitable methods and materials similar or equivalent to those described herein find use in the practice of the present invention, exemplary methods and materials are described herein. It is understood that the present invention is not limited to the particular methodology, protocols, and reagents described, as such may vary depending on the context in which they are used by those of skill in the art. Accordingly, the terms defined immediately below are more fully described by reference to this application as a whole.

[0065] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0066] As used herein, the term "comprising" and its cognates are used in their inclusive sense (i.e., equivalent to the term "including" and its corresponding cognates).

[0067] Additionally, where the description of embodiments uses the term "comprising" and its cognates, it should be understood that the embodiments can also be described using the language "consisting essentially of" or "consisting of."

[0068] Moreover, numerical ranges are inclusive of the numbers defining that range. Accordingly, every numerical range disclosed herein is intended to include 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 limitation disclosed herein is also intended to include every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.

[0069] As used herein, the term "about" refers to a tolerance for a particular value. In some instances, "about" refers to a range of within 0.05%, 0.5%, 1.0%, or 2.0% of a given value. In some instances, "about" refers to within 1, 2, 3, or 4 standard deviations of a given value.

[0070] Furthermore, the headings provided herein are not limitations of the various aspects or embodiments of the present invention that can be had by reference to this application as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to this application as a whole.

[0071] "EC" numbers refer to the Enzym 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.

[0072] "ATCC" refers to the American Type Culture Collection, whose biorepository collection includes genes and strains.

[0073] "NCBI" refers to the National Center for Biological Information and the sequence databases it hosts.

[0074] "Protein," "polypeptide," and "peptide" are used interchangeably and refer to a polymer of at least two amino acids covalently joined by amide bonds, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

[0075] "Amino acids" are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes. Abbreviations used for genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine ​​(Cys or C), glutamate (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). Unless specifically preceded by "L" or "D" or otherwise apparent from the context in which the abbreviation is used, when three-letter abbreviations are used, the amino acids may be in either the L- or D-configuration about the α-carbon (Cα). For example, "Ala" designates alanine without specifying the configuration about the α-carbon, whereas "D-Ala" and "L-Ala" designate D-alanine and L-alanine, respectively. When single-letter abbreviations are used, an uppercase letter designates an amino acid in the L-configuration about the α-carbon, and a lowercase letter designates an amino acid in the D-configuration about the α-carbon. For example, "A" designates L-alanine and "a" designates D-alanine. When polypeptide sequences are presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequence is presented in the amino (N) to carboxy (C) orientation, according to common convention.

[0076] "Fusion protein" and "chimeric protein" and "chimera" refer to a hybrid protein created by the joining of two or more polynucleotides that originally encode separate proteins. In some embodiments, fusion proteins are created by recombinant techniques (e.g., molecular biology techniques known in the art).

[0077] "Polymerase" refers to a class of enzymes that polymerize nucleoside triphosphates. In some embodiments, a polymerase uses a template nucleic acid strand to synthesize a complementary nucleic acid strand. The template strand and the synthesized nucleic acid strand can independently be either DNA or RNA. Polymerases known in the art include, but are not limited to, DNA polymerases (e.g., E. coli DNA pol I, T. aquaticus DNA polymerase (Taq)), DNA-dependent RNA polymerases, and reverse transcriptases. As used herein, a polymerase is a polypeptide or protein that contains sufficient amino acids to perform the desired enzymatic function of the polymerase. In some embodiments, a polymerase does not contain all of the amino acids found in the native enzyme, but contains only sufficient amino acids to enable 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 comprises a DNA polymerase in classification EC 2.7.7.7.

[0078] "DNA polymerase activity," "synthetic activity," and "polymerase activity" are used interchangeably herein and refer to the ability of an enzyme to synthesize new DNA strands by incorporation of deoxynucleoside triphosphates or analogs thereof.

[0079] "Polynucleotide" is used herein to refer to a polymer containing at least two nucleotides, where the nucleotides are either deoxyribonucleotides or ribonucleotides, or a mixture of deoxyribonucleotides and ribonucleotides. In some embodiments, the abbreviations used for genetically encoded nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless otherwise specified, the abbreviated nucleoside may be either a ribonucleoside or a 2'-deoxyribonucleoside. Nucleosides can be identified as either ribonucleosides or 2'-deoxyribonucleosides on an individual or collective basis. When a nucleic acid sequence is presented as a series of one-letter abbreviations, the sequence is presented in the 5' to 3' direction, according to common convention, and phosphates are not indicated. The term "DNA" refers to deoxyribonucleic acid. The term "RNA" refers to ribonucleic acid.

[0080] "Duplex" and "ds" refer to a double-stranded nucleic acid (e.g., DNA or RNA) molecule composed of two single-stranded polynucleotides whose sequences are complementary (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]).

[0081] "Engineered," "recombinant," "non-naturally occurring," and "variant," when used with respect to a cell, polynucleotide, or polypeptide, refer to a material or material that corresponds to a natural or native form of the material that has been modified in a way that does not otherwise occur in nature, or a material that is identical to a natural or native form of the material but made or obtained from synthetic materials and / or by manipulation using recombinant techniques.

[0082] "Wild-type" and "naturally-occurring" refer to forms found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence that can be isolated from a natural source and is present in an organism without being intentionally modified by human manipulation.

[0083] "Coding sequence" refers to a portion of a nucleic acid (eg, a gene) that codes for the amino acid sequence of a protein.

[0084] "Percent (%) sequence identity" refers to a comparison between polynucleotides and polypeptides and is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence due to optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions where the identical nucleic acid base or amino acid residue appears in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage sequence identity. Alternatively, the percentage can be calculated by determining the number of positions where either the identical nucleic acid base or amino acid residue appears in both sequences, or the number of positions where the nucleic acid base or amino acid residue is aligned with a gap, to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage sequence identity. Those skilled in the art will appreciate that there are many established algorithms available for aligning two sequences. Optimal alignment of sequences for comparison can be achieved, for example, as known in the art, by the Smith and Waterman local homology algorithm (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by the Needleman and Wunsch homology alignment algorithm (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), by the Pearson and Lipman similarity search method (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85:2444), by computer implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection.Examples of algorithms that are suitable for determining percent sequence identity and percent sequence similarity include, but are not limited to, the 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 a query sequence that match or meet some positive threshold score "T" when aligned with words of the same length in a database sequence. T is referred to as 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 for as far as possible to increase the cumulative alignment score. Cumulative scores are calculated using, for nucleotide sequences, the parameters "M" (reward score for a matching residue pair; always >0) and "N" (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score falls by an amount "X" from its maximum achieved value; when the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments; or when 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 as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program can use as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). Exemplary sequence alignments and determination of percent sequence identity can utilize the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison, WI) using the default parameters provided.

[0085] A "reference sequence" refers to a defined sequence used as a basis for sequence comparison. A reference sequence can also be a subset of a larger sequence, e.g., a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotides or amino acid residues long, at least 25 residues long, at least 50 residues long, at least 100 residues long, or the full length of a nucleic acid or polypeptide. Because two polynucleotides or polypeptides can each contain (1) sequences that are similar between the two sequences (i.e., a portion of the complete sequence) and (2) additional sequences that differ between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is 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, in which case the reference sequence can have one or more changes in the primary sequence. For example, the phrase "a reference sequence corresponding to SEQ ID NO: 2 with a leucine at the residue corresponding to X478" (or "a reference sequence corresponding to SEQ ID NO: 2 with a leucine at the residue corresponding to position 478") refers to a reference sequence in which the corresponding residue at position X478 of SEQ ID NO: 2 (e.g., methionine) has been changed to leucine.

[0086] A "comparison window" refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues within which a sequence can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and the portion of the sequence within the comparison window may contain no more than 20 percent additions or deletions (i.e., gaps) compared to the reference sequence (which contains no additions or deletions) for optimal alignment of the two sequences. The comparison window may be longer than 20 contiguous residues, and optionally includes windows of 30, 40, 50, 100, or longer.

[0087] When used in the context of the numbering of a given amino acid or polynucleotide sequence, "corresponding to," "with respect to," and "toward" refer to the numbering of residues in a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, residue numbers or residue positions in a given polymer are specified relative to the reference sequence, rather than by the actual numbered positions of the residues in the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as the amino acid sequence of an engineered DNA polymerase, can be aligned with a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although gaps exist, the numbering of residues in a given amino acid or polynucleotide sequence is relative to the reference sequence to which it is aligned. In some embodiments, the sequence is tagged (e.g., has a histidine tag).

[0088] "Mutation" refers to an alteration in a nucleic acid sequence. In some embodiments, a mutation results in a change in the encoded polypeptide sequence (i.e., compared to the original sequence without the mutation). In some embodiments, a mutation comprises a substitution, such that a different amino acid is produced. In some alternative embodiments, a mutation comprises an addition, such that an amino acid is added (e.g., inserted) to the original polypeptide sequence. In some further embodiments, a mutation comprises a deletion, such that an amino acid is deleted from the original polypeptide sequence. Any number of mutations may be present in a given sequence.

[0089] "Amino acid difference" and "residue difference" refer to the difference in an amino acid residue at a position in a polypeptide sequence compared to the amino acid residue at the corresponding position in a reference sequence. The position of an amino acid difference is generally referred to herein as "Xn," where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, "residue difference at position X478 compared to SEQ ID NO:2" (or "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 a reference polypeptide of SEQ ID NO:2 has a methionine at position 478, then "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 examples herein, a specific amino acid residue difference at a position is designated as "XnY," where "Xn" designates the corresponding residue and position in the reference polypeptide (as described above), and "Y" is the single-letter identifier of the amino acid found in the engineered polypeptide (i.e., the residue that differs in the reference polypeptide). In some instances (e.g., in the Tables in the Examples), the disclosure also provides specific amino acid differences, denoted by the conventional notation "AnB," where A is the single-letter identifier of the residue in the reference sequence, "n" is the number of the residue position in the reference sequence, and B is the single-letter identifier of the residue substitution in the sequence of the engineered polypeptide. In some embodiments, a "substitution" includes a deletion of an amino acid and may be indicated by a "-" symbol. In some embodiments, the phrase "amino acid residue nB" denotes the presence of an amino acid residue in the engineered polypeptide at a defined amino acid position, which may or may not be a substitution in the context of the reference polypeptide or amino acid sequence.

[0090] In some embodiments, polypeptides of the present disclosure can contain one or more amino acid residue differences relative to a reference sequence, as indicated by a list of the specific positions at which the residue difference occurs relative to the reference sequence. In some embodiments, when more than one amino acid can be used at a particular residue position in a polypeptide, the various amino acid residues that can be used are separated by a " / " (e.g., X478L / X478I, X478L / I, or 478L / I). The present disclosure includes engineered polypeptide sequences that contain one or more amino acid differences, including either or both conservative and non-conservative amino acid substitutions, as well as insertions and deletions of amino acids in the sequence.

[0091] "Amino acid substitution set" and "substitution set" refer to a group of amino acid substitutions within a polypeptide sequence. In some embodiments, a substitution set includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions. In some embodiments, a substitution set refers to a set of amino acid substitutions present in any of the variant DNA polymerase polypeptides listed in any of the tables in the Examples. In these substitution sets, individual substitutions are separated by a semicolon (";"; e.g., M478L; R516K; E541D) or a slash (" / "; e.g., M478L / R516K / E541D or 478L / 516K / 541D). In some embodiments, the phrase "mutation set" is used.

[0092] "Conservative amino acid substitution" refers to the replacement of a residue with a different residue having a similar side chain, and thus typically includes the replacement of an amino acid in a polypeptide with an amino acid within the same or similar defined class of amino acids. By way of example, and not limitation, an amino acid with an aliphatic side chain can be replaced with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain can be replaced with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain can be replaced with another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain can be replaced with another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain can be replaced with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and a hydrophobic or hydrophilic amino acid can be replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0093] A "non-conservative substitution" refers to the substitution 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 defined groups rather than within them, and affect (a) the structure of the peptide backbone in the area of ​​the substitution (e.g., substitution of glycine with proline); (b) the charge or hydrophobicity; and / or (c) the bulk of the side chain. By way of example, and not limitation, exemplary non-conservative substitutions include an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0094] "Deletion" refers to the modification of a polypeptide by the removal of one or more amino acids from a reference polypeptide. Deletions can include 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, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids comprising the reference enzyme, while retaining the enzymatic activity and / or improving properties of the engineered polymerase enzyme. Deletions can involve internal and / or terminal portions of the polypeptide. In various embodiments, deletions can comprise a contiguous segment or can be discontinuous. As noted above, in some embodiments, deletions are indicated by "-" and can be present in a substitution or set of substitutions.

[0095] "Insertion" refers to the modification of a polypeptide by the addition of one or more amino acids from a reference polypeptide. The insertion may be in an internal portion of the polypeptide or at the carboxy or amino terminus. As used herein, an insertion includes fusion proteins as known in the art. An insertion may be a contiguous segment of amino acids in a naturally occurring polypeptide or may be separated by one or more amino acids.

[0096] "Combination(s)" or "combination," in the context of any list of mutations (e.g., substitutions) or amino acid positions of mutations, refers to any combination of the listed mutations and / or amino acid positions. In some embodiments, "combination(s)" 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.

[0097] "Functional fragment" and "biologically active fragment" are used interchangeably herein and refer to a polypeptide that has amino-terminal and / or carboxy-terminal deletion(s) and / or internal deletion(s), but where 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 which retains substantially all of the activity of the full-length polypeptide.

[0098] An "isolated polypeptide" refers to a polypeptide that has been substantially separated from other contaminants that naturally accompany it (e.g., proteins, lipids, and polynucleotides). The term encompasses polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., a host cell or in vitro synthesis). Recombinant DNA polymerase polypeptides may be present within a cell, in cell culture medium, or prepared in various forms, such as a lysate or isolated preparation. Thus, in some embodiments, the recombinant DNA polymerase polypeptides provided herein are isolated polypeptides.

[0099] A "substantially pure polypeptide" or "purified polypeptide" refers to a composition in which the polypeptide species is the predominant species present (i.e., it is more abundant than any other individual macromolecular species in the composition, on a molar or weight basis); generally, a composition is substantially purified when the target species constitutes at least about 50 percent of the macromolecular species present, on a molar or weight percent basis. Generally, a substantially pure DNA polymerase composition constitutes 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 the total macromolecular species present in the composition, on a molar or weight percent basis. In some embodiments, the target species is purified to essential homogeneity, such that the composition consists essentially of a single macromolecular species (i.e., contaminants cannot be detected in the composition by conventional detection methods). Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, an isolated recombinant DNA polymerase polypeptide is a substantially pure polypeptide composition.

[0100] "Improved enzymatic properties" refers to an engineered DNA polymerase polypeptide that exhibits an improved enzymatic property compared to a reference DNA polymerase polypeptide, such as 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, properties such as increased protein expression, increased thermal activity, increased thermostability, 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 processivity, increased inhibitor resistance or tolerance, and altered temperature profile.

[0101] "Increased enzymatic activity" and "enhanced catalytic activity" refer to improved properties of an engineered DNA polymerase polypeptide, which may be represented by an increase in specific activity (e.g., product produced / time / weight of protein) and / or an increase in the percent conversion of substrate to product (e.g., the percent conversion of a starting amount of substrate to product within a specified period of time using a specified amount of DNA polymerase) compared to a reference DNA polymerase enzyme (e.g., a wild-type DNA polymerase and / or another engineered DNA polymerase). Exemplary methods for determining enzymatic activity are provided in the Examples. m , V max or k cat Any property related to enzymatic activity can be affected, including classical enzymatic properties of (these changes can lead to increased enzymatic activity.) Improved 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 higher enzymatic activity than the naturally occurring DNA polymerase or another engineered DNA polymerase from which the DNA polymerase polypeptide is derived.

[0102] " Hybridization stringency " refers to the hybridization conditions, such as washing conditions, during nucleic acid hybridization. Generally, hybridization reactions are carried out under conditions of lower stringency, followed by washing under various but higher stringency conditions (see, for example, 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 the conditions that allow target DNA to bind to the complementary nucleic acid that has about 60% identity with target DNA, preferably about 75% identity, about 85% identity, and about 90% identity or more with target polynucleotide. Exemplary moderately stringent conditions are those equivalent to hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by a wash in 0.2x SSPE, 0.2% SDS at 42°C. "High stringency hybridization" refers to hybridization at a temperature higher than the thermal melting temperature, T, determined under solution conditions for a defined polynucleotide sequence. mHigh stringency conditions generally refer to conditions at or below about 10°C. In some embodiments, high stringency conditions refer to conditions that allow hybridization of only 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 will not be stable under high stringency conditions as contemplated herein). High stringency conditions can be provided, for example, by hybridization under conditions equivalent to 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.1x 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 washing in 0.1x SSC containing 0.1% SDS at 65°C. Other high stringency hybridization conditions and moderately stringent conditions are described in the references above.

[0103] "Codon-optimized" refers to changing the codons in a polynucleotide encoding a protein to those preferentially used in that particular organism, such that the encoded protein is more efficiently expressed in that organism. While the genetic code is degenerate, with most amino acids represented by several codons called "synonymous" or "synonymous" codons, it is well known that codon usage by a particular organism is not random but is biased toward certain codon triplets. This codon usage bias can be higher for a given gene, genes of common function or ancestral origin, highly expressed proteins relative to low copy number proteins, and aggregated protein-coding regions of an organism's genome. In some embodiments, a polynucleotide encoding a DNA polymerase enzyme is codon-optimized for optimal production from the host organism selected for expression.

[0104] The term "control sequences" is used herein to include all components necessary or advantageous for the expression of 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, polyadenylation sequence, propeptide sequence, promoter sequence, signal peptide sequence, initiation sequence, and transcription terminator. In some embodiments, at least, control sequences include a promoter and transcriptional and translational stop signals. In some embodiments, control sequences are provided with linkers to introduce specific restriction sites facilitating ligation of the control sequences with the coding region of a nucleic acid sequence encoding a polypeptide.

[0105] "Operably linked" refers to a configuration in which a control sequence is suitably positioned relative to a polynucleotide of interest (i.e., in a functional relationship) so that the control sequence directs or regulates the expression of the polynucleotide and, if necessary, the encoded polypeptide of interest.

[0106] A "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. A promoter can be any nucleic acid sequence that shows transcriptional activity in the host cell of choice, including mutant, truncated, and hybrid promoters, and can be derived from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0107] "Suitable reaction conditions" or "suitable conditions" refer to conditions in an enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffers, co-solvents, etc.) under which a DNA polymerase polypeptide of the disclosure can convert a substrate into a desired product compound. Exemplary "suitable reaction conditions" are provided herein (see Examples).

[0108] "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.

[0109] "Culturing" refers to growing a population of cells, eg, microbial cells, under suitable conditions using any suitable medium (eg, liquid, gel, or solid).

[0110] A "vector" is a polynucleotide construct for introducing a polynucleotide sequence into a cell. In some embodiments, the vector is an expression vector operably linked to suitable control sequences capable of effecting expression of a polynucleotide of interest, and optionally 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, also includes a transcription terminator sequence.

[0111] "Expression" includes any step involved in producing a 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.

[0112] "Produce" refers to the production of a protein and / or other compound by a cell. The term is intended to encompass any step involved in producing a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of a polypeptide from the cell.

[0113] "Heterologous" or "recombinant" refers to the relationship between two or more nucleic acid or polypeptide sequences (e.g., promoter sequences, signal peptides, terminator sequences, etc.) that are derived from different sources and are not naturally associated.

[0114] "Host cell" and "host strain" refer to suitable hosts for expression vectors containing DNA (e.g., polynucleotide sequences encoding at least one DNA polymerase variant) provided herein. In some embodiments, host cells are prokaryotic or eukaryotic cells transformed or transfected with vectors constructed using recombinant DNA techniques known in the art.

[0115] "Analog" refers to a polypeptide having greater than 70% sequence identity but less than 100% sequence identity (e.g., greater 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, an analog comprises non-naturally occurring amino acid residues as well as naturally occurring amino acids, including, but not limited to, homoarginine, ornithine, and norvaline. In some embodiments, an analog also comprises one or more D-amino acid residues and a non-peptide bond between two or more amino acid residues.

[0116] An "effective amount" means an amount sufficient to produce a desired result. One of ordinary skill in the art can determine what an effective amount is using routine experimentation.

[0117] "Cell-free DNA" or "cfDNA" refers to DNA that circulates freely in the bloodstream and is not contained in or associated with cells. In some embodiments, cell-free DNA includes DNA originally obtained and released from normal somatic or germline cells, cancer cells, fetal cells, microbial cells, or viruses.

[0118] "Cell-free RNA" or "cfRNA" refers to RNA that circulates freely in the bloodstream and is not contained in or associated with cells. In some embodiments, cell-free RNA includes RNA originally obtained and released from normal somatic or germline cells, cancer cells, fetal cells, microbial cells, or viruses.

[0119] "Amplification" refers to nucleic acid replication. In some embodiments, the term refers to the replication of a specific template nucleic acid.

[0120] "Polymerase chain reaction" and "PCR" refer to methods for generating multiple copies of a nucleic acid template of interest in the presence of nucleic acid primers by repeated cycles of denaturation, annealing, and primer extension with a polymerase, such as those 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. Each series of denaturation, annealing, and extension constitutes a "cycle." The steps of denaturation, primer annealing, and polymerase extension can be repeated (i.e., multiple cycles are used) to amplify the nucleic acid template. In some embodiments, the annealing and extension process occurs in one step, sometimes referred to as two-step PCR. Thus, as used herein, the term "PCR" encompasses many variations of the method, including, inter alia, qPCR, hot start PCR, touchdown PCR, asymmetric PCR, multiplex PCR, long or long range PCR, assembly PCR and inverse PCR.

[0121] "Target," when used in reference to a method using a DNA polymerase, refers to a region of nucleic acid for preparation of complementary DNA. A "target" is selected from other nucleic acids present in a method using a DNA polymerase. In some embodiments, a "segment" is a region of nucleic acid within a target sequence.

[0122] "Target DNA," when used in the context of a DNA polymerase, refers to DNA, all or a portion thereof, that is the target for the preparation of a complementary DNA copy. The target DNA can be the entire DNA sequence or a portion thereof, e.g., a segment of the DNA sequence.

[0123] "Target RNA" refers to all or a portion of an RNA that is a target for the preparation of a complementary DNA copy. The target RNA can be an entire RNA sequence or a portion thereof, e.g., a segment of an RNA sequence.

[0124] "Sample template" refers to nucleic acid derived from a sample being analyzed for the presence of a target nucleic acid. 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 accidentally included in a sample, which may result from carryover contamination 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 as background in a test sample. However, it is not intended that the present invention be limited to any particular nucleic acid sample or template.

[0125] "Amplifiable nucleic acid" is used in reference to a nucleic acid that can be amplified by any amplification method, including, but not limited to, PCR. In most embodiments, the amplifiable nucleic acid comprises a sample template.

[0126] "PCR product," "PCR fragment," and "amplification product" refer to the resulting compound obtained after two or more cycles of PCR amplification (or other amplification methods indicated by the context), which typically include the steps of denaturing, annealing, and extension. The terms encompass situations in which there has been amplification of one or more segments of one or more target sequences.

[0127] "Amplification reagents" and "PCR reagents" refer to the reagents needed for amplification (e.g., deoxyribonucleotide triphosphates, buffers, etc.), excluding primers, nucleic acid template, and amplification enzyme. Typically, amplification reagents, along with other reaction components, are placed and contained within a reaction vessel (e.g., test tube, microwell, etc.). It is not intended that the present invention be limited to any particular amplification reagents, as any suitable reagents will find use in the present invention.

[0128] "Primer" refers to an oligonucleotide (i.e., a sequence of nucleotides), whether naturally occurring or produced synthetically, recombinantly, or by amplification, that can act as a point of initiation of nucleic acid synthesis when placed under conditions that induce synthesis of a primer extension product complementary to the nucleic acid strand (i.e., in the presence of nucleotides and an inducing agent, e.g., a DNA polymerase, and at a suitable temperature and pH). In some embodiments, a primer is single-stranded, while in some embodiments, a primer is double-stranded. In some embodiments, a primer is sufficiently long to prime the synthesis of an extension product in the presence of a nucleic acid polymerase. The exact length of a primer will depend on many factors, as known to those of skill in the art.

[0129] "Probe" refers to an oligonucleotide (i.e., a sequence of nucleotides) capable of hybridizing to another oligonucleotide of interest, whether naturally occurring or produced synthetically, recombinantly, or by amplification. Probes find use in the detection, identification, and / or isolation of specific gene sequences of interest. In some embodiments, probes are labeled with a "reporter molecule" (also called a "label") that facilitates detection of the probe in a suitable detection system (e.g., fluorescent, radioactive, luminescent, enzymatic, and other systems). It is not intended that the present invention be limited to any particular detection system or label. Primers, deoxyribonucleotides, and deoxyribonucleosides can contain labels. Indeed, it is not intended that the labeled compositions of the present invention be limited to any particular components. Exemplary labels include:32 P, 35 and fluorescent molecules (e.g., fluorescent dyes, including, but not limited to, green fluorescent protein).

[0130] "Fidelity," when used in reference to a polymerase, is intended to refer to the accuracy of template-directed incorporation of a complementary base in a synthesized DNA strand compared to a template strand. Typically, fidelity is measured based on the frequency of incorrect base incorporation in a newly synthesized nucleic acid strand. Incorrect base incorporation can result in point mutations, insertions, or deletions. Fidelity can be calculated according to any method known in the art (see, for example, Tindall and Kunkel, Biochem., 1988, 27:6008-6013; and Barnes, Gene, 1992, 112:29-35). A polymerase or polymerase variant can exhibit either high or low fidelity. As used herein, "high fidelity" refers to a polymerase having a frequency of correct base incorporation above a predetermined value. As used herein, the term "low fidelity" refers to a polymerase having a frequency of correct base incorporation below a predetermined value. In some embodiments, the predetermined value is a desired frequency or fidelity of correct base incorporation of a known polymerase (i.e., a reference polymerase).

[0131] "Altered fidelity" refers to a fidelity of a polymerase variant that differs from the fidelity of the parent 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. Altered fidelity can be determined by assaying the parent and variant polymerases and comparing their activities using any suitable assay known in the art.

[0132] "Processivity" refers to the ability of a nucleic acid modifying enzyme, such as a DNA polymerase, to remain bound to a template or substrate and carry out multiple modification reactions. Processivity is generally measured by the number of catalytic events that occur per binding event.

[0133] "Altered processivity" refers to a processivity of a polymerase or variant thereof that differs from the processivity of the parent or reference polymerase from which the variant is derived. In some embodiments, the altered processivity is higher than the processivity of the parent or reference enzyme, while in some other embodiments, the altered processivity is lower than the processivity of the parent or reference enzyme. Altered processivity can be determined by assaying the parent / reference and variant enzymes and comparing their activities using any suitable assay known in the art.

[0134] "Subject" includes mammals, such as humans, non-human primates, livestock, companion animals, and laboratory animals (e.g., rodents and lagamorphs). The term is intended to include males as well as females.

[0135] "Patient" means any subject being evaluated for, treated for, or experiencing a disease. In some embodiments, a "sample" for reaction with a DNA polymerase is obtained from a patient.

[0136] As used herein, a "sample" refers to a material or substance for reaction with a nucleic acid polymerase, e.g., to detect the presence of a target nucleic acid or to prepare DNA copies of a target nucleic acid for sequencing or generation of a cDNA library. In some embodiments, the sample is a "biological sample," which refers to a sample of biological tissue or fluid. Such samples are typically derived from humans, but also include tissues isolated from non-human primates or rodents, e.g., mice and rats, and include sections of tissue such as biopsy and autopsy samples, frozen sections taken for histological purposes, blood, plasma, serum, sputum, feces, tears, mucus, hair, skin, and the like. A "biological sample" also refers to a cell or cell population or a tissue or fluid content from an organism. In some embodiments, a biological sample has been removed from an animal, although the term "biological sample" can also refer to cells or tissues that are analyzed in vivo, i.e., without being removed from the 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 material, 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, buccal scrapings, saliva samples, or nipple secretions. As used herein, "tissue biopsy" refers to a quantity of tissue removed from an animal, preferably a human, for diagnostic analysis. In patients with cancer, tissue can be removed from a tumor to allow for analysis of cells within the tumor. "Tissue biopsy" can refer to any type of biopsy, such as needle biopsy, fine needle biopsy, surgical biopsy, etc. In some embodiments, samples can be obtained from environmental sources, including, but not limited to, water (e.g., oceans, rivers, garbage / sewage, etc.), soil, air, vents, or surfaces (e.g., floors, machines, counters, etc.).

[0137] Engineered DNA polymerase polypeptides In one aspect, the present disclosure provides DNA polymerases, including engineered DNA polymerase polypeptide variants. In some embodiments, DNA polymerases and engineered DNA polymerase polypeptide variants are useful for performing polymerase reactions, including preparing complementary DNA for a target DNA target / template. In some embodiments, the engineered DNA polymerase variants of the present disclosure find use in efficiently generating complementary DNA, in whole or in part, from a DNA template, such as in sequencing (e.g., NGS sequencing), amplification (e.g., PCR), and diagnostic methods, such as for detecting target nucleic acids. These engineered DNA polymerase variants can be used in solution as well as in immobilized embodiments. In some embodiments, the engineered DNA polymerases can be prepared and used as non-fusion or fusion polypeptides.

[0138] In some embodiments herein, when a particular DNA polymerase variant (i.e., an engineered DNA polymerase polypeptide) is referenced by reference to a modification of a particular amino acid residue in the sequence of a wild-type or reference DNA polymerase polypeptide, it is understood that this also encompasses variants of another DNA polymerase modified at the equivalent position(s) (as determined from an optional amino acid sequence alignment between the respective amino acid sequences). For example, for a substitution at a particular amino acid position(s) numbered with reference to SEQ ID NO: 2, the equivalent amino acid position(s) can be readily ascertained for another sequence, such as the reference sequence of SEQ ID NO: 220, 226, 272, 328, 546, 710, or 1208.

[0139] In some embodiments, the engineered DNA polymerase has a sequence similar to or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 1080%, 109%, 1109, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1211, 1220, 1230, 1221, 1231, 1240, 1241, 1250, 1 and amino acid sequences having 3%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, wherein the amino acid sequence contains one or more mutations 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.

[0140] 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 12-844 of SEQ ID NO:2 or a reference sequence corresponding to SEQ ID NO:2, wherein the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 of SEQ ID NO:2 or a reference sequence corresponding to SEQ ID NO:2.

[0141] 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 12-844 of SEQ ID NO:2 or 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 12-844 of SEQ ID NO:2 or a reference sequence corresponding to SEQ ID NO:2.

[0142] 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 12-844 of SEQ ID NO:2 or a reference sequence corresponding to SEQ ID NO:2, wherein the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 of SEQ ID NO:2 or a reference sequence corresponding to SEQ ID NO:2.

[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 12-844 of SEQ ID NO:2 or a corresponding reference sequence corresponding to an even-numbered SEQ ID NO:2, wherein the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 of SEQ ID NO:2 or a reference sequence corresponding to SEQ ID NO:2.

[0144] In some embodiments, the amino acid sequence of the engineered DNA polymerase is at 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, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 430, 431, 432, 433, 434, 6, 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, 70 704, 707, 808, 809, 812, 815, 820, 824, 825, or 831, or a combination thereof, wherein the positions are relative to the reference sequence corresponding to SEQ ID NO:2.

[0145] One of the specific ingredients is a DNA scaffold 5. 2-3N, 21A / G / N, 39F, and 1.0 1T、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 336E 338V 341H / T / Y 342G 351H 356I / S 359G / V 362M / P / S 5T、368H、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、39 3G / R / S / W、395G / H / S、396F / L / P / R / S、398G / Q、399G / T、402R / Y、403I、404T、4 09S、410V / W、412M / Q / R / V、413T、416S、417R / W、418K、420V、425T、426T、427 D / 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 44M, 545L, 550C / T, 554G / N, 557I, 558G / L / T, 559R, 562K, 569M / Q, 575W, 577 S, 578A, 579L / Q, 589G, 594A, 601T, 603M / R, 604H, 612D, 613H / R, 641A, 644Q651Q, 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 , 704L / 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, 73 5L, 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 combinations thereof, wherein the positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

[0146] One of the specific ingredients is a DNA scaffold Means 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、V3 71L, L372V / Y, A373G / T, L374S / V / Y, E376F / S, G377C, V378C / F / L / S, A379G. L380I, D381E / K, P382C / S, L388C / M / P / V, A390S / T, L392A / C / S / T / V, L393G / R / S / W、P395G / H / S、A396F / L / P / R / S、T398G / Q、N399G / T、G402R / Y、V403I、A40 4T、G409S、E410V / W、T412M / Q / R / V、E413T、A416S、E417R / W、R418K、L420V、L4 25T、F426T、Q427D / F / H / I / L / R / T / Y、L429F、F430G、P431G / L / R / V、R432T、S4 34N、E435K / 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、R461 L / Q / S, P465Y, L466Y, A469T / V, E473N, L474A / C / G, E475A / S / V / Y, E477V, M47 8I / L, E479L, E482S, G483D, L488W, G490W, K503R, E508K, V514I, G515H / T, R 516K, A526S, 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, A5 79L / 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, Y697A / I / L / V, K698E, E699V, E701M, A702S / V, I704L / V, R706I / S / V, Y707W, F708L, K713G, V714I, R715 H / L, A716E / L, I718F / Q, T721A / M / N, E723Y, E724F / L, G725V, R728S, V731I / T, E732L / Q, F735L, Y740S / V, P742K, D743H / V, A745S / T / V, R747S, V748G, R749M, E753A, M758G, F760T / 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 the reference sequence corresponding to SEQ ID NO: 2.

[0147] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one 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 one mutation 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 position is 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 one mutation 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 position is 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 one mutation 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 position is relative to a reference sequence corresponding to SEQ ID NO:2.

[0148] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one mutation at amino acid position 559, the position being 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 one substitution 559R, the position being 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 one substitution P559R, the position being relative to the reference sequence corresponding to SEQ ID NO: 2.

[0149] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one mutation at amino acid position 514, 515, or 760, or a combination thereof, where 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 one mutation 514I, 515T, or 760W, or a combination thereof, where 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 one mutation V514I, G515T, or F760W, or a combination thereof, where the position is relative to the reference sequence corresponding to SEQ ID NO: 2.

[0150] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one mutation at amino acid position 402, 604, or 660, or a combination thereof, where 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 one mutation 402R, 604H, or 660V, or a combination thereof, where 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 one mutation G402R, R604H, or L660V, or a combination thereof, where the position is relative to the reference sequence corresponding to SEQ ID NO: 2.

[0151] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one mutation at amino acid position 291, 395, 687, or 745, or a combination thereof, where 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 one mutation 291L, 395H, 687A, or 745T, or a combination thereof, where 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 one mutation F291L, P395H, H687A, or A745T, or a combination thereof, where the position is relative to the reference sequence corresponding to SEQ ID NO: 2.

[0152] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one mutation at amino acid position 257, 395, 474, 475, 536, or 662, or a combination thereof, where 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 one mutation 257V, 395S, 474A, 475A, 536V, or 662S, or a combination thereof, where 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 one mutation A257V, H395S, L474A, E475A, A536V, or P662S, or a combination thereof, where 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 one mutation at amino acid position 478, 515, 526, 527, or 528, or a combination thereof, where 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 one mutation 478L, 515T, 526S, 527A, or 528A, or a combination thereof, where 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 one mutation M478L, G515T, A526S, Q527A, or V528A, or a combination thereof, where the position is relative to the reference sequence corresponding to SEQ ID NO: 2.

[0154] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one 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 at least two, three, four, five, six, seven, eight, nine, 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 one 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 two, three, four, five, six, seven, eight, nine, 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 one mutation 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 two, three, four, five, six, seven, eight, nine or all of the mutations G402R, M478L, G515T, A526S, Q527A, G528A, P559R, R604H, L660V or F760W.

[0155] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one 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 mutations at at least two, three, four, five, six, 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 one 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 two, three, four, five, six, or all of the mutations 478L, 515T, 526S, 527A, 528A, 559R, and 760W. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one mutation M478L, G515T, A526S, Q527A, G528A, P559R, or F760W, or a combination thereof, the positions being relative to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least two, three, four, five, six, or all of the mutations M478L, G515T, A526S, Q527A, G528A, P559R, and F760W.

[0156] 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, where 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, where 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, where the positions are relative to SEQ ID NO: 2.

[0157] 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, where 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 each of amino acid positions 402R, 478L, 515T, 526S, 527A, 528A, 559R, 604H, 660V, and 760W, where 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 each of amino acid positions G402R, M478L, G515T, A526S, 527A, 528A, 559R, 604H, 660V, and 760W, where the positions are relative to SEQ ID NO: 2.

[0158] 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, where 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 147H, 257V, 291L, 395S, 402R, 474A, 475A, 478L, 515T, 526S, 527A, 528A, 536V, 559R, 604H, 660V, 662S, 687A, 745T, and 760W, where 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, where the positions are relative to SEQ ID NO:2.

[0159] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one mutation at amino acid position 147, where 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 one mutation 147H, where 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 one mutation C147H, where the position is relative to the reference sequence corresponding to SEQ ID NO: 2.

[0160] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises amino acid position(s) 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 / 516 / 541, 478 / 541, 2 88 / 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, 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 / 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 / 51 6 / 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, 4 78 / 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 / 6 98, 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 / 541, 288 / 399 / 478 / 514 / 554 / 557, at least one mutation or set of mutations at 288 / 399 / 514 / 516 / 528, 478 / 554, 399 / 514 / 516, 514, 399 / 550 / 554, 288 / 478 / 514 / 528 / 541 / 554, 478 / 514 / 516 / 554, 478 / 514 / 541, 288 / 541 / 554 / 557, 698 or 478 / 514 / 526 / 527 / 528, where 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 one mutation or 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、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 / 52 6S / 550T / 554N、478L / 541D / 550T / 557I、541D、288R / 514I / 526S / 541D、554N / 557I, 399T / 478L, 288R / 514I / 554N / 557I, 478L / 514I / 516K / 526S / 527A / 529V / 557I, 399T / 541D / 550T / 554N, 516K / 529V / 541D / 554G / 557I, 478I / 541D / 554G / 557I, 288R / 478L / 526S / 528S / 529V / 554N / 557I, 288R / 514I / 516K / 529V / 541D, 478I / 514I / 516K / 541D, 478L / 514I / 557I, 399T / 541D / 557I, 399T / 541D, 288R / 399T / 516K / 527A / 541D / 557I, 288R / 478I / 514I / 541D, 288R / 514I / 516K, 664A / 698E, 399T / 478L / 516K / 541D, 399T / 528A / 550T, 399T / 478I / 514I / 541D / 557I、288R / 478L / 514I / 516K、224L / 664A、478L / 526S / 527A / 52 8S、288R / 478L / 514I / 516K / 541D / 550T / 554N、478L / 514I / 541D / 554N、478L / 516K、478L / 514I / 516K / 541D / 554N / 557I、288R / 399T / 478L / 516K / 541D、28 8R / 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 / 541D399T / 478L / 554N, 516K / 528A / 541D / 554N / 557I, 39F / 199P / 698E, 399T / 478L / 526S / 528S / 529V, 288R / 399T / 514I / 541D / 550T / 554N, 288R / 541D, 288R / 478I / 514I / 550T / 554N / 557I、288R / 399T / 478L / 516K、550T / 554N、526S / 529V / 554G / 557I、288R / 478I / 557I、288R / 478L / 514I / 550T / 554N、478L / 541 D / 550T / 554N, 516K / 526S / 528S, 288R / 526S / 528S / 550T / 554N / 557I, 199P / 664A / 698E, 514I / 526S / 528S / 529V / 550T / 557I, 288R, 399T / 554N / 557I, 2 88R / 554G / 557I, 399T / 514I / 541D, 288R / 478L / 514I / 527A / 554N / 557I, 399T / 478I / 516K / 541D / 554N, 87V / 541D / 554N, 288R / 550T / 554N / 557I, 527A / 5 29V / 541D, 528S / 529V / 554N, 478L / 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 / 478 L / 526S, 399T / 478L / 514I / 516K / 526S / 527A / 528S / 529V / 541D, 288R / 399T / 478I / 514I / 554G / 557I, 288R / 399T / 514I / 516K / 528S, 478L / 554N, 399T / 5 14I / 516K、514I、399T / 550T / 554N、478I / 516K、288R / 478L / 514I / 528A / 541 D / 554N、478I / 514I / 516K / 554N、478L / 514I / 541D、288R / 541D / 554N / 557I、698E or 478L / 514I / 526S / 527A / 528A, the positions being 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 one mutation or set 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 / S554G / L55 7I, S550T / S554N / L557I, M478L, N399T, V514I / R516K, N399T / R516K / A 526S / G528A / A529V / S554N / L557I, M478L / R516K / E541D, M478I / E541D , E288R / N399T / V514I / S554G / L557I, M478L / V514I / S554N / L557I, E28 8R / M478L / A526S / S550T / S554N, M478L / E541D / S550T / L557I, E541D, E2 88R / V514I / A526S / E541D, S554N / L557I, N399T / M478L, E288R / V514I / S554N / L557I, M478L / V514I / R516K / A526S / Q527A / A529V / L557I, N399 T / E541D / S550T / S554N, R516K / A529V / E541D / S554G / L557I, M478I / E5 41D / S554G / L557I, E288R / M478L / A526S / G528S / A529V / S554N / L557I, E 288R / V514I / R516K / A529V / E541D, M478I / V514I / R516K / E541D, M478L / V514I / L557I, N399T / E541D / L557I, N399T / E541D, E288R / N399T / R51 6K / Q527A / E541D / L557I, E288R / M478I / V514I / E541D, E288R / V514I / R 516K, L664A / K698E, N399T / M478L / R516K / E541D, N399T / G528A / S550T,N399T / M478I / V514I / E541D / L557I、E288R / M478L / V514I / R516K、V224L / L664A、M478L / A526S / Q527A / G528S、E 288R / M478L / V514I / R516K / E541D / S550T / S554N、M478L / V514I / E541D / S554N、M478L / R516K、M478L / V514I / R、 516K / E541D / S554N / L557I、E288R / N399T / M478L / R516K / E541D、E288R / M4 78L / V514I / S554N / L557I、E288R / M478I / R516K、E288R / N399T / R516K / S554 G / L557I、S550T / S554G、E288R / S550T / S554N、E288R / N399T / M478L / R516K / S550T / S554N、N399T / V514I / E541D / S550T / S554G、E288R / M478L / V514I / S 554N, 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 / M 478I / V514I / S550T / S554N / L557I、E288R / N399T / M478L / R516K、S550T / S55 4N、A526S / A529V / S554G / L557I、E288R / M478I / L557I、E288R / M478L / V514 I / 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 / L557 I、N399T / V514I / E541D、E288R / M478L / V514I / Q527A / S554N / L557I、N399T / M478I / R516K / E541D / S554N、A87V / E541D / S554N、E288R / S550T / S554N / L5 57I, Q527A / A529V / E541D, G528S / A529V / S554N, M478L / S554G / L557I, M478L / V514I / R516K / E541D / S550T / S554G / L557I, R199P / R216K / R644Q / L664AA396S / M478I / V514I / S554G / L557I / G694D, M478I, R241E / R644Q / A663E / L664A, N3 99T / M478I / S554N / L557I, E288R / N399T / S550T, E288R / R516K / A526S / Q527A / G528A / L557I, N399T / A526S / Q527A / G528S / A529V, E288R / M478L / A526S, N399T / M478L / V 514I / R516K / A526S / Q527A / G528S / A529V / E541D, E288R / N399T / M478I / V514I / S554 G / L557I, E288R / N399T / V514I / R516K / G528S, M478L / S554N, N399T / V514I / R516K, V514I, N399T / S550T / S554N, M478I / R516K, E288R / M478L / V514I / G528A / E541D / S55 4N, M478I / V514I / R516K / S554N, M478L / V514I / E541D, E288R / E541D / S554N / L557I, K698E or M478L / V514I / A526S / Q527A / G528A, where the positions are relative to the reference sequence corresponding to SEQ ID NO: 2.

[0161] In some embodiments, the amino acid sequence of the engineered DNA polymerase is 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, 402 / 478 / 514 / 526 / 527 / 528, 398 / 478 / 514 / 526 / 527 / 528, 478 / 514 / 526 / 527 / 528 / 758, 399 / 478 / 514 / 526 / 527 / 528, 478 / 514 / 526 / 527 / 528 / 575, 257 / 478 / 514 / 526 / 527 / 528 / 758, 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 the reference sequence corresponding to SEQ ID NO:2.In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one of the following mutation sets: 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 ...41S, 478L / 514I / 526S / 527A / 528A / 559R, 478L / 514I / 526S / 527A / 528A / 560W, 478L / 514I / 526S / 527A / 528A / 561S, 478L / 514I / 526S / 527A / 528A / 561R, 478L / 514I / 526S / 527A / 528A / 561R, 478L / 514I / 526S / 527A / 528A / 561R, 478L / 514I / 526S / 527A / 514I / 526S / 527A / 528A / 541N, 478L / 514I / 526S / 527A / 528A / 753A, 402R / 478L / 514I / 526S / 527A / 528A, 478L / 514I / 526S / 527A / 528A / 760T, 398Q / 478L / 514I / 526S / 527A / 528A, 478L / 514I / 526S / 527A / 528A / 758G, 478L / 514I / 526S / 527A / 528A / 7 60Y, 399G / 478L / 514I / 526S / 527A / 528A, 478L / 514I / 526S / 527A / 528A / 575W, 257V / 478L / 514I / 526S / 527A / 528A / 758G, 4 78L / 514I / 526S / 527A / 528A / 558L, 402Y / 478L / 514I / 526S / 527A / 528A, 478L / 514I / 526S / 527A / 528A / 558T, 478L / 514I / 52 6S / 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, where 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 one mutation or set of mutations provided in Tables 5.1 and 5.2 relative to the reference sequence corresponding to SEQ ID NO: 2.

[0162] In some embodiments, the amino acid sequence of the engineered DNA polymerase is 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, 402 / 478 / 514 / 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 / 52 and at least one set of mutations in: 6 / 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, where 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 one of the following mutation sets: 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, 402Y / 478L / 514I / 515T ... S / 527A / 528A / 559R, 377C / 478L / 514I / 515T / 526S / 527A / 528A / 559R, 478L / 514I / 515T / 526S / 527A / 528A / 559R, 478L / 515T / 526S / 527A / 528A / 559R, 402 Y / 478L / 514I / 526S / 527A / 528A / 559R, 478L / 503R / 514I / 526S / 527A / 528A / 559R, 239V / 478L / 514I / 526S / 527A / 528A / 559R, 73I / 478L / 514I / 526S / 527A / 528A / 559R, 478L / 514I / 526S / 527A / 541N / 559R, 59K / 478L / 514I / 526S / 52 7A / 528A / 559R, 478L / 514I / 515T / 526S / 527A / 528A / 559R / 575W, 3N / 478L / 51 4I / 515T / 526S / 527A / 528A, 377C / 478L / 514I / 515H / 526S / 527A / 541N / 554N / 557I / 559R, 402R / 478L / 515T / 526S / 527A / 528A / 541N, 478L / 515T / 526S / 52 7A / 528A / 541G, 478L / 515T / 526S / 527A / 528A, 478L / 514I / 515T / 526S / 527A / 528A, 478L / 514I / 515T / 526S / 527A / 528A, 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, where 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 one set of mutations provided in Tables 6.1 and 6.2 relative to the reference sequence corresponding to SEQ ID NO:2.

[0163] In some embodiments, the amino acid sequence of the engineered DNA polymerase is 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 / 541 / 559 / 660 / 760 60, 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 / 52 6 / 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 / 483 / 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 / 526 / 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、and at least one set of mutations at: 403 / 478 / 515 / 526 / 527 / 528 / 541 / 559 / 604 / 660 / 760, 257 / 377 / 403 / 478 / 483 / 515 / 526 / 527 / 528 / 541 / 559 / 601 / 604 / 760, 478 / 515 / 526 / 527 / 528 / 559 / 760, 402 / 478 / 515 / 526 / 527 / 541 / 559 / 601 / 760 or 257 / 377 / 478 / 515 / 526 / 527 / 528 / 559 / 760, where 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 one of the following 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 / 5 26S / 527A / 528A / 541G / 559R / 660V / 760W, 377C / 402Y / 478L / 515T / 526S / 527A / 528A / 541N / 559R / 660V / 760W, 25 7V / 377C / 402Y / 478L / 515T / 526S / 527A / 528A / 559R / 660V / 760W, 478L / 483D / 515T / 526S / 527A / 528A / 559R / 66 0V / 760W, 257V / 402Y / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 257V / 478L / 515T / 526S / 527A / 528A / 559R / 760 W, 257V / 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,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 / 760W478L / 515T / 526S / 527A / 528A / 559R / 743V / 760W, 396R / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 478L / 479L / 515T / 526S / 527A / 528A / 559R / 760W, 377C / 403I / 478L / 515T / 526S / 527A / 528A / 541D / 559R / 604H / 760W, 475A / 478L / 51 5T / 526S / 527A / 528A / 559R / 760W, 178L / 257V / 403I / 478L / 515T / 526S / 527A / 559R / 760W, 448I / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 478L / 515T / 5 26S / 527A / 528A / 559R / 577S / 760W, 395G / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 478L / 515T / 526S / 527A / 528A / 559R / 760W / 769Q, 475V / 478L / 515T / 5 26S / 527A / 528A / 559R / 760W, 448C / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 478L / 515T / 526S / 527A / 528A / 559R / 691W / 760W, 478L / 515T / 526S / 527A / 5 28A / 559R / 677L / 760W, 478L / 515T / 526S / 527A / 528A / 559R / 704L / 760W, 377C / 478L / 515T / 526S / 527A / 528A / 541N / 559R / 575W / 604H / 660V / 760W, 178L / 2 57V / 478L / 515T / 526S / 527A / 528A / 559R / 601T / 604H / 760W, 478L / 515T / 526S / 527A / 528A / 559R / 685R / 760W, 377C / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W, 478L / 515T / 526S / 527A / 528A / 559R / 749M / 760W, 396F / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 395H / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 475S / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 474C / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 478L / 482S / 515T / 526S / 527A / 528A / 559R / 760W, 291L / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 448V / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 478L / 515T / 526S / 527A / 528A / 559R / 579Q / 760W178L / 478L / 515T / 526S / 527A / 528A / 541G / 550C / 559R / 604H / 760W、477V / 478L / 515T / 526S / 527A / 528A / 559R / 760W、178L / 257V / 478L / 515T / 526S / 5 27A / 528A / 559R / 760W, 257V / 402R / 478L / 490W / 515T / 526S / 527A / 541G / 559R / 660V / 760W, 328L / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 395S / 478 L / 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 / 515T / 526S / 527A / 528A / 559R / 579L / 760W、368H / 478L / 515T / 526S / 527A / 528A / 559R / 760W、47 8L / 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 / 515T / 526S / 527A / 528A / 559R / 578A / 760W 478L / 515T / 526S / 527A / 528A / 559R / 687A / 760W 4 78L / 515T / 526S / 527A / 528A / 541D / 559R / 601T / 604H / 760W, 328V / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 396L / 478L / 515T / 526S / 527A / 528A / 559 R / 760W, 475Y / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 395G / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 257V / 478L / 515T / 526S / 527A / 559R / 760W,257V / 478L / 515T / 526S / 527A / 554N / 559R / 760W, 478L / 515T / 526S / 527A / 528A / 559R / 760W / 768S, 257V / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 25 7V / 478L / 515T / 526S / 527A / 528A / 559R / 760W、396P / 478L / 515T / 526S / 527 A / 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 / 52 7A / 528A / 559R / 760W, 257V / 403I / 478L / 515T / 526S / 527A / 528A / 541N / 559R / 760W, 474G / 478L / 515T / 526S / 527A / 528A / 559R / 760W, 478L / 515T / 526S / 527A / 528A / 559R / 604H / 760W, 402Y / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 760W, 478L / 515T / 526S / 527A / 528A / 559R / 691S / 760W, 257V / 316G / 47 8L / 515T / 526S / 527A / 528A / 541D / 559R / 575W / 604H / 760W, 327L / 478L / 515T / 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 / 60 4H / 760W、478L / 515T / 526S / 527A / 528A / 559R / 740S / 760W、478L / 515T / 526 S / 527A / 528A / 559R / 760W、257V / 478L / 515T / 526S / 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, where 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 one set of mutations provided in Tables 7.1 and 7.2 relative to the reference sequence corresponding to SEQ ID NO: 2.

[0164] In some embodiments, the amino acid sequence of the engineered DNA polymerase is 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 / 677 / 760, 402 / 478 / 515 / 526 / 527 / 528 / 559 / 562 / 604 / 660 / 760, 178 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 562 / 604 / 660 / 760, 8 / 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 / 67 7 / 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 / 760 , 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 / 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 / 4 02 / 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 / 5 28 / 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 / 31 6 / 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 / 47 8 / 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,In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one of the following 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 / 677L / 760W, 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 677L / 760W, 402R / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 660V / 760W, 178L / 395H / 402R / 478L / 51 5T / 526S / 527A / 528A / 559R / 604H / 660V / 745T / 760W, 291L / 395H / 402R / 4 78L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 257V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 677L / 760 W, 303T / 338V / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 7 60W, 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W, 79R / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W , 359G / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W, 79T / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W, 402R / 47 4A / 475S / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 677L / 704V / 7 60W, 79M / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 760W, 3 95S / 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 / 528、 A / 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, where 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 one set of mutations provided in Tables 8.1 and 8.2 relative to the reference sequence corresponding to SEQ ID NO: 2.

[0165] In some embodiments, the amino acid sequence of the engineered DNA polymerase is 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, 291 / 395 / 402 / 478 / 515 / 526 / 527 / 528 / 559 / 604 / 660 / 687 / 745 / 760, 26 / 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 / 40 2 / 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, 29 1 / 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 / 52、 7 / 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, 29 1 / 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, where 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 one of the following mutation sets: 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 / 745T / 760W, 0V / 687A / 697I / 745T / 760W, 291L / 395H / 402R / A457W / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687 A / 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 / 745 T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 6 60V / 687A / 708L / 745T / 760W、291L / 395H / 402R / 431G / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395S / 402R / 47 4A / 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 / 7 60W, 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 / 760W291L / 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 / 4 02R / 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 / 4 02R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 699V / 745T / 760W、291L / L372V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 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 / 760W79R / 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 / 687A / 745T / 7 60W, 291L / 395H / 402R / 448V / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 381E / 395H / 402R / 478L / 515T / 526S / 527A / 5 28A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 707W / 745T / 760W, 291L / 381K / 39 5H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 395H / 402R / 457Y / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 66 0V / 687A / 745T / 760W, 291L / 395H / 402R / 416S / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 395H / 402R / 474A / 475A / 478 L / 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 / 760W291L / 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 / 515T / 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 / 74 5T / 760W / 792F, 79R / 291L / 395H / 402R / 474A / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 662S / 687A / 699V / 745T / 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 / 74 5T / 760W, 291L / 395H / 402R / 447R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 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、29 1L / 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 / 528A / 559R / 604H / 660V / 687A / 702V / 745T / 7 60W, 291L / 342G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 716L / 745T / 760W, 233K / 291L / 395H / 402R / 478L / 515T / 526S / 527 A / 528A / 559R / 562K / 604H / 660V / 687A / 713G / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 704V / 745T / 760W、291L / 395H / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 562K / 604H / 660V / 662S / 687A / 745T / 760W、291L / 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 / 68 7A / 745T / 760W, 257V / 291L / 316G / 395H / 402R / 474A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 745T / 760W / 792F, 291L / 395H / 402R / 427D / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 370G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 395H / 402R / 417R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 395H / 402R / 425T / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 562K / 604H / 660V / 68 7A / 745T / 760W, 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 714I / 745T / 760W, 233H / 291L / 395H / 402R / 478L / 515T / 5 26S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 392T / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W291L / 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 / 660V / 687A / 697A / 745T / 760W、291L / 378L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 3 78L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 395H / 402R / 427H / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 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 / 687A / 745T / 760W、79R / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 660V / 687A / 745T / 760W、291L / 39 5H / 402R / 435P / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 257V / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 699V / 745T / 760W / 792F, 79R / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 745T / 760W291L / 395H / 402R / A453S / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / P667T / 687A / 745T / 760W、79A / 291L / 303T / 338V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 613H / 660V / 687A / 745T / 760W / 804R / A812S、79R / 257V / 291L / 395S / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 792F, 79A / 291L / A338V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 660V / 687A / 745T / 760W, 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 5H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 704V / 745T / 760W、79R / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 562K / 604H / 660V / 687A / 699V / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、21A / 79A / 291L / 338V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / R641A / 660V / 687A / 699V / 745T / 760W、291L / 395H / 402R / 478L / 515T / 5 26S / 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、79R / 178L / 257V / 291L / 3 95H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 662S / 687A / 699V / 745T / 760W, 79R / 257V / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 562K / 604H / 660V / 662S / 687A / 745T / 760W, 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 660V / 687A / 699V / 745 T / 760W、291L / 359G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604 H / 613H / 660V / 687A / 742K / 745T / 760W / 808Y / A812S、21N / 79A / 291L / 338V / A379G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 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 / 760W21N / 79R / 291L / 359G / 395H / 402R / 442G / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 662S / 687A / 745T / 760W、79A / 291L / A338V / 359V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 660V / 6 87A / 745T / 760W, 21N / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 613H / 660V / 662S / 687A / 742K / 745T / 760W, 21A / 79A / 291L / 303T / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660 V / 662S / 687A / 699V / 742K / 745T / 760W / 808Y, 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 / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 613H / 660V / 662S / 687A / 745T / 760W, 291 L / 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 / 6 62S / 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 / 760W79A / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 713G / 745T / 760W、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, 291L / 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 / 604H / 660V / 662S / 687A / 745T / 760W, 257V / 291L / 316G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 745T / 760W, 21N / 79R / 291L / 303T / 395H / 402R / 442G / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 662S / 687A / 745T / 760W, 21N / 79R / 233K / 291L / 303T / 338V / A379G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 660V / 687A / 713G / 745T / 760W, 291L / 338V / 395H / 402R / 442G / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 699V / 745T / 760W291L / 395H / 402R / 426T / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 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 / 5 62K / 604H / 660V / 687A / 745T / 760W / 812S, 79R / 291L / 395H / 402R / 474A / 478L / 515T / 526S / 527A / 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 / 52 6S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、21A / 79A / 291L / 33 8V / 379G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 6 62S / 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, 291L / 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 / 760W291L / 395H / 402R / R461L / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 395H / 402R / V451L / 478L / 515T / 526S / 527A / 52 8A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 715L / 745T / 760W, 79R / 291L / 303 T / 338V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 641A / 660 V / 687A / 713G / 745T / 760W、291L / 393W / 395H / 402R / 478L / 515T / 526S / 527 A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 372Y / 395H / 402R / 478 L / 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 / 760W 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 / 760W、79R / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 395H / 402R / 437S / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W257V / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 562K / 604H / 660V / 662S / 687A / 745T / 760W, 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / V731T / 745T / 760W, 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / R706V / 745T / 760W 291L / 395H / 402R / 448T / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W 21A / 79A / 291L / 379G / 395H / 402R / 442G / 478L / 515T / 52 6S / 527A / 528A / 559R / 562K / 604H / 660V / 662S / 687A / 745T / 760W、291L / 39 5H / 402R / 457Y / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745 T / 760W、291L / 362M / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604 H / 660V / 687A / 745T / 760W、291L / 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 / 528 A / 559R / 604H / 660V / 687A / 745T / 760W、21A / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 808Y / 812S、291L / 395H / 402R / 435K / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、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 / 559 R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 418K / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 338V / 395H / 402R / 442G / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / T721N / 745T / 760W、291L / 395H / 402R / 449A / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、291L / 395H / 402R / 457W / 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 / 760W、291L / 379G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 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、291L / 316G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 562K / 604H / 660V / 687A / 745T / 760W、257V / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 745T / 760W / 792F、21N / 79R / 291L / 395H / 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 / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 792F, 291L / 395H / 402R / 431V / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760 W, 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 / 7 45T / 760W、291L / 395H / 402R / 412V / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 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 / 6 99V / 745T / 760W, 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 / 760W257V / 291L / 316G / 395H / 402R / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 745T / 760W / 792F, 291L / 395H / 402R / L438S / 478L / 5 15T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 388C / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W 291L / 380I / 395H / 402R / 454L / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W 291L / 395H / 402R / 447R / 478L / 515T / 526S / 527A / 5 28A / 559R / 604H / 660V / 687A / 745T / 760W, 21N / 79R / 233K / 291L / 303T / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 613H / 660V / 687A / 699V / 7 45T / 760W / 809K, 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 724F / 745T / 760W, 291L / 395H / 402R / 461Q / 478L / 515T / 526 S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、21A / 79R / 291L / 338V / 3 95H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 613H / 660V / 687A / 745 T / 760W、79R / 178L / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 687A / 745T / 760W / 792F、291L / 395H / 402R / 478L / 515T / 52 6S / 527A / 528A / 559R / 604H / 660V / 687A / 715H / 745T / 760W、291L / 390T / 395 H / 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 / 5 28A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 374S / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 362S / 395 H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W 、21A / 79R / 291L / 379G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 60 4H / 613H / 660V / 687A / 713G / 745T / 760W、79R / 291L / 316G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 699V / 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 / 52 8A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 376F / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 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 / 66 0V / 662S / 687A / 745T / 760W, 79R / 257V / 262N / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 562K / 604H / 660V / 687A / 699V / 745T / 760W291L / 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 291 L / 388M / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 79R / 233K / 291L / 338V / 359G / 379G / 395H / 402R / 442G / 478L / 515 T / 526S / 527A / 528A / 559R / 562K / 604H / 613H / 660V / 687A / 745T / 760W / 812S、 233K / 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 68 7A / 745T / 760W, 291L / 371L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 376S / 395H / 402R / 478L / 515T / 526S / 5 27A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 316G / 395H / 402R / 475A / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 660V / 662S / 687A / 745T / 760W / 792F, 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 702S / 745T / 760W, 79R / 257V / 291L / 316G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W / 792F, 291L / 373G / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W291L / 395H / 402R / 454W / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W H / 660V / 687A / 728S / 745T / 760W、291L / 395H / 402R / 447W / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W、79R / 233K / 291L / 338V / 39、 5H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 395H / 402R / L437P / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 6 60V / 687A / 745T / 760W, 291L / 395H / 402R / 432T / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 79R / 178L / 257V / 291L / 316G / 39 5S / 402R / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 68 7A / 699V / 745T / 760W、79R / 291L / 303T / 395H / 402R / 478L / 515T / 526S / 527 A / 528A / 559R / 604H / 660V / 687A / 713G / 745T / 760W、291L / 395H / 402R / 478 L / 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 / 291L / 379G / 395H / 402R / 442G / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 613H / 660V / 662S / 687A / 713 G / 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 / 687A / 732L / 745T / 760W 291L / 392S / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W291L / 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 / 745T / 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, 291L / 395H / 402R / 412M / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 79R / 257V / 291L / 395S / 402R / 478L / 51 5T / 526S / 527A / 528A / 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 / 604H / 660V / 687A / 745T / 760W 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 562K / 604H / 660V / 687A / 699V / 745T / 760WH2- / 291L / L374Y / 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 / 723Y / 745T / 760W, 291L / 395H / 402R / 434N / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 388 V / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 395H / 402 R / 420V / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W, 291L / 395H / 402R / 47 8L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 699V / 745T / 760W, 291L / 395H / 402R / 427I / 47 8L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 745T / 760W or 291L / 395H / 402R / 478L / 515T / 526S / 527A / 528A / 559R / 604H / 660V / 687A / 724L / 745T / 760W, where 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 one set of mutations provided in Tables 9.1, 9.2, and 9.3 relative to the reference sequence corresponding to SEQ ID NO: 2.

[0166] In some embodiments, the amino acid sequence of the engineered DNA polymerase is 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, 257 / 291 / 395 / 402 / 466 / 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 / 807, 257 / 291 / 395 / 402 / 4 74 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 612 / 660 / 662 / 687 / 745 / 760, 147 / 257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 604 / 66 0 / 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 / 395 / 402 / 474 / 475 / 47 8 / 488 / 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 / 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, 257 / 291 / 395 / 402 / 47 4 / 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 / 474 / 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、and at least one set of mutations in 257 / 291 / 395 / 402 / 474 / 475 / 478 / 515 / 526 / 527 / 528 / 536 / 559 / 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, where 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 one of the following mutation sets: 68R / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W, 78S ... 15T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W, 68K / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W, 257V / 291L / 395S / 402R / 466Y / 474 A / 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 / 395 S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 612D / 660V / 662S / 687A / 745T / 760W, 68E / 25 7V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W,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 / 787N257V / 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 / 291、 L / 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 / 5 62K / 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 / 687 A / 745T / 760W / 820T、257V / 275T / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 7 60W、257V / 291L / 378F / 381E / 395S / 402R / 474A / 475A / 478L / 515T / 526 S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 697L / 745T / 760W、257V / 291L / 395S / 402R / 469V / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W W / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 697L / 745T / 760W, 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 798I, 79R / 257V / 291L / 388V / 395S / 402R / 474A / 475A / 478L / 51 5T / 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, 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 / 760W257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 787M, 257V / 291L / 370V / 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 / 52 7A / 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 / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745S / 760W, 257V / 291L / 395S / 402R / 465Y / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、257V / 291L / 395S / 402R / 474A / 47 5A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 603M / 604H / 660V / 662S / 687A / 745T / 760W, 78N / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 52 7A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W, 257V / 291L / 317R / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 6 60V / 662S / 687A / 745T / 760W, 251A / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W257V / 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 / 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 / 824M 78V / 257V / 291L / 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, 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 / 760W257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 820E, 181Q / 257V / 291L / 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 / 760W257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 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, where 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 one set of mutations provided in Tables 10.1 and 10.2 relative to the reference sequence corresponding to SEQ ID NO:2.

[0167] In some embodiments, the amino acid sequence of the engineered DNA polymerase is 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, 68 / 78 / 147 / 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 / 370 / 395 / 402 / 474 / 47 5 / 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 / 559 / 603 / 604 / 612 / 660 / 662 / 687 515T / 526S / 527A / 528A / 536V / 559R / 603M / 604H / 612D / 68R / 147H / 251I / 257V / 291L / 395S / 402R / 474A / 475A / 478L / ... / 660V / 662S / 687A / 745T / 760W / 798I, 68R / 147H / 257V / 291L / 395S / 402R / 474A / 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、147H / 257V / 291L / 370V / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、147H / 251I / 2 57V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W / 798I, 68G / 147H / 257V / 291L / 370V / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 603M / 604H / 612D / 660V / 662S / 687A / 745T / 760W, 51T / 147H / 25 7V / 291L / 370V / 395S / 402R / 412R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 603M / 604H / 612D / 660V / 662S / 687A / 745T / 760W、1 47H / 257V / 291L / 395S / 402R / 412R / 474A / 475A / 478L / 515T / 526S / 527 A / 528A / 536V / 559R / 603M / 604H / 660V / 662S / 687A / 745T / 760W / 798I、1 47H / 251I / 257V / 291L / 370V / 395S / 402R / 412R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 612D / 660V / 662S / 687A / 745T / 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、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 / 251I / 257V / 291L / 370V / 395S / 402R / 474A / 475A / 478L / 508K / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W 78N / 14 7H / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 508K / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、78N / 147H / 2 57V / 291L / 370V / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528 A / 536V / 559R / 604H / 660V / 662S / 687A / 697L / 745T / 760W / 798I、51T / 1 47H / 251I / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 604H / 660V / 662S / 687A / 745T / 760W、68G / 147H / 257V / 291L / 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、7 8N / 147H / 257V / 291L / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 603M / 604H / 660V / 662S / 687A / 745T / 760W / 798I / 831G、147H / 257 V / 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 / 604 H / 660V / 662S / 687A / 745T / 760W / 798I, 68G / 78N / 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 or 51T / 68T / 147H / 251I / 257V / 291L / 370V / 395S / 402R / 474A / 475A / 478L / 515T / 526S / 527A / 528A / 536V / 559R / 603M / 604H / 660V / 662S / 687A / 745T / 760W, where 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 one set of mutations provided in Tables 11.1 and 11.2 relative to the reference sequence corresponding to SEQ ID NO:2.

[0168] 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 12-844 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 a reference sequence corresponding to residues 12-844 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.

[0169] 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 12-844 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 a reference sequence corresponding to residues 12-844 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.

[0170] In some embodiments, the amino acid sequence of the engineered DNA polymerase is at 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, 49 0, 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, 7 710, 720, 726, 730, 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: 220, 226, 272, 328, 546, 710, or 1208.

[0171] In some embodiments, the amino acid sequence of the engineered DNA polymerase has at least one mutation 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, 328L / V, 336E, 338V, 341H / T / Y, 342G, 351H, 356I / S, 359G / V, 362M / P / S, 365T, 368H, 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, 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 / M, 479L, 482S, 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, 5613H / 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, 704L / V, 706I / S / V, 707W, 708L, 713G, 714I, 715H / L, 716E / L, 7 18F / Q, 721A / M / N, 723Y, 724F / L, 725V, 728S, 731I / T, 732L / Q, 735L, 74 0S / 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 combinations thereof, wherein the positions are relative to a reference sequence corresponding to SEQ ID NOs: 220, 226, 272, 328, 546, 710, or 1208.

[0172] 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 12-844 of SEQ ID NO:220 or a reference sequence corresponding to SEQ ID NO:220, wherein the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 of SEQ ID NO:220 or a reference sequence corresponding to SEQ ID NO:220.

[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 12-844 of SEQ ID NO:220 or a reference sequence corresponding to an even-numbered SEQ ID NO:220, wherein the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 of SEQ ID NO:220.

[0174] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one mutation or set of mutations at amino acid position(s) 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 one 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 one 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.

[0175] 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 12-844 of SEQ ID NO:226 or a reference sequence corresponding to SEQ ID NO:226, wherein the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 of SEQ ID NO:226 or a reference sequence corresponding to SEQ ID NO:226.

[0176] 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 12-844 of SEQ ID NO:226 or a reference sequence corresponding to SEQ ID NO:226, wherein the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 of SEQ ID NO:226 or a reference sequence corresponding to SEQ ID NO:226.

[0177] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises amino acid position(s) 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 / 5 at least one mutation or set of mutations at 41 / 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, where 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 one mutation or set of mutations 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, 3 77C / 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 the reference sequence corresponding to SEQ ID NO: 226.In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one 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, G37 7C / 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.

[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 12-844 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 a reference sequence corresponding to residues 12-844 of SEQ ID NO:272 or a reference sequence corresponding to SEQ ID NO:272.

[0179] 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 12-844 of SEQ ID NO: 322-534 or a reference sequence corresponding to even-numbered SEQ ID NO: 322-534, and the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 of SEQ ID NO: 272 or a reference sequence corresponding to SEQ ID NO: 272.

[0180] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises amino acid position(s) 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, 257 / 377, 178 / 660, 257 / 377 / 402, 660, 178 / 257 / 377 / 402 / 403 / 4 83 / 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 / 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 / 541 / 660, 328, 740, 368, 721 / 745, 578, 687, 5 In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one mutation or set of mutations in: 41 / 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, where the positions are relative to a reference sequence corresponding to SEQ ID NO: 272.At least one mutation or set of mutations 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 / 40 3I / 483D / 604H / 660V, 377C, 178L / 402R / 403I / 528G / 541G / 660V, 377C / 601T / 660V, 178L / 377C, 377C / 490W / 660V, 402R / 403I / 483D / 554N / 557I, 178L / 2 57V / 377C / 558T / 660V, 257V / 402Y / 403I / 483D / 660V, 178L / 554N / 557I / 558 T / 660V, 554N / 557I / 660V, 178L / 483D / 541N / 545L / 554N / 557I / 604H / 660V, 7 92F, 402Y / 403I / 483D / 604H / 660V, 178L, 474A, 257V / 377C / 541D / 604H, 743 H, 396L, 743V, 396R, 479L, 377C / 403I / 541D / 604H, 475A, 178L / 257V / 403I / 528G, 448I, 577S, 395G, 769Q, 475V, 448C, 691W, 677L, 704L, 377C / 541N / 57 5W / 604H / 660V, 178L / 257V / 601T / 604H, 685R, 377C / 604H / 660V, 749M, 396F, 395H, 475S, 474C, 482S, 291L, 448V, 579Q, 178L / 541G / 550C / 604H, 477V, 17 8L / 257V, 257V / 402R / 490W / 528G / 541G / 660V, 328L, 395S, 740V, 483D / 660V, 257V / 377C, 579L, 368H, 792Y, 721A / 745T, 474A, 743H, 578A, 687A, 541D / 60 1T / 604H, 328V, 396L, 475Y, 395G, 257V / 528G, 257V / 528G / 554N, 768S, 257V,and 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 the reference sequence corresponding to SEQ ID NO: 272. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one mutation or set of mutations 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 / L 660V, A257V / G377C / G402Y, L660V, V178L / A257V / G377C / G402R / V403I / G483D / R604H / L660V, G377C, V178L / G4 02R / V403I / A528G / E541G / L660V, G377C / L601T / L660V, V178L / G377C, G377C / G490W / L660V, G402R / V403I / G48 3D / S554N / L557I, V178L / A257V / G377C / D558T / L660V, A257V / G402Y / V403I / G483D / L660V, V178L / S554N / L557 I / D558T / L660V, S554N / L557I / L660V, V178L / G483D / E541N / Q545L / S554N / L557I / R604H / L660V, L792F, G402Y / V403I / G483D / R604H / L660V, V178L, L474A, A257V / G377C / E541D / R604H, D743H, A396L, D743V, A396R, E479L,G377C / V403I / E541D / R604H, E475A, V178L / A257V / V403I / A528G, L448I, Q577S, P395G, A769Q, E475V, L448C, Q691W, N677 L, I704L, G377C / E541N / F575W / R604H / L660V, V178L / A257V / L601T / R604H, S685R, G377C / R604H / L660V, R749M, A396F, P3 95H, E475S, L474C, E482S, F291L, L448V, A579Q, V178L / E541G / S550C / R604H, E477V, V178L / A257V, A257V / G402R / G490W / A528G / E541G / L660V, E328L, P395S, Y740V, G483D / L660V, A257V / G377C, A579L, D368H, L792Y, T721A / A745T, L474A, D743H , T578A, H687A, E541D / L601T / R604H, E328V, A396L, E475Y, P395G, A257V / A528G, A257V / A528G / S554N, A768S, A257V, A25 7V, A396P, D770E, A257V / G377C / G483D / E541N, L542R, A257V / G402Y / V403I, A257V / V403I / E541N, L474G, R604H, G402Y / R 604H, Q691S, 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 the reference sequence corresponding to SEQ ID NO: 272.

[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 12-844 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 a reference sequence corresponding to residues 12-844 of SEQ ID NO: 328 or a reference sequence corresponding to SEQ ID NO: 328.

[0182] 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 12-844 of SEQ ID NO: 536-674 or a reference sequence corresponding to even-numbered SEQ ID NO: 536-674, wherein the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 of SEQ ID NO: 328 or a reference sequence corresponding to SEQ ID NO: 328.

[0183] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises amino acid position(s) 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 / 68 7, 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, 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 / 3 at least one mutation or set of mutations at 95 / 745, 185, 257 / 316 / 687, 291 / 316 / 475 / 687 / 745, 257 / 291 / 395 / 740 / 745, 450, 473, 124, 430, 413, 66 or 365, where 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 one mutation or 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, 395 S / 687A, 671W, 233K, 699V, 379G, 257V / 792F, 257V / 395S, 21A, 257V / 448C / 474A / 475S, 713G, 641A, 442G, 656S, 4 75S / 704V, 742K, 667W, 21N, 257V / 448I, 257V / 316G / 395H / 482S / 687A / 745T, 804R, 704V, 178L / 257V / 395S / 482S / 579L / 687A / 745T, 257V / 579L, 257V / 291L / 395S / 475V / 541N / 687A, 257V / 768S, 667S, 812S, 475A, 662S, 536V, 7 48G, 808W, 291L / 316G / 395H / 687A, 613H, 257V / 291L / 316G, 257V / 291L / 579L, 257V / 316G / 541N / 745T, 351H, 257 V / 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, where 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 one 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 / H68 7A, R671W, R233K, E699V, A379G, A257V / L792F, A257V / P395S, P21A, A257V / L448C / L474A / E475S, K713G, R641A, Q442G, W656S, E475 S / I704V, P742K, P667W, P21N, A257V / L448I, A257V / E316G / P395H / E482S / H687A / A745T, E804R, I704V, V178L / A257V / P395S / E482S / 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 / A745T, P351H, A 257V / 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, where the positions are relative to the reference sequence corresponding to SEQ ID NO: 328.

[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 12-844 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 a reference sequence corresponding to residues 12-844 of SEQ ID NO: 546 or a reference sequence corresponding to SEQ ID NO: 546.

[0185] 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 12-844 of 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 a reference sequence corresponding to residues 12-844 of SEQ ID NO: 546 or a reference sequence corresponding to SEQ ID NO: 546.

[0186] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises amino acid position(s) 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 / 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 / 56 2 / 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, 4 53 / 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、at least one mutation or set of mutations at 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, where the positions are relative to the reference sequence corresponding to SEQ ID NO: 546. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one mutation or set of mutations: 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 / 66 2S, 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 / 4 75A / 792F, 79R / 474A / 662S / 699V, 725V, 706I, 447R, 697L, 79R / 257V / 699V, 701M, 257V / 474A / 475A / 536V / 562K / 662S, 702 V, 342G / 716L, 233K / 562K / 713G, 704V, 474A / 475A / 536V / 562K / 662S, 454F, 327E / 427T, 257V / 316G / 474A / 536V / 792F, 427 D, 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, 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 / 662S, 79R / 178L / 257V / 662S / 699V, 79R / 257V / 536V / 562K / 662S, 562K / 699V, 359G / 613H / 742K / 808Y / 812S, 21N / 79A / 338V / 379G / 562K / 662S / 804R / 812S, 448V, 731I, 21N / 79R / 359G / 442G / 662S, 79A / 338V / 359V / 562K, 21N / 613H / 662S / 742K, 21A / 79A / 303T / 662S / 69 9V / 742K / 808Y、21A / 79A / 338V / 442G / 613H / 699V、79A / 303T / 338V / 562K / 61 3H / 662S、303T / 338V / 562K / 699V / 808W / 812S、79R / 257V、257V / 662S / 792F、 79R / 536V / 699V, 79A / 359G / 713G / 742K / 808W, 21A / 359G / 442G / 808W / 812S, 79R / 257V / 475A / 536V / 662S, 21N / 79A / 338V, 79A / 713G, 457R, 79A / 562K / 64 1A, 392C, 373T, 79R / 178L / 257V / 316G / 536V / 662S, 21N / 233K / 338V / 562K / 662S, 257V / 316G / 536V, 21N / 79R / 303T / 442G / 662S, 21N / 79R / 233K / 303T / 33 8V / 379G / 562K / 713G、338V / 442G / 699V、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, 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, 431R, 454G, 79R, 437S, 257V / 536V / 562K / 662S, 731T, 706V, 448T, 21A / 79A / 379G / 442G / 562K / 662S, 457Y, 362M, 410W, 45 4D、338V / 812S、21N / 79R / 338V、21A / 662S / 808Y / 812S、435K、21A / 303T / 338 V、378S、378C、427L、418K、338V / 442G、721N、449A、457W、449G、379G / 641A、 410V、431L、316G / 536V / 562K、257V / 536V / 792F、21N / 79R / 442G / 613H、536V 、257V / 536V / 662S / 792F、431V、362P、257V / 474A / 529S / 536V / 662S、382S、4 12V, 447S, 79R / 257V / 536V / 662S / 699V, 392T, 461S, 412Q, 257V / 316G / 475A / 536V / 792F, 438S, 388C, 380I / 454L, 447R, 21N / 79R / 233K / 303T / 613H / 699 V / 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 / 536 V / 662S / 699V、370S、417W、376F、393R、21N / 562K / 662S、79R / 257V / 262N / 53 6V / 562K / 699V、718Q、395S / 475A / 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, 447W79R / 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, 79R / 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, where the positions are relative to the reference sequence corresponding to SEQ ID NO: 546. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one 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 / E699V, V378F, A370V, R706S, A 257V / H395S / L474A / E475A / A536V / P662S, L474A / A536V / R562K, A257V / H395S / L474A / A536V, K79R / A257V / H395S / L474A / E475A / A536V / R562K, V378S, K79R / H395S / A536V / E699V, L372V, A257V / H395S / L792F, A390S, Q427L, K79R / L474A / A536V / E699V / L792F, G409S, E443A, L448V, D381E, Y707W, D381K, A457Y, A416S, L474A / E475A / P662S, K79R / H395S / L474A / E475 A / A536V / L792F, L474A / E475A / A536V / P662S, P382C, A257V / L474A / A536V / R562K / P662S, A257V / H395S / L474A / E475A / L7 92F, K79R / L474A / P662S / E699V, G725V, R706I, P447R, Y697L, K79R / A257V / E699V, E701M, A257V / L474A / E475A / A536V / R5 62K / 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, P21A / K79 A / G303T / R562K / E613H、A370S、A457N、K79R / R562K、E435P、A257V / A536V / E699V / L792F、K79R / A536V、A453S / P667T、K79A / G303T / A338V / E613H / E804 R / A812S, K79R / A257V / H395S / A536V / P662S / L792F, K79A / A338V / R562K, K79R / A257V / E699V, K79R / R233K / G303T / P662S, K79R / P662S / E699V, K79R / H395S / E475A / P662S, I704V, K79R / A536V / R562K / E699V, A536V / P662S, P21A / K79A / A338V / R641A / E699V, P662S, P21A / K79A / Q442G / E699V, K79A / G30 3T / P662S、K79R / V178L / A257V / P662S / E699V、K79R / A257V / A536V / R562K / P662S、R562K / E699V、K359G / E613H / P742K / E808Y / A812S、P21N / K79A / A33 8V / 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、G303T / A338V / R562K / E699V / E808W / A812S、K79R / A257V、A257V / P662S / L792F、K79R / A536V / E699V、K79A / K359G / K713G / P742K / E808W、P21A / K359G / Q442G / E808W / A812S、K79R / A257V / E475A / A536V / P662S, P21N / K79A / A338V, K79A / K713G, A457R, K79A / R562K / R641A, L392C, A373T, K79R / V178L / A257V / E316G / A536V / P66 2S, P21N / R233K / A338V / R562K / P662S, A257V / E316G / A536V, P21N / K79R / G303T / Q442G / P662S, P21N / K79R / R233K / G303T / A338V / A379G / 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, R715L, K79R / G 303T / A338V / R641A / K713G, L393W, L372Y, K79R / A257V / L474A / A536V / E699V, P431R, H454G, K79R, L437S, A257V / A536V / R562K / P662S, V731T, R706V L448T、P21A / K79A / A379G / Q442G / R562K / P662S、A457Y、R362M、E410W、H45 4D、A338V / A812S、P21N / K79R / A338V、P21A / P662S / E808Y / A812S、E435K、P2 1A / G303T / A338V、V378S、V378C、Q427L、R418K、A338V / Q442G、T721N、S449 A、A457W、S449G、A379G / R641A、E410V、P431L、E316G / A536V / R562K、A257V / A536V / L792F, P21N / K79R / Q442G / E613H, A536V, A257V / A536V / P662S / L792F, P431V, R362P, A257V / L474A / A529S / A536V / P662S, P382S, T412V, P447SK79R / A257V / A536V / P662S / E699V, L392T, R461S, T412Q, A257V / E316G / E4 75A / A536V / L792F, L438S, L388C, L380I / H454L, P447R, P21N / K79R / R233K / G303T / E613H / E699V / E809K, E724F, R461Q, P21A / K79R / A338V / E613H, K7 9R / V178L / A536V / L792F, R715H, A390T, S449G / R747S, S449V, L374S, R362 S, P21A / K79R / A379G / E613H / K713G, K79R / E316G / A536V / P662S / E699V, A3 70S, E417W, E376F, L393R, P21N / R562K / P662S, K79R / A257V / D262N / A536V / R562K / E699V, I718Q, H395S / E475A / A536V / R562K / P662S / E699V, S449L, L388M, K79R / R233K / A338V / K359G / A379G / Q442G / R562K / E613H / A812S, R2 33K, V371L, E376S, E316G / E475A / R562K / P662S / L792F, A702S, K79R / A257 V / E316G / L792F, A373G, H454W, R728S, P447W, K79R / R233K / A338V, L437P, R432T, K79R / V178L / A257V / E316G / H395S / A536V / P662S / E699V, K79R / G30 3T / K713G, I718F, A453L, Q427F, R233K / A379G / Q442G / R562K / E613H / P662 S / K713G, P21A / P742K, E732L, L392S, L388P, L393S, L437N, L392A, L393G, T412M, K79R / A257V / H395S / R562K / P662S / E699V, P21A / R233K / A338V / A379G / Q442G / R562K / E613H / P662S / A812S, Q427Y, E732Q, L429F, R562K / E699V, H2- / L374Y, E723Y, S434N, L388V, L420V, E699V, Q427I or E724L, and the positionrelative to the reference sequence corresponding to SEQ ID NO: 546.

[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 12-844 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 a reference sequence corresponding to residues 12-844 of SEQ ID NO: 710 or a reference sequence corresponding to SEQ ID NO: 710.

[0188] 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 12-844 of SEQ ID NO: 1192-1326 or a reference sequence corresponding to even-numbered SEQ ID NO: 1192-1326, wherein the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 of SEQ ID NO: 710 or a reference sequence corresponding to SEQ ID NO: 710.

[0189] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one 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. In some embodiments, the amino acid sequence of the engineered DNA polymerase contains at least one mutation or set 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, 412R / 434N / 427H ... 17W / 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 one 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 / E4 17W / Y697L, L798I, K79R / L388V, A469T, T651Q, D825E, V51T, G787M, A370V, T745 V, K815T, T745S, P465Y, Q603M, A78N, G317R, R251A, K815R, D68T, G569Q, C147A, A 341T, L824M, A78V, D68Q, R251L, K181R, N820E, K181Q, C147M, R275A, G569M, F735L, A341Y, A336E, A341H, K815L or K815S, where the positions are relative to the reference sequence corresponding to SEQ ID NO: 710.

[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 12-844 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 a reference sequence corresponding to residues 12-844 of SEQ ID NO: 1208 or a reference sequence corresponding to SEQ ID NO: 1208.

[0191] 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 12-844 of 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 a reference sequence corresponding to residues 12-844 of SEQ ID NO: 1208 or a reference sequence corresponding to SEQ ID NO: 1208.

[0192] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises amino acid position(s) 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, 78 / 508, 78 / 370 / 697 / 798, 5 and at least one mutation or set of mutations in: 1 / 251, 68 / 370 / 508 / 603 / 697, 78 / 251 / 370 / 697 / 798, 78 / 603 / 798 / 831, 370 / 412 / 508 / 612, 72 / 508 / 798, 51 / 251 / 412 / 798, 51 / 251 / 370 / 508 / 603 / 798, 68 / 78 / 251 / 370 / 603 / 612 / 697 or 51 / 68 / 251 / 370 / 603, wherein the positions are relative to the reference sequence corresponding to SEQ ID NO: 1208. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one 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, 78N / 37 and 0V / 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 reference sequence corresponding to SEQ 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 / A3 70V / T412R / Q603M / E612D, T412R / Q603M / L798I, R251I / A370V / T412R / E612D, A370V / L798I, A 78N / R251I, R251I, D68R / A370V / E508K / L798I, R251I / A370V / E508K, A78N / E508K, A78N / A370 V / Y697L / L798I, V51T / R251I, D68G / A370V / E508K / Q603M / Y697L, A78N / R251I / A370V / Y697L / L798I, A78N / Q603M / L798I / E831G, A370V / T412R / E508K / E612D, V72A / E508K / L798I, V51T / R2 51I / T412R / L798I, V51T / R251I / A370V / E508K / Q603M / L798I, D68G / A78N / R251I / A370V / Q603M / E612D / Y697L or V51T / D68T / R251I / A370V / Q603M, where the positions are relative to the reference sequence corresponding to SEQ ID NO: 1208.

[0193] 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.

[0194] In some embodiments, the amino acid sequence of the engineered DNA polymerase 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 relative to a reference sequence corresponding to SEQ ID NO: 2, 220, 226, 272, 328, 546, 710 or 1208.

[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 an amino acid sequence comprising 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.

[0196] In some embodiments, the engineered DNA polymerase comprises a sequence comprising residues 12-844 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 12-844 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. The amino acid sequence has 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 comprising the engineered DNA polymerase set forth in any of the preceding paragraphs 8.1, 8.2, 9.1, 9.2, 9.3, 10.1, 10.2, 11.1 and 11.2.

[0197] 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 sequence corresponding to residues 12-844 of an even-numbered SEQ ID NO:4-1378 or a sequence corresponding to an even-numbered SEQ ID NO:2-1378.

[0198] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence comprising residues 12-844 of an even-numbered SEQ ID NO:4-1378 or an amino acid sequence comprising an even-numbered 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, 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, substitutions include non-conservative substitutions, hi some embodiments, guidance regarding non-conservative and conservative substitutions is provided by the variants disclosed herein.

[0199] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence comprising residues 12 to 844 of SEQ ID NOs: 4-220, 222-268, 270-320, 322-538, 540-674, 676-1190, 1192-1326, or an even-numbered SEQ ID NO: 1328-1378, or an amino acid sequence comprising SEQ ID NOs: 2-220, 222-268, 270-320, 322-538, 540-674, 676-1190, 1192-1326, and an even-numbered SEQ ID NO: 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 substitutions comprise non-conservative substitutions or conservative substitutions. In some embodiments, the substitutions comprise conservative substitutions. In some embodiments, the substitutions comprise non-conservative substitutions.

[0200] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence comprising residues 12-844 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, 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.

[0201] In some embodiments, the engineered DNA polymerase is provided as a fusion protein. In some embodiments, the engineered DNA polymerases described herein can be fused to various polypeptide sequences, such as, by way of example and not limitation, polypeptide tags that can be used for detection and / or purification. In some embodiments, the engineered DNA polymerase fusion protein comprises a glycine-histidine or histidine-tag (His-tag). In some embodiments, the engineered DNA polymerase fusion protein comprises an epitope tag, such as c-myc, FLAG, V5, or hemagglutinin (HA). In some embodiments, the engineered DNA polymerase fusion protein comprises a GST, SUMO, Strep, MBP, or GFP tag. In some embodiments, the fusion is to the amino (N-) terminus of the engineered DNA polymerase polypeptide. In some embodiments, the fusion is to the carboxy (C-) terminus of the engineered DNA polymerase polypeptide.

[0202] In some embodiments, the engineered DNA polymerases of the present disclosure have DNA polymerase activity, hi some embodiments, the engineered DNA polymerase has at least one improved or enhanced property compared to a reference DNA polymerase.

[0203] In some embodiments, the engineered DNA polymerase has increased activity compared to a reference DNA polymerase, ie, the engineered DNA polymerase has at least about a 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 increased activity over the reference DNA polymerase.

[0204] In some embodiments, the engineered DNA polymerase has increased stability compared to the reference DNA polymerase, hi some embodiments, the engineered DNA polymerase has increased thermostability compared to the reference DNA polymerase.

[0205] In some embodiments, the engineered DNA polymerase has increased processivity compared to the reference DNA polymerase, hi some embodiments, the engineered DNA polymerase has increased fidelity compared to the reference DNA polymerase.

[0206] In some embodiments, the engineered DNA polymerase has increased input DNA template sensitivity compared to a reference DNA polymerase. In some embodiments, the engineered DNA polymerase has increased product yield, for example, in a PCR reaction, compared to a reference DNA polymerase. In some embodiments, the engineered DNA polymerase has 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 input target DNA compared to a reference DNA polymerase (e.g., as provided in the Examples).

[0207] In some embodiments, the engineered DNA polymerase has increased resistance or tolerance to an inhibitor(s), such as guanidine isothiocyanate (GITC), compared to a reference DNA polymerase.

[0208] In some embodiments, the reference DNA polymerase comprises a sequence corresponding to residues 12-844 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 12-844 of SEQ ID NO:2, or an amino acid sequence corresponding to SEQ ID NO:2.

[0209] In some embodiments, the engineered DNA polymerase has one or more improved properties 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 inhibitor(s), or any combination of i), ii), iii), vi), v), vi), vii), and viii), relative to a reference DNA polymerase. In some embodiments, the reference DNA polymerase has an amino acid sequence corresponding to residues 12-844 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 12-844 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.

[0210] 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.

[0211] In some embodiments, the present disclosure further provides functional or biologically active fragments of the engineered DNA polymerase polypeptides described herein. Thus, for each and every embodiment of an engineered DNA polymerase herein, a functional or biologically active fragment of the engineered DNA polymerase is provided herein. In some embodiments, a functional or biologically active fragment of an engineered DNA polymerase comprises 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 the DNA polymerase polypeptide (i.e., the parent DNA polymerase) from which it is derived. In some embodiments, a functional or biologically active fragment comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the DNA polymerase. In some embodiments, the functional fragment is truncated by fewer than 5, fewer than 10, fewer than 15, fewer than 10, fewer than 25, fewer than 30, fewer than 35, fewer than 40, fewer than 45, fewer than 50 amino acids, fewer than 55 amino acids, fewer than 60 amino acids, fewer than 65 amino acids, or fewer than 70 amino acids.

[0212] In some embodiments, functional or biologically active fragments of the engineered DNA polymerase polypeptides described herein comprise at least one mutation or set of mutations in the amino acid sequence of an engineered DNA polymerase described herein, and thus, in some embodiments, functional or biologically active fragments of the engineered DNA polymerase exhibit enhanced or improved properties relative to the mutation or set of mutations in the parent DNA polymerase.

[0213] Polynucleotides encoding engineered polypeptides, expression vectors and host cells In another aspect, the present disclosure provides a recombinant polynucleotide encoding the engineered DNA polymerase described herein. In some embodiments, the recombinant polynucleotide is operably linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide construct capable of expressing the DNA polymerase. In some embodiments, an expression construct comprising at least one heterologous polynucleotide encoding an engineered DNA polymerase polypeptide(s) is introduced into a suitable host cell to express the corresponding DNA polymerase polypeptide(s).

[0214] As will be apparent to those skilled in the art, the availability of protein sequences and knowledge of the codons corresponding to various amino acids provides a description of all polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, in which the same amino acid is coded for by alternative or synonymous codons, allows for an extremely large number of nucleic acids to be generated, all of which encode the engineered DNA polymerases of the present disclosure. Thus, the present disclosure provides methods and compositions for the production of each and every possible variation of polynucleotides that can be made that encode the engineered DNA polymerase polypeptides described herein by selecting combinations based on possible codon choices, and all such variations of polynucleotides should be considered 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 Sequence Listing.

[0215] In some embodiments, codons are preferably optimized for utilization by a selected host cell for protein production. In some embodiments, bacterially preferred codons are used for expression in bacteria. In some embodiments, fungal cell preferred codons are used for expression in fungi. In some embodiments, mammalian cell preferred codons are used for expression in mammalian cells. In some embodiments, codon-optimized polynucleotides encoding engineered DNA polymerase polypeptides described herein contain preferred codons at about 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90% of the codon positions in the full-length coding region.

[0216] In some embodiments, the recombinant polynucleotide has a sequence similar to or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108% or 109% of the sequence corresponding to a reference sequence corresponding to residues 12-844 of SEQ ID NO: 2, 220, 226, 272, 328, 546, 710, or 1208. The present invention also includes polynucleotide sequences encoding engineered DNA polymerases, including amino acid sequences with 8%, 99% or more sequence identity, wherein the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 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.

[0217] 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 the reference sequence corresponding to residues 12-844 of SEQ ID NO:2 or the 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 12-844 of SEQ ID NO:2 or the reference sequence corresponding to SEQ ID NO:2.

[0218] 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 12-844 of SEQ ID NO:2 or 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 12-844 of SEQ ID NO:2.

[0219] 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 12-844 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 12-844 of SEQ ID NO:2, or a reference sequence corresponding to SEQ ID NO:2.

[0220] In some embodiments, the recombinant polynucleotide comprises a sequence identical to that at 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, 38 0, 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.

[0221] 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 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.

[0222] 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 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.

[0223] 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 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.

[0224] 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 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.

[0225] In some embodiments, the recombinant polynucleotide comprises a sequence encoding an amino acid sequence at amino acid position(s) 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 / 516 / 541, 478 / 541, 288 / 399 / 514 / 55 4 / 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, 478 / 514 / 516 / 52 6 / 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 / 529 / 541, 478 / 514 / 51 6 / 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 / 55 0, 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 / 51 6 / 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 / 55 0 / 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 / 55 7, 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 / 55 4 / 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 / 541, 288 / 399 / 478 / 514 / 554 / 557, 288 / 399 / 514 / 516 / 528, 478 / 554, 399 / 514 / 5 and a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least one mutation or set of mutations in: 16, 514, 399 / 550 / 554, 288 / 478 / 514 / 528 / 541 / 554, 478 / 514 / 516 / 554, 478 / 514 / 541, 288 / 541 / 554 / 557, 698, or 478 / 514 / 526 / 527 / 528, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0226] In some embodiments, the recombinant polynucleotide comprises a sequence identical to or identical to 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, 402 / 478 / 514 / 526 / 527 / 528, 398 / 478 / 514 / 526 / 527 / 528, 478 / 514 / 526 / 527 / 528 / 758, 399 / 478 / 514 / 526 / 527 / 5 28, 478 / 514 / 526 / 527 / 528 / 575, 257 / 478 / 514 / 526 / 527 / 528 / 758, 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.

[0227] 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 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.

[0228] 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 or set of mutations provided in Tables 7.1 and 7.2, the positions being 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 one mutation or set of mutations provided in Tables 8.1 and 8.2, wherein the position is relative to a reference sequence corresponding 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 one mutation or set of mutations provided in Tables 9.1, 9.2, and 9.3, wherein the position is relative to a reference sequence corresponding 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 one mutation or set of mutations provided in Tables 10.1 and 10.2, wherein the position is 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 one mutation or set of mutations provided in Tables 11.1 and 11.2, wherein the position is relative to a reference sequence corresponding to SEQ ID NO:2.

[0233] In some embodiments, the recombinant polynucleotide has a sequence similar to or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108% or more similar to a reference sequence corresponding to residues 12-844 of SEQ ID NO: 220, 226, 272, 328, 546, 710, or 1208. and polynucleotide sequences encoding engineered DNA polymerases comprising an amino acid sequence with 8%, 99% or more sequence identity, wherein the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 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.

[0234] 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 12-844 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 a reference sequence corresponding to residues 12-844 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.

[0235] In some embodiments, the recombinant polynucleotide comprises a sequence identical to that at 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, 7 31, 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 a reference sequence of SEQ ID NO: 220, 226, 272, 328, 546, 710, or 1208.

[0236] 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 12-844 of SEQ ID NO: 220 or 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 12-844 of SEQ ID NO: 220 or a reference sequence corresponding to SEQ ID NO: 220.

[0237] 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 12-844 of SEQ ID NO:220 or a reference sequence corresponding to an even-numbered SEQ ID NO:220, wherein the amino acid sequence comprises one or more mutations relative to the reference sequence corresponding to residues 12-844 of SEQ ID NO:220.

[0238] 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 or set of mutations at amino acid position(s) 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.

[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 12-844 of SEQ ID NO: 226 or 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 12-844 of SEQ ID NO: 226 or a reference sequence corresponding to SEQ ID NO: 226.

[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 12-844 of SEQ ID NO:226 or a reference sequence corresponding to SEQ ID NO:226, wherein the amino acid sequence comprises one or more mutations relative to a reference sequence corresponding to residues 12-844 of SEQ ID NO:226.

[0241] In some embodiments, the recombinant polynucleotide comprises a sequence identical to that of amino acid position(s) 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

[0023] The present invention also includes a polynucleotide encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least one mutation or set of mutations in 514 / 515 / 541 / 559, 514 / 515 / 541 / 559, 514 / 515 / 559, 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.

[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 12-844 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 12-844 of SEQ ID NO:272 or a reference sequence corresponding to SEQ ID NO:272.

[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 12-844 of 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 12-844 of SEQ ID NO: 272, or the reference sequence corresponding to SEQ ID NO: 272.

[0244] In some embodiments, the recombinant polynucleotide comprises a sequence encoding an amino acid sequence at amino acid position(s) 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, 257 / 377, 178 / 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, 792, 402 / 403 / 483 / 604 / 660, 178, 474, 257 / 377 / 541 / 604, 743, 396, 479, 377 / 40 3 / 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, and a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least one mutation or set of mutations in 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.

[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 12-844 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 12-844 of SEQ ID NO: 328 or a reference sequence corresponding to SEQ ID NO: 328.

[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 12-844 of 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 12-844 of SEQ ID NO: 328, or the reference sequence corresponding to SEQ ID NO: 328.

[0247] In some embodiments, the recombinant polynucleotide comprises a sequence encoding an amino acid sequence at amino acid position(s) 475 / 704, 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, 69 9, 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, 704, 178 / 257 / 395 / 482 / 579 / 687 / 745, 257 / 579, 257 / 29 1 / 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 / 4 The polynucleotide sequence encoding an engineered DNA polymerase comprises an amino acid sequence comprising at least one mutation or set of mutations at 75 / 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.

[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 12-844 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 12-844 of SEQ ID NO: 546 or a reference sequence corresponding to SEQ ID NO: 546.

[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 12-844 of 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 12-844 of SEQ ID NO: 546, or the reference sequence corresponding to SEQ ID NO: 546.

[0250] In some embodiments, the recombinant polynucleotide comprises a sequence encoding an amino acid sequence at amino acid position(s) 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 / 5 36 / 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 / 42 7, 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 / 6 99, 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 / 8 12, 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、and a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least one mutation or set of mutations at 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.

[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 12-844 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 12-844 of SEQ ID NO: 710.

[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 12-844 of 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 a reference sequence corresponding to residues 12-844 of SEQ ID NO: 710, or a reference sequence corresponding to SEQ ID NO: 710.

[0253] 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 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.

[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 the reference sequence corresponding to residues 12-844 of SEQ ID NO: 1208 or the 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 12-844 of SEQ ID NO: 1208 or the reference sequence corresponding to SEQ ID NO: 1208.

[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 12-844 of 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 12-844 of SEQ ID NO: 1208, or the reference sequence corresponding to SEQ ID NO: 1208.

[0256] In some embodiments, the recombinant polynucleotide comprises a sequence encoding an amino acid sequence at amino acid position(s) 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, 78 / 508, 78 / 370 / 697 / 798, 51 / 251, 68 / 370 / 508 / 603 / 697, 78

[0023] The present invention also includes a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least one mutation or set of mutations in: 51 / 251 / 370 / 697 / 798, 78 / 603 / 798 / 831, 370 / 412 / 508 / 612, 72 / 508 / 798, 51 / 251 / 412 / 798, 51 / 251 / 370 / 508 / 603 / 798, 68 / 78 / 251 / 370 / 603 / 612 / 697, or 51 / 68 / 251 / 370 / 603, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 1208.

[0257] In some embodiments, for each of the foregoing embodiments of recombinant polynucleotides, the specific amino acid mutations described herein for each mutation or set of mutations may be used in the encoded engineered DNA polymerase polypeptide.

[0258] 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 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, wherein the amino acid positions are relative to a reference sequence of SEQ ID NO: 2, 220, 226, 272, 328, 546, 710 or 1208.

[0259] 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 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.

[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 an amino acid sequence comprising 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.

[0261] In some embodiments, the recombinant polynucleotide comprises a sequence comprising residues 12 to 844 of an engineered DNA polymerase set forth 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 set forth 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 The present invention also includes a polynucleotide sequence encoding an engineered DNA polymerase, which 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 sequence comprising the engineered DNA polymerase set forth in 10.1, 10.2, 11.1 and 11.2.

[0262] 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 12-844 of an even-numbered SEQ ID NO:4-1378, or a sequence corresponding to an even-numbered SEQ ID NO:2-1378.

[0263] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising residues 12-844 of an even-numbered SEQ ID NO:4-1378 or an amino acid sequence comprising an even-numbered SEQ ID NO:2-1378. In some embodiments, the encoded 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 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 substitutions comprise non-conservative substitutions or conservative substitutions. In some embodiments, the substitutions comprise conservative substitutions. In some embodiments, the substitutions comprise non-conservative substitutions.

[0264] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising residues 12-844 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, or 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.

[0265] 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 34-2532 of an odd-numbered SEQ ID NO: 1-1377, or a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO: 1-1377, and the recombinant polynucleotide encodes a DNA polymerase.

[0266] In some embodiments, the recombinant polynucleotide comprises 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 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 SEQ ID NO: 1, 219, 225, 271, 327, 545, 709, or 1207, and the recombinant polynucleotide encodes a DNA polymerase.

[0267] 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: 1-1377 or a polynucleotide sequence comprising an odd-numbered SEQ ID NO: 1-1377.

[0268] 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.

[0269] In some embodiments, the present disclosure provides recombinant polynucleotides that can hybridize under highly stringent conditions to reference polynucleotides encoding the engineered DNA polymerase polypeptides 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 highly stringent conditions to the reference polynucleotide sequences described herein that encode the engineered DNA polymerase. In some embodiments, the recombinant polynucleotide hybridizes under highly stringent 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 highly stringent conditions to a reference polynucleotide corresponding to nucleotide residues 34 to 2532 of an odd-numbered SEQ ID NO: 1-1377, or a reference polynucleotide comprising a sequence corresponding to an odd-numbered SEQ ID NO: 1-1377, or a reverse complement thereof.

[0270] In some embodiments, a polynucleotide capable of hybridizing under highly stringent 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 set forth 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, polynucleotides that hybridize under highly stringent conditions comprise a reference sequence corresponding to residues 34 to 2532 of SEQ ID NO: 1, 219, 225, 271, 327, 545, 709, or 1207, or a polynucleotide sequence having 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 SEQ ID NO: 1, 219, 225, 271, 327, 545, 709, or 1207. In some additional embodiments, polynucleotides that hybridize under highly stringent conditions are 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 the 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. 9.1, 9.2, 9.3, 10.1, 10.2, 11.1 and 11.2.

[0271] In some embodiments, a recombinant polynucleotide encoding any of the DNA polymerases herein is manipulated in various ways to facilitate expression of the DNA polymerase polypeptide. In some embodiments, the recombinant polynucleotide encoding the DNA polymerase comprises an expression vector in which one or more control sequences are present to regulate expression of the DNA polymerase polynucleotide and / or the encoded polypeptide. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector utilized. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. In some embodiments, control sequences include, among others, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators.

[0272] In some embodiments, a suitable promoter is selected based on the choice of host cell. For bacterial host cells, promoters suitable for directing transcription of the nucleic acid constructs of the present disclosure include the E. coli lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyM), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyL), the Bacillus amyloliquefaciens alpha-am ...L), the Bacillus amyloliquefaciens alpha-amylase gene (amyM), the Bacillus amyloliquefaciens alpha Examples of promoters that can be used include, but are not limited to, promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase genes (see, e.g., Villa-Kamaroff et al., Proc. Natl. Acad. Sci. USA, 1978, 75:3727-3731), and the tac promoter (see, e.g., DeBoer et al., Proc. Natl. Acad. Sci. USA, 1983, 80:21-25).Exemplary promoters for filamentous fungal host cells include 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 protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum Examples of promoters include, but are not limited to, promoters obtained from the gene for A. oxysporum trypsin-like protease (see, e.g., WO 96 / 00787) and the NA2-tpi promoter (a hybrid of promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), as well as mutants, truncations, and hybrid promoters thereof. Exemplary yeast cell promoters can be derived 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, for example, Romanos et al., Yeast, 1992, 8:423-488).

[0273] In some embodiments, the control sequence is a suitable transcription terminator sequence (i.e., a sequence recognized by a host cell to terminate transcription). In some embodiments, the terminator 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 host cell of choice finds use in the present invention. For bacterial expression, the transcription terminator can be a Rho-dependent terminator that relies on Rho transcription factors, or a Rho-independent or endogenous terminator that does not require transcription factors. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (see, e.g., Romanos et al., supra).

[0274] In some embodiments, the control sequence is a suitable leader sequence (i.e., a nontranslated region of an mRNA important for translation by the host cell). In some embodiments, the leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the DNA polymerase polypeptide. Any suitable leader sequence that is functional in the host cell of choice finds use in the present invention. Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0275] In some embodiments, the regulatory sequence is a polyadenylation sequence (i.e., a sequence operably linked to the 3' end of a nucleic acid sequence that, upon transcription, is recognized by a host cell as a signal for the addition of polyadenosine residues to the transcribed mRNA). Any suitable polyadenylation sequence that is functional in the selected host cell finds use in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are known (see, e.g., Guo and Sherman, Mol. Cell. Biol., 1995, 15:5983-5990).

[0276] In some embodiments, a control sequence is also a signal peptide (i.e., a coding region that encodes an amino acid sequence linked to the amino terminus of a polypeptide that directs the encoded polypeptide into the secretory pathway of a cell). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted 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 into the secretory pathway of a selected host cell finds use for expressing an engineered polypeptide(s). Useful signal peptide coding regions for bacterial host cells include, but are not limited to, those obtained from the genes for Bacillus NClB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional 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, signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase.Useful signal peptides for yeast host cells include, but are not limited to, those derived from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase.

[0277] In some embodiments, the control sequence is a propeptide coding region that encodes an amino acid sequence positioned at the amino terminus of a polypeptide. The resulting polypeptide is referred to as a "proenzyme," "propolypeptide," or "zymogen." A propolypeptide can be converted to a mature, active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region can be obtained from any suitable source, including, but not limited to, the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase (see, e.g., WO 95 / 33836). When both a signal peptide and a propeptide region are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of the polypeptide, and the signal peptide region is positioned next to the propeptide region.

[0278] In some embodiments, regulatory sequences are also utilized. These sequences facilitate regulation of polypeptide expression relative to the growth of the host cell. Examples of regulatory systems are those that turn gene expression on or off in response to chemical or physical stimuli, including the presence of a regulatory compound. 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 alpha-amylase promoter, the Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter.

[0279] In another aspect, the disclosure provides recombinant expression vectors that include a recombinant polynucleotide encoding an engineered DNA polymerase polypeptide and, depending on the type of host into which it will be introduced, one or more expression control regions, e.g., a promoter and terminator, an origin of replication, etc. In some embodiments, the various nucleic acid and control sequences described herein are joined together (i.e., operably linked) to produce a recombinant expression vector that includes one or more convenient restriction sites to allow for insertion or substitution of a nucleic acid sequence encoding a DNA polymerase polypeptide at such sites. Alternatively, in some embodiments, a nucleic acid sequence of the invention is expressed by inserting the nucleic acid sequence or a nucleic acid construct containing the sequence into an appropriate vector for expression. In some embodiments involving the creation of an expression vector, a coding sequence is placed within the vector such that the coding sequence is operably linked to appropriate control sequences for expression.

[0280] In embodiments herein, the recombinant expression vector may be any suitable vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and result in expression of a polynucleotide encoding a DNA polymerase. The choice of vector typically depends on the compatibility of the vector with a host cell into which the vector will be introduced. The vector may be a linear or closed circular plasmid.

[0281] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome, the replication of which is independent of chromosomal replication). The vector can contain any means for ensuring self-replication. In some alternative embodiments, the vector is one that, when introduced into a host cell, integrates into the genome and is replicated together with the chromosome(s) into which it has been integrated. Furthermore, some embodiments utilize a single vector or plasmid, or two or more vectors or plasmids and / or transposons that together contain the total DNA to be introduced into the genome of the host cell.

[0282] In some embodiments, the expression vector contains one or more selectable markers to allow easy selection of transformed cells. A "selectable marker" is a gene the product of which provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc. 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 A. nidulans or A. orzyae), 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 A. nidulans or A. oryzae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase) and equivalents thereof.

[0283] 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 invention, wherein the recombinant polynucleotide(s) is / are operably linked to one or more control sequences for expression of the engineered DNA polymerase enzyme(s) in the host cell. Host cells suitable for use in expressing polypeptides encoded by the expression vectors of the invention are well known in the art and include, but are not limited to, bacterial cells such as E. 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 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 Escherichia coli strains (e.g., W3110(ΔfhuA) and BL21).

[0284] Thus, in another aspect, the disclosure provides methods of producing an engineered DNA polymerase polypeptide, the methods comprising culturing a host cell capable of expressing a polynucleotide encoding the engineered DNA polymerase polypeptide under conditions suitable for expression or production of the encoded polypeptide. In some embodiments, the methods further comprise step(s) of isolating the DNA polymerase polypeptide, such as from the medium and / or the host cell. In some embodiments, the methods further comprise purifying the DNA polymerase polypeptide, as described herein. In some embodiments, the host cell produces more than one engineered DNA polymerase polypeptide.

[0285] Suitable culture media and growth conditions for host cells are well known in the art. Any suitable method for introducing a polynucleotide into cells for expression of a DNA polymerase polypeptide is intended to find use in the present invention. Suitable techniques include, but are not limited to, electroporation, gene gun method, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0286] Recombinant polypeptides (e.g., DNA polymerase enzyme variants) herein can be produced using any suitable method known in the art. For example, there are a wide variety of different mutagenesis techniques well known to those of skill in the art. In addition, mutagenesis kits are available from many commercial molecular biology suppliers. Methods are available for making specific substitutions at defined amino acids (site-directed), specific or random mutations in localized regions of a gene (position-directed), or random mutagenesis throughout a gene (e.g., saturation mutagenesis). Numerous suitable methods for generating enzyme variants are known to those of skill in the art, including, but not limited to, site-directed mutagenesis of single- or double-stranded DNA using PCR, cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, and chemical saturation mutagenesis, or any other suitable method known in the art. Non-limiting examples of methods used in 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 produced, they can be screened for any desired property (e.g., high or increased activity or low or reduced activity, increased thermal activity, increased stability, increased processivity, increased fidelity, increased inhibitor resistance or tolerance, and / or pH stability, etc.).

[0287] In some embodiments, engineered DNA polymerase polypeptides having the properties disclosed herein can be obtained by subjecting a polynucleotide encoding a naturally occurring or engineered DNA polymerase polypeptide to any suitable mutagenesis and / or directed evolution method 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. Pat. No. 6,537,746). Other directed evolution procedures that can be used include, among others, 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).

[0288] 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 method finds use in the present invention and is known in the art (e.g., U.S. Pat. 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, 6,309,883, 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, 6,3 91,552, 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, 6,573,098, 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-US 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; European Patent No. 3049973; International Publication No. WO 95 / 22625; International Publication No. WO 97 / 0078; International Publication No. WO 97 / 35966; International Publication No. WO 98 / 27230; International Publication No. WO 00 / 42651; International Publication No. WO 01 / 75767; International Publication No. WO 2009 / 152336; and International Publication No. WO 2015 / 048573, all of which are incorporated herein by reference.

[0289] In some embodiments, clones expressing enzyme variants obtained after mutagenesis treatment are screened by subjecting enzyme preparations to defined treatment or assay conditions (e.g., temperature, pH, input template concentration, nucleotides, etc.) and measuring the amount of enzyme activity remaining after treatment or other suitable assay conditions. Clones containing polynucleotides encoding DNA polymerase polypeptides are then isolated from the gene, sequenced to identify nucleotide sequence changes (if any), and used to express the enzyme in host cells. Measurement of enzyme activity from an expression library can be performed using any suitable method known in the art and as described in the Examples.

[0290] For engineered polypeptides of known sequence, polynucleotides encoding the enzymes can be prepared by standard solid-phase methods according to known synthesis methods. In some embodiments, fragments of up to about 100 bases can be synthesized individually and then joined (e.g., by enzymatic or chemical ligation methods, or polymerase-mediated methods) to form any desired contiguous sequence. For example, the polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using the classical phosphoramidite method (see, e.g., Beaucage et al., Tet. Lett., 1981, 22:1859-69; and Matthes et al., EMBO J., 1984, 3:801-05), as typically performed in automated synthesis methods. According to the phosphoramidite method, oligonucleotides are synthesized (e.g., in an automated DNA synthesizer, purified, annealed, ligated, and cloned into an appropriate vector).

[0291] In some embodiments, methods for preparing an engineered DNA polymerase polypeptide ...

Claims

1. at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more relative to a reference sequence corresponding to residues 12-844 of SEQ ID NO: 2, 220, 226, 272, 328, 546, 710, or 1208 or higher sequence identity, 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, 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.

2. 10. 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 12-844 of SEQ ID NO:2 or 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 12-844 of SEQ ID NO:2 or a reference sequence corresponding to SEQ ID NO:

2.

3. 10. 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 12-844 of SEQ ID NO:2 or 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 12-844 of SEQ ID NO:2 or a reference sequence corresponding to SEQ ID NO:

2.

4. 10. 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 12-844 of 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 12-844 of SEQ ID NO:2 or a reference sequence corresponding to SEQ ID NO:

2.

5. The amino acid sequence of the engineered DNA polymerase is shown at 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, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435 80, 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, 5 08, 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, 7 5. The engineered DNA polymerase of any one of claims 1-4, wherein the engineered DNA polymerase comprises at least one mutation at position 16, 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, and the positions are relative to the reference sequence corresponding to SEQ ID NO:

2.

6. The amino acid sequence of the engineered DNA polymerase has at least one mutation: 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, 336E, 338V, 341H / T / Y, 342G, 351H, 356I / S, 359G / V, 362M / P / S, 365T, 368H, 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, 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, 550C / 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, 704L / 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 6. The engineered DNA polymerase of any one of claims 1 to 5, wherein the engineered DNA polymerase comprises a nucleotide sequence selected from the group consisting of nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 80, 80, 80, 80, 80, 81, 81, 82, 83, 84, 85, 86, 87, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 110, 111, 112, 11

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

2.

9. 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 one 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. 10. The engineered DNA polymerase of any one of claims 1 to 5 and 9, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one mutation 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 position is relative to a reference sequence corresponding to SEQ ID NO:

2.

11. The amino acid sequence of the engineered DNA polymerase is shown at 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 / 516 / 541, 478 / 541, 288 / 399 / 514 / 554 / 55 7, 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, 478 / 514 / 516 / 526 / 5 27 / 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 / 529 / 541, 478 / 514 / 516 / 5 41, 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 / 5 14 / 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 / 541, 288 / 399 / 478 / 514 / 554 / 557, 288 / 399 / 514 / 516 / 528, 478 / 554, 3 6. The engineered DNA polymerase of any one of claims 2 to 5, comprising at least one mutation or set of mutations at 99 / 514 / 516, 514, 399 / 550 / 554, 288 / 478 / 514 / 528 / 541 / 554, 478 / 514 / 516 / 554, 478 / 514 / 541, 288 / 541 / 554 / 557, 698 or 478 / 514 / 526 / 527 / 528, the positions being relative to a reference sequence corresponding to SEQ ID NO:

2.

12. 10. 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 12-844 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 12-844 of SEQ ID NO: 220, 226, 272, 328, 546, 710, or 1208.

13. 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 12-844 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 12-844 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.

14. The amino acid sequence of the engineered DNA polymerase is shown at 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, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435 81, 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, 5 16, 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, 7 25, 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, and the position is relative to a reference sequence corresponding to SEQ ID NO: 220, 226, 272, 328, 546, 710, or 1208.

15. The amino acid sequence of the engineered DNA polymerase has at least one mutation 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, 328L / V, 336E, 338V, 341H / T / Y, 342G, 351H, 356I / S, 359G / V, 362M / P / S, 365T, 368H, 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, 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 / M, 479L, 482S, 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, 558G / 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, 697A / 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, 7 24F / L, 725V, 728S, 731I / T, 732L / Q, 735L, 740S / V, 742K, 743H / V, 745S / T / 15. The engineered DNA polymerase of any one of claims 12-14, comprising a nucleotide sequence selected from the group consisting of 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 position is relative to a reference sequence corresponding to SEQ ID NO: 220, 226, 272, 328, 546, 710, or 1208.

16. 13. 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 12-844 of SEQ ID NO:220, or 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 12-844 of SEQ ID NO:220, or a reference sequence corresponding to SEQ ID NO:

220.

17. 14. 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 12-844 of SEQ ID NO:220 or a reference sequence corresponding to even-numbered SEQ ID NO:

220.

18. 18. The engineered DNA polymerase of claim 16 or 17, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one mutation or set of mutations at amino acid position(s) 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. 13. 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 12-844 of SEQ ID NO:226, or 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 12-844 of SEQ ID NO:226, or a reference sequence corresponding to SEQ ID NO:

226.

20. 14. 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 12-844 of SEQ ID NO:226 or a reference sequence corresponding to SEQ ID NO:

226.

21. The amino acid sequence of the engineered DNA polymerase may be selected from the amino acid position(s) 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 / 51 21. The engineered DNA polymerase of claim 19 or 20, comprising one or a set of mutations at 4 / 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. 13. 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 12-844 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 12-844 of SEQ ID NO:272, or a reference sequence corresponding to SEQ ID NO:

272.

23. 14. 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 12-844 of SEQ ID NO:322-534 or a reference sequence corresponding to 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 12-844 of SEQ ID NO:272 or the reference sequence corresponding to SEQ ID NO:

272.

24. The amino acid sequence of the engineered DNA polymerase may be selected from the amino acid position(s) 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, 257 / 377, 178 / 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, 792, 402 / 403 / 483 / 604 / 660, 178, 474, 257 / 377 / 541 / 604, 743, 396, 47 9, 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 / 24. The engineered DNA polymerase of claim 22 or 23, comprising at least one mutation or set of mutations in 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.

25. 13. 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 12-844 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 12-844 of SEQ ID NO: 328, or a reference sequence corresponding to SEQ ID NO:

328.

26. 14. 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 12-844 of SEQ ID NO:536-674, or a reference sequence corresponding to 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 12-844 of SEQ ID NO:328, or the reference sequence corresponding to SEQ ID NO:

328.

27. The amino acid sequence of the engineered DNA polymerase may be selected from the amino acid position(s) 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 / 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, 27. The engineered DNA polymerase of claim 25 or 26, comprising at least one mutation or set of mutations at 291 / 316 / 475 / 687 / 745, 257 / 291 / 395 / 740 / 745, 450, 473, 124, 430, 413, 66 or 365, wherein the positions are relative to the reference sequence corresponding to SEQ ID NO:

328.

28. 13. 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 12-844 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 12-844 of SEQ ID NO:546, or a reference sequence corresponding to SEQ ID NO:

546.

29. 14. 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 12-844 of SEQ ID NO:676-1190, or a reference sequence corresponding to 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 12-844 of SEQ ID NO:546, or the reference sequence corresponding to SEQ ID NO:

546.

30. The amino acid sequence of the engineered DNA polymerase may be selected from the group consisting of 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 / 5 36 / 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 / 42 7, 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 / 6 99, 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 / 8 12, 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、30. The engineered DNA polymerase of claim 28 or 29, comprising at least one mutation or set of mutations at 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. 13. 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 12-844 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 12-844 of SEQ ID NO:710, or a reference sequence corresponding to SEQ ID NO:

710.

32. 14. 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 12-844 of SEQ ID NO: 1192-1326, or a reference sequence corresponding to 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 12-844 of SEQ ID NO: 710, or the reference sequence corresponding to SEQ ID NO:

710.

33. 33. The engineered DNA polymerase of claim 31 or 32, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one 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.

34. 13. 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 12-844 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 12-844 of SEQ ID NO: 1208, or a reference sequence corresponding to SEQ ID NO: 1208.

35. 14. 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 12-844 of SEQ ID NO: 1328-1378, or a reference sequence corresponding to 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 12-844 of SEQ ID NO: 1208, or the reference sequence corresponding to SEQ ID NO: 1208.

36. The amino acid sequence of the engineered DNA polymerase may be selected from the amino acid position(s) 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, 78 / 508, 78 / 370 / 697 / 798, 51 / 251, 68 / 370 / 508 / 603 / 69 36. The engineered DNA polymerase of claim 34 or 35, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 1208.

37. 2. 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. 10. 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 12-844 of an even-numbered SEQ ID NO:4-1378 or a sequence corresponding to an even-numbered SEQ ID NO:2-1378.

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

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

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

42. 2. 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. 43. The engineered DNA polymerase of any one of claims 1 to 42, wherein the engineered DNA polymerase further comprises a fusion polypeptide.

44. 44. The engineered DNA polymerase of any one of claims 1 to 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. 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 inhibitor(s), or any combination of i), ii), iii), vi), v), vi), vii), and vii), and viii), relative to a reference DNA polymerase having a sequence corresponding to residues 12-844 of SEQ ID NO:2, or a sequence corresponding to SEQ ID NOs:2, 220, 226, 272, 328, 546, 710, or 1208.

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

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

49. 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. 50. The recombinant polynucleotide of claim 48 or 49, comprising 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 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 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 to 50, wherein the polynucleotide sequence is codon optimized.

52. 49. The recombinant polynucleotide 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. 49. The recombinant polynucleotide of claim 48, wherein the polynucleotide sequence comprises residues 34 to 2532 of an odd-numbered SEQ ID NO: 1-1377 or an odd-numbered SEQ ID NO: 1-1377.

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

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

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

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

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

59. 60. 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 so that at least one engineered DNA polymerase is produced.

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

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

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

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

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

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

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

67. 100. A method for preparing a complementary DNA to a target DNA, the method comprising contacting the target DNA with a DNA polymerase comprising an amino acid sequence comprising or including residues 12 to 844 of SEQ ID NO:2, or an engineered DNA polymerase of 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. 100. A method for detecting the presence of target DNA, comprising contacting a sample suspected of containing target DNA with a DNA polymerase comprising an amino acid sequence comprising or comprising residues 12 to 844 of SEQ ID NO:2, or an amino acid sequence comprising SEQ ID NO:2, in the presence of a substrate under conditions suitable for DNA polymerase-mediated production of DNA complementary to all or a portion of the target DNA, and detecting the presence of the complementary DNA.

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

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

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

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

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

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

75. 100. A method for sequencing a target DNA, comprising contacting the target DNA with a DNA polymerase comprising an amino acid sequence comprising or including residues 12 to 844 of SEQ ID NO:2, or an engineered DNA polymerase of 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 DNA complementary to the target DNA, and determining the sequence of the target DNA.

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

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

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

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

Images