Engineered DNA polymerase variants

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 375,371, filed on September 12, 2022, and U.S. Provisional Application No. 63 / 494,211, filed on April 4, 2023, the contents of which are incorporated herein by reference.

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

[0004] The sequence listing filed concurrently herewith as file name CX9-226WO2_ST26.xml, created on September 12, 2023, with a file size of 1,322,734 bytes, is part of this specification and is incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure provides engineered DNA polymerase polypeptides and compositions thereof, and polynucleotides encoding engineered DNA polymerase polypeptides. The present disclosure also provides methods of using engineered DNA polymerases or compositions thereof for diagnosis, molecular biology, and other purposes.

[0006] background

[0007] DNA polymerase is an enzyme (EC 2.7.7.7) that synthesizes complementary DNA chains using deoxyribonucleotide substrates. There are multiple types of DNA polymerases showing different properties in each organism, and there are different DNA polymerases in different organisms. DNA polymerase plays an important role in DNA replication and repair, and these functions are essential for maintaining genetic integrity. In addition to their biological effects, DNA polymerase is a basic tool for DNA manipulation, which includes DNA cloning, sequencing, labeling, mutagenesis, detection and diagnosis, and other. Although all DNA polymerases have the ability to synthesize deoxyribonucleotide chains, different polymerases have different properties, including differences in stability (e.g., thermal and / or chemical stability), processivity, fidelity, nucleotide selectivity, sensitivity, and template selectivity (e.g., DNA, RNA, etc.).

[0008] Although many different DNA polymerases are available, it is desirable to have a DNA polymerase that exhibits certain properties, such as certain levels of stability, processivity, fidelity, nucleotide selectivity, sensitivity, and template selectivity that are suitable for different uses, such as use as tools in molecular biology and diagnostics. For example, some DNA polymerases cannot effectively use RNA as a template, so a separate enzyme (reverse transcriptase) is required to synthesize DNA that is complementary to the RNA template, which can then be recognized by the DNA polymerase. The requirement to use another enzyme along with the DNA polymerase inserts additional steps and complexity into the diagnostics for detecting the target RNA.

[0009] Overview

[0010] The present disclosure provides engineered DNA polymerase polypeptides and compositions thereof, and polynucleotides encoding engineered DNA polymerase polypeptides, wherein the engineered DNA polymerase has DNA polymerase and reverse transcriptase activity, i.e., can use DNA and RNA as templates. The present disclosure also provides methods for using the engineered DNA polymerase polypeptides and compositions thereof for diagnosis and other purposes.

[0011] The engineered DNA polymerases of the present disclosure are based on a large fragment (SEQ ID NO: 2) of the full-length wild-type DNA polymerase of Parageobacillus genomosp 1 (SEQ ID NO: 540), wherein the large fragment includes a DNA polymerase domain but lacks a 5'-exonuclease domain.

[0012] In one aspect, the present disclosure provides an engineered DNA polymerase or a functional fragment thereof, comprising an amino acid 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 amino acid residues 12 to 604 of SEQ ID NO: 2, 10, 80, 224 or 366, or to a reference sequence corresponding to SEQ ID NO: 2, 10, 80, 224 or 366, wherein the amino acid sequence is 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 the reference sequence corresponding to amino acid residues 12 to 604 of SEQ ID NO: 2, 10, 80, 224 or 366, or to a reference sequence corresponding to SEQ ID NO: The reference sequence of ID NO: 2, 10, 80, 224 or 366 comprises one or more substitutions.

[0013] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 amino acid residues 12 to 604 of SEQ ID NO:2, or to a reference sequence corresponding to amino acid residues 12 to 604 of SEQ ID NO:2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12 to 604 of SEQ ID NO:2, or to the reference sequence corresponding to SEQ ID NO:2.

[0014] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 amino acid residues 12 to 604 of SEQ ID NO: 10, 80, 224 or 366, or to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12 to 604 of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2.

[0015] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 amino acid residues 12 to 604 of SEQ ID NO: 4-488 or to a reference sequence corresponding to an even numbered SEQ ID NO: 4-488, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12 to 604 of SEQ ID NO: 2 or 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 substitution at the following amino acid positions: 21, 24, 25, 25, 34, 36, 52, 58, 66, 68, 81, 84, 92, 101, 105, 114, 115, 124, 133, 133, 134, 136, 144, 152, 154, 168, 183, 184, 191, 192, 210, 212, 221, 226, 230, 241, 252 , 253, 276, 287, 290, 294, 295, 300, 304, 322, 325, 372, 373, 374, 393, 427, 432, 452, 454, 456, 462, 470, 483, 486, 495, 505, 509, 541, 541, 545, 547, 551, 552, 573, 575, 578, 584, 585 or 593 or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:2.

[0017] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at the following amino acid positions: 36, 52, 101, 124, 134, 136, 154, 212, 241, 253, 294, 300, 372, 393, 452, 454, 456, 470, 483, 505, 509, 547, 573 or 584 or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0018] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 36, 241, 372, or 470, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:2.

[0019] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions at one or more of the following amino acid positions: 509, 300, 452, 36 / 241 / 372 / 470, 124 / 192 / 210 / 372 / 427 / 456 / 552, 124, 52, 483, 372, 393, 212, 52 / 66, 133, 454, 154, 593, 462, 541, 21, 573, 505, 152, 294, 545, 101 / 241 / 470, 584, 304, 295, 578, 456 / 470, 253, 290, 192 / 241 / 372 / 456, or 252, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:2.

[0020] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution of the engineered DNA polymerase variants listed in Tables 5.1, 6.1, 7.1, 8.1, and 9.1, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:2.

[0021] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions of the engineered DNA polymerase variants listed in Tables 5.1, 6.1, 7.1, 8.1, and 9.1, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:2.

[0022] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of SEQ ID NO: 10, 80, 224 or 366, or to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366.

[0023] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of an even numbered SEQ ID NO:4-488, or to a reference sequence corresponding to an even numbered SEQ ID NO:4-488.

[0024] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of SEQ ID NO: 10, 80, 224 or 366, or to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 10, 80, 224 or 366, or to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366.

[0025] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of even numbered SEQ ID NOs in SEQ ID NOs: 4-488, or to a reference sequence corresponding to even numbered SEQ ID NOs in SEQ ID NOs: 4-488, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NOs: 10, 80, 224, or 366, or relative to the reference sequence corresponding to SEQ ID NOs: 4-488.

[0026] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at the following amino acid positions: 21, 24, 25, 25, 34, 36, 52, 58, 66, 68, 81, 84, 92, 101, 105, 114, 115, 124, 133, 133, 134, 136, 144, 152, 154, 168, 183, 184, 191, 192, 210, 212, 221, 226, 230, 241, 252 , 253, 276, 287, 290, 294, 295, 300, 304, 322, 325, 372, 373, 374, 393, 427, 432, 452, 454, 456, 462, 470, 483, 486, 495, 505, 509, 541, 541, 545, 547, 551, 552, 573, 575, 578, 584, 585 or 593 or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366.

[0027] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at the following amino acid positions: 36, 52, 101, 124, 134, 136, 154, 212, 241, 253, 294, 300, 372, 393, 452, 454, 456, 470, 483, 505, 509, 547, 573 or 584 or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366.

[0028] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 36, 241, 372, or 470, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 10, 80, 224, or 366.

[0029] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of SEQ ID NO: 10, or to a reference sequence corresponding to SEQ ID NO: 10, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 10, or to a reference sequence corresponding to SEQ ID NO: 10.

[0030] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of SEQ ID NOs: 10-218 or to a reference sequence corresponding to an even numbered SEQ ID NOs: 10-218, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 10 or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0031] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one set of substitutions at the following amino acid positions: 52 / 101 / 124 / 212 / 294 / 372 / 393 / 452 / 483 / 509, 52 / 124 / 300 / 393 / 452, 154 / 212 / 294 / 300 / 372 / 393 / 452 / 483 / 509, 52 / 101 / 154 / 294 / 300 / 452 / 509 / 593, 52 / 154 / 212 / 294 / 300 / 393 / 452, 212 / 300 / 393 / 452 / 483 / 509, 09 / 593、52 / 124 / 294 / 300 / 452 / 509、124 / 300 / 393 / 452 / 483 / 509 / 593、101 / 124 / 212 / 452 / 483 / 509、52 / 393 / 452 / 509 / 593、212 / 300 / 452 / 509 / 593、52 / 452 / 509 / 593、154 / 212 / 300 / 372 / 452 / 509 / 593、52 / 124 / 212 / 294 / 393 / 452 / 593、124 / 452 / 509、152 / 253 / 287 / 505 / 541 / 573 / 584、154 / 212 / 300 / 452、 152 / 253 / 287 / 304 / 454 / 545 / 573 / 584、52 / 124 / 154 / 294 / 300 / 372 / 452 / 509、294 / 300 / 372 / 452 / 509 / 593、52 / 212 / 452、124 / 294 / 393 / 452、52 / 300 / 50 9 / 593、152 / 253 / 287 / 454 / 541 / 584、52 / 101 / 452 / 509、52 / 154 / 212 / 300 / 372 / 393 / 509、21 / 253 / 287 / 454 / 584、52 / 393 / 452、52 / 124 / 154 / 294 / 509、101 / 124 / 212 / 300 / 393、124 / 294 / 300 / 372 / 452 / 509 / 593、52 / 124 / 154 / 294 / 483 / 593、52 / 372 / 393 / 452 / 509 / 593、154 / 300 / 393 / 452 / 593、52 / 101 / 294 / 3 72 / 393 / 452 / 509、21 / 152 / 253 / 287 / 304 / 454 / 505、52 / 300 / 393、101 / 124 / 154 / 294 / 452 / 509、154 / 212 / 372 / 393 / 509 / 593、21 / 253 / 287 / 454 / 505 / 541、287 / 454 / 505 / 584、52 / 124 / 294 / 300 / 372 / 452、287 / 505 / 541 / 573、253 / 505 / 584、84 / 253 / 287 / 456 / 573、52 / 372 / 509 / 593、509 / 593、154 / 212 / 300 / 372 / 509、52 / 154 / 294 / 509 / 593、152 / 295 / 505 / 545 / 573、253 / 541 / 584、253 / 295 / 304 / 454 / 462 / 573 / 584、253 / 454 / 505 / 573 / 584、154 / 212 / 300、287 / 454 / 54 or 152 / 253 / 454 / 584, wherein the amino acid positions are relative to the amino acid positions corresponding to SEQ ID NO: 5 / 573, 21 / 152 / 287 / 304 / 454, 152 / 253 / 295 / 304 / 541 / 573, 152 / 253 / 287 / 304 / 454, 152 / 454 / 573, 124 / 294 / 372 / 509 / 593, 152 / 253 / 295 / 454, 154 / 300 / 452, 287 / 304 / 505, 152 / 253 / 454 / 505, 253 / 454 / 505, 21 / 253 / 454 / 505 / 545, 152 / 253 / 287 / 505, 505 / 541 / 584 or 152 / 253 / 454 / 584, wherein the amino acid positions are relative to the amino acid positions corresponding to SEQ ID NO: 5 / 573, 21 / 152 / 287 / 304 / 454, 152 / 253 / 295 / 304 / 541 Reference sequence of ID NO:10.

[0032] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of SEQ ID NO:80, or to a reference sequence corresponding to SEQ ID NO:80, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO:80, or to a reference sequence corresponding to SEQ ID NO:80.

[0033] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of SEQ ID NOs: 220-258, or to a reference sequence corresponding to an even numbered SEQ ID NOs: 220-258, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 80, or relative to the reference sequence corresponding to SEQ ID NO: 80.

[0034] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions at one or more of the following amino acid positions: 300 / 454 / 456 / 541 / 584, 21 / 300 / 454 / 545 / 584, 253 / 300 / 454 / 456 / 584, 154 / 253 / 300 / 454 / 456 / 505 / 573, 21 / 154 / 300 / 454 / 456 / 573, 152 / 300 / 454 / 456 / 505 / 584 / 593, 154 / 253 / 300 / 456 / 505 / 541 / 584, 21 / 300 / 454 / 545 / 584, 253 / 300 / 454 / 456 / 505 / 573, 154 / 253 / 300 / 454 / 456 / 541 / 573 / 584、154 / 300 / 454 / 505、21 / 253 / 300 / 454 / 456 / 545 / 573 / 593、168 / 300 / 454 / 456 / 545 / 573、253 / 300 / 454 / 505 / 573、 154 / 300 / 505 / 545 / 584、154 / 253 / 300 / 456 / 541 / 573 / 584、21 / 154 / 300 / 454 / 456 / 505 / 545、154 / 253 / 300 / 454 / 505、21 / 154 / 253 / 300 / 454 / 573、300 / 45 4 / 505 / 541 / 584、300 / 454 / 505 / 541 / 573、154 / 253 / 300 / 454、154 / 300 / 454 / 573、300 / 454 / 456 / 545 / 573、253 / 300 / 454 / 456、154 / 253 / 300 / 454 / 545 / 5 73、21 / 152 / 253 / 300 / 454 / 456 / 541 / 573、154 / 253 / 300 / 454 / 456 / 573、152 / 300 / 454、154 / 253 / 300 / 454 / 505 / 541 / 593、21 / 154 / 300 / 454 / 573 / 584、15 4 / 300 / 456 / 573 / 593、152 / 253 / 300 / 454 / 545 / 573、21 / 300 / 456 / 495 / 505 / 541 / 584、154 / 300 / 454 / 541 / 584、253 / 300 / 505 / 541 / 573 / 584、253 / 300 / 58 4. 253 / 300 / 454 / 456 / 545, 21 / 253 / 300 / 573, 300 / 454 / 545, 454 / 456 / 541 / 545 / 573 / 584, 152 / 253 / 454 / 505 / 545 / 573, 21 / 300 / 454 / 456 / 495, 300 / 573,154 / 300 / 454 / 545, 154 / 253 / 300 / 545 / 584, 21 / 300 / 505, 21 / 152 / 253 / 300 / 454 / 456 / 545, 154 / 253 / 300 / 456 / 541 / 573, 154 / 300 / 454, 253 / 300 / 454 / 541, 253 / 454 / 456 / 541 / 584, 21 / 300 / 573, 300 / 454 / 456, 300 / 454, 154 / 253 / 300 / 454 4 / 456, 154 / 456 / 505 / 593, 454 / 456 / 505, 253 / 454 / 456 / 584, 154 / 253 / 300, 253 / 456 / 505 / 584, 454 / 456, 154 / 454, 584, 432 / 505 / 573, 456 / 584, 21 / 505 / 545 / 584, 21 / 154 / 545 / 573, 454 / 584 or 154 / 253 / 505 or 505, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 80.

[0035] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of SEQ ID NO: 224, or to a reference sequence corresponding to SEQ ID NO: 224, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 224, or to a reference sequence corresponding to SEQ ID NO: 224.

[0036] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of SEQ ID NO: 360-400, or to a reference sequence corresponding to an even numbered SEQ ID NO: 360-400, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 224, or relative to the reference sequence corresponding to SEQ ID NO: 224.

[0037] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one set of substitutions at the following amino acid positions: 144 / 154 / 505 / 547 / 573 / 584, 154 / 191 / 325 / 505 / 573 / 584, 144 / 154 / 373 / 374 / 505 / 573 / 584, 134 / 136 / 154 / 505 / 547 / 573 / 584, 81 / 144 / 154 / 505 / 547 / 573 / 584、154 / 505 / 573 / 584、144 / 154 / 191 / 230 / 322 / 505 / 573 / 584、68 / 144 / 154 / 505 / 573 / 584、144 / 154 / 226 / 230 / 505 / 573 / 584、144 / 154 / 374 / 486 / 505 / 573 / 584、81 / 114 / 144 / 154 / 505 / 573 / 584、144 / 154 / 276 / 505 / 573 / 584、134 / 144 / 154 / 505 / 573 / 584、144 / 154 / 505 / 573 / 584、81 / 144 / 154 / 183 / 505 / 547 / 573 / 584、68 / 81 / 133 / 134 / 144 / 154 / 505 / 547 / 573 / 584、144 / 154 / 374 / 505 / 573 / 584, 92 / 133 / 144 / 154 / 505 / 573 / 584, 144 / 154 / 373 / 505 / 573 / 584, 114 / 144 / 154 / 505 / 573 / 584, 154 / 505 / 551 / 573 / 584 / 585 or 81 / 154 / 505 / 547 / 573 / 584, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:224.

[0038] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of SEQ ID NO: 366, or to a reference sequence corresponding to SEQ ID NO: 366, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 366, or to a reference sequence corresponding to SEQ ID NO: 366.

[0039] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of SEQ ID NO: 402-488, or to a reference sequence corresponding to an even numbered SEQ ID NO: 402-488, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 366, or relative to the reference sequence corresponding to SEQ ID NO: 366.

[0040] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions at one or more of the following amino acid positions: 24 / 58 / 432, 58 / 432 / 575, 58 / 115 / 432 / 575, 24 / 115 / 432 / 575, 24 / 25 / 221 / 432, 25 / 58 / 432 / 575, 24 / 25 / 58 / 432 / 575, 221 / 432 / 575, 432 / 575, 24 / 221 / 432, 24 / 115 / 221 / 432, 24 / 115 / 221 / 432 / 575, 432, 24 / 34 / 432, 24 / 432 / 575, 184 / 221 / 432 / 575, 24 / 432, 24 / 25 / 58 / 115 / 432 / 575, 25 / 221 / 432, 25 / 58 / 115 / 432, 221 / 575, 221 / 432, 24 / 58 / 115 / 221 / 575, 24 / 25 / 221 / 432 / 575, 24 / 25 / 58 / 221 / 432, 34 / 58 / 105 / 432, 24 / 25 / 115 / 432, 24 / 221 / 575, 115 / 432, 58 / 221, 58 / 575, 25 / 432 / 575, 25 / 432, 24 / 221 / 432 / 575, 24 / 115 / 432, 24 / 25 / 105 / 432, 25 / 34 / 432, 25 / 105 / 221 / 432, 24 / 25 / 432, 24 / 58 / 575 or 24 / 25 / 105 / 432 / 575, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:366.

[0041] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution of the engineered DNA polymerase variants listed in Tables 5.1, 6.1, 7.1, 8.1, and 9.1, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 2, 10, 80, 224, or 366.

[0042] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions of the engineered DNA polymerase variants listed in Tables 5.1, 6.1, 7.1, 8.1, and 9.1, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 2, 10, 80, 224, or 366.

[0043] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 amino acid sequence comprising a substitution or set of substitutions of the engineered DNA polymerase variants listed in Tables 5.1, 6.1, 7.1, 8.1, and 9.1.

[0044] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence comprising residues 12 to 604 of an even-numbered SEQ ID NO. in SEQ ID NO: 4-488 or an amino acid sequence comprising an even-numbered SEQ ID NO. in SEQ ID NO: 4-488, optionally wherein the polypeptide has 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions in the polypeptide sequence. In some embodiments, the amino acid sequence of the engineered DNA polymerase has 1, 2, 3, 4, up to 5 substitutions in the amino acid sequence. In some embodiments, the substitutions include conservative substitutions.

[0045] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence comprising residues 12 to 604 of SEQ ID NO: 10, 80, 224, or 366 or an amino acid sequence comprising SEQ ID NO: 10, 80, 224, or 366, optionally wherein the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions in the amino acid sequence. In some embodiments, the amino acid sequence has 1, 2, 3, 4, up to 5 substitutions in the amino acid sequence. In some embodiments, the substitutions comprise conservative substitutions.

[0046] In some embodiments, the engineered DNA polymerase has DNA polymerase activity, including reverse transcriptase activity, and exhibits one or more improved properties compared to a reference DNA polymerase or a reference engineered DNA polymerase. In some embodiments, the engineered DNA polymerase has an improved property selected from the following compared to a reference DNA polymerase: i) increased activity, ii) increased stability, iii) increased thermal stability, iv) increased processivity, v) increased fidelity, vi) increased sensitivity to input target RNA or DNA, vii) increased product yield in isothermal amplification reactions, viii) increased salt tolerance, and ix) increased resistance to inhibitors, or any combination of i), ii), iii), iv), v), vii), vii), viii), and ix). In some embodiments, the reference DNA polymerase has an amino acid sequence corresponding to residues 12 to 604 of SEQ ID NO: 2, 10, 80, 224, or 366 or an amino acid sequence corresponding to SEQ ID NO: 2, 10, 80, 224, or 366. In some embodiments, the reference DNA polymerase has an amino acid sequence corresponding to residues 12 to 604 of SEQ ID NO:2 or corresponds to an amino acid sequence of SEQ ID NO:2.

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

[0048] In another aspect, the present disclosure provides a recombinant polynucleotide comprising a polynucleotide sequence encoding any of the engineered DNA polymerases disclosed herein.

[0049] In some embodiments, the recombinant polynucleotide comprises 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 polynucleotide sequence corresponding to nucleotide residues 34 to 1812 of SEQ ID NO: 1, 9, 79, 223 or 365, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 1, 9, 79, 224 or 365, wherein the recombinant polynucleotide encodes an engineered DNA polymerase.

[0050] In some embodiments, the recombinant polynucleotide comprises 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 polynucleotide sequence corresponding to nucleotide residues 34 to 1812 of an odd-numbered SEQ ID NO. in SEQ ID NOs: 3-487, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. in SEQ ID NOs: 3-487, wherein the recombinant polynucleotide encodes an engineered DNA polymerase.

[0051] In some embodiments, the polynucleotide sequence of the recombinant polynucleotide encoding the engineered DNA polymerase is codon-optimized for expression in an organism or a cell type thereof, such as a bacterial cell, a fungal cell, an insect cell, or a mammalian cell.

[0052] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 1812 of SEQ ID NO: 9, 79, 223, or 365 or comprises a polynucleotide sequence comprising SEQ ID NO: 9, 79, 223, or 365.

[0053] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 1812 of an odd-numbered SEQ ID NO: 3-487 or comprises a polynucleotide sequence comprising an odd-numbered SEQ ID NO: 3-487.

[0054] On the other hand, the disclosure provides an expression vector comprising a recombinant polynucleotide encoding an engineered DNA polymerase provided herein. In some embodiments, the recombinant polynucleotide of the expression vector is operably linked to a control sequence. In some embodiments, the control sequence includes a promoter, particularly a heterologous promoter.

[0055] On the other hand, the disclosure also provides host cells transformed with expression vectors or recombinant polynucleotides provided herein. In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the host cell is a bacterial cell, a fungal cell, an insect cell, or a mammalian cell. In some embodiments, the host cell is a bacterial cell, such as Escherichia coli.

[0056] In other aspects, the disclosure provides a method for producing an engineered DNA polymerase polypeptide, the method comprising culturing a host cell described herein under suitable culture conditions such as producing at least one engineered DNA polymerase. In some embodiments, the method further comprises recovering or separating the engineered DNA polymerase from culture medium and / or host cell. In some embodiments, the method further comprises purifying the engineered DNA polymerase.

[0057] In other aspects, the disclosure provides compositions comprising at least one engineered DNA polymerase disclosed herein, particularly compositions for performing in vitro polymerase reactions. In some embodiments, the compositions include one or more of the following: buffer, DNA polymerase substrate, for example, one or more nucleotide substrates (e.g., dNTP or its analog) and / or oligonucleotide primer substrates. In some embodiments, the compositions include at least one DNA or RNA template, such as a target DNA or RNA in a sample.

[0058] In another aspect, the disclosure provides the use of an engineered DNA polymerase in a method for preparing a complementary DNA copy of a target nucleic acid (in whole or in part). In some embodiments, the method for preparing a complementary DNA of a target DNA or RNA (in whole or in part) comprises contacting the target DNA or RNA with an engineered DNA polymerase described herein in the presence of an appropriate substrate under conditions suitable for DNA polymerase-mediated production of DNA complementary to the target DNA or RNA.

[0059] In some embodiments, engineered DNA polymerase is used to detect target nucleic acid, the method comprising contacting a sample suspected of containing target nucleic acid with an engineered DNA polymerase of the disclosure under conditions suitable for DNA polymerase-mediated production of DNA complementary to target nucleic acid (in whole or in part) in the presence of an appropriate substrate, and detecting the presence of complementary DNA. In some embodiments, the target nucleic acid is DNA or RNA. In some embodiments, the sample is a biological sample or an environmental sample. In some embodiments, detection of complementary DNA is performed by amplifying complementary DNA, such as by isothermal amplification or polymerase chain reaction.

[0060] In other aspects, the disclosure also provides a test kit comprising at least one engineered DNA polymerase disclosed herein. In some embodiments, the test kit also comprises one or more of a buffer, a nucleotide substrate and / or an oligonucleotide primer substrate. In some embodiments, the test kit may also comprise a nucleic acid template, such as a DNA or RNA template. In some embodiments, the test kit may comprise a second DNA polymerase, such as a thermostable DNA polymerase.

[0061] Details

[0062] The present disclosure provides engineered DNA polymerase polypeptides and compositions thereof and recombinant polynucleotides encoding engineered DNA polymerase polypeptides. The present disclosure also provides methods for using engineered DNA polymerase polypeptides and compositions thereof for diagnosis, molecular biology and other purposes. In some embodiments, the engineered DNA polymerase polypeptides described herein can use DNA as a template or RNA as a template (i.e., reverse transcriptase activity), and the engineered DNA polymerase polypeptides have one or more improved properties, including improved polymerization activity, improved replication fidelity, improved continuous synthesis ability (particularly under conditions involving low concentrations of target DNA or RNA input), increased resistance to inhibitors and increased salt tolerance and other.

[0063] In some embodiments, the engineered DNA polymerases of the present disclosure are particularly useful in diagnostic and research applications using small amounts of DNA or RNA from samples, including cell-free DNA or RNA, circulating tumor DNA or RNA, DNA or RNA isolated from circulating tumor cells, circulating fetal DNA or RNA, DNA or RNA isolated from virally infected cells, fine needle aspirates, single cells isolated by FACS (fluorescence activated cell sorting), and environmental samples (e.g., water, sewer eluate, air, etc.). However, the samples used in the present disclosure are not intended to be limited to any particular sample type, as any suitable sample, including those with low target DNA or RNA concentrations, can be used with the engineered DNA polymerases herein.

[0064] Abbreviations and definitions

[0065] Unless otherwise defined, all technical and scientific terms used herein generally have the same meanings as those generally understood by those of ordinary skill in the art to which the invention belongs. Typically, the nomenclature used herein and the experimental procedures in 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 well known and described in many textbooks and reference works well known to those skilled in the art. Standard techniques or their modified forms are used for chemical synthesis and chemical analysis.

[0066] Additionally, the headings provided herein should not be construed as limitations of the various aspects or embodiments of the invention, but rather should be viewed with reference to the application as a whole.Accordingly, the terms defined immediately below are more fully described by reference to the application as a whole.

[0067] Furthermore, as used herein, the singular "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0068] As used herein, the term "comprising" and its cognates are used in their inclusive sense (ie, equivalent to the term "including" and its corresponding cognates).

[0069] It will be further understood that where the description of embodiments uses the term "comprising" and its cognates, embodiments may also be described using the language "consisting essentially of" or "consisting of.

[0070] Numerical ranges include the numbers that define the ranges. Therefore, each numerical range disclosed herein is intended to include each narrower numerical range that falls within such a wider numerical range, as if such a narrower numerical range is fully and clearly written herein. It is also intended that each maximum (or minimum) numerical limit disclosed herein includes each lower (or higher) numerical limit, as if such lower (or higher) numerical limits are clearly written herein.

[0071] As used herein, the term "about" means an acceptable error for a particular value. In some instances, "about" means within 0.05%, 0.5%, 1.0%, or 2.0% of a given value. In some instances, "about" means within 1, 2, 3, or 4 standard deviations of a given value.

[0072] "EC" numbers refer to the enzyme nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions that the enzymes catalyze.

[0073] "ATCC" refers to the American Type Culture Collection, whose biological collection includes genes and strains.

[0074] "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

[0075] "Protein," "polypeptide," and "peptide" are used interchangeably to refer to a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modifications (eg, glycosylation or phosphorylation).

[0076] "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. Abbreviations for genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glutamine (Gln or Q), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). When three-letter abbreviations are used, unless specifically preceded by "L" or "D", or it is clear from the context in which the abbreviation is used, an amino acid can be in the L-configuration or the D-configuration about the α-carbon (Cα). For example, "Ala" represents alanine without specifying the configuration about the α-carbon, while "D-Ala" and "L-Ala" represent D-alanine and L-alanine, respectively. When single-letter abbreviations are used, capital letters represent amino acids in the L-configuration about the α-carbon, and lowercase letters represent amino acids in the D-configuration about the α-carbon. For example, "A" represents L-alanine and "a" represents D-alanine. When a polypeptide sequence is presented as a string of single-letter or three-letter abbreviations (or a mixture thereof), the sequence is presented as an amino (N) to carboxyl (C) direction according to conventional practice.

[0077] "Fusion protein", "fusion polypeptide", "chimeric protein" and "chimera" refer to a hybrid protein produced by connecting two or more polynucleotides that originally encoded separate proteins. In some embodiments, the fusion protein is produced by recombinant technology (e.g., molecular biology techniques known in the art).

[0078] "Polymerase" refers to a class of enzymes (e.g., EC 2.7.7.7) that polymerize nucleoside triphosphates to form polynucleotides. In this article, a polymerase uses a template nucleic acid chain to synthesize a complementary nucleic acid chain. In some embodiments, the template chain and the synthesized nucleic acid chain can be independently DNA or RNA, depending on the substrate and template specificity of the polymerase. Polymerases known in the art include, but are not limited to, DNA polymerases (e.g., Escherichia coli DNA polI, Thermus aquaticus (T.aquaticus) DNA polymerase (Taq), Bst polymerase), DNA-dependent RNA polymerase, and reverse transcriptase. As used herein, a polymerase is a polypeptide or protein containing enough amino acids to play the desired enzyme function of a polymerase. In some embodiments, a polymerase does not include all the amino acids found in a natural enzyme, but only includes amino acids sufficient to allow a polymerase to play the desired catalytic activity, including but not limited to amino acids that play 5'-3' polymerization, 5'-3' exonuclease, and 3'-5' exonuclease activity. In some embodiments, a polymerase is limited to a polymerase domain and does not include an exonuclease function.

[0079] "DNA polymerase activity", "synthetic activity" and "polymerase activity" are used interchangeably herein and refer to the ability of an enzyme to synthesize a new DNA chain by incorporating deoxynucleoside triphosphates or their analogs. In some embodiments, the DNA polymerase can use DNA and / or RNA as a template.

[0080] "Reverse transcriptase activity" refers to the ability of a DNA polymerase to synthesize a new DNA strand by incorporating deoxynucleoside triphosphates or their analogs using RNA as a template.

[0081] "Polynucleotide", "nucleic acid" or "oligonucleotide" is used herein to refer to a polymer comprising at least two nucleotides, wherein the nucleotide is a deoxyribonucleotide or a ribonucleotide or a mixture of deoxyribonucleotides and ribonucleotides. In some embodiments, the abbreviations for genetically encoded nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically described, the abbreviated nucleosides can be ribonucleosides or 2'-deoxyribonucleosides. Nucleosides can be designated as ribonucleosides or 2'-deoxyribonucleosides on an individual or overall basis. When a polynucleotide, nucleic acid or oligonucleotide sequence is presented as a string of single-letter abbreviations, according to common practice, the sequence is presented in the 5' to 3' direction and phosphates are not indicated. The term "DNA" refers to deoxyribonucleic acid. The term "RNA" refers to ribonucleic acid. A polynucleotide or nucleic acid can be single-stranded or double-stranded, or can include both single-stranded and double-stranded regions.

[0082] In some embodiments, "polynucleotide", "nucleic acid" and "oligonucleotide" include polynucleotides or nucleic acid or oligonucleotide analogs, which include nucleosides linked together via non-standard linkages other than standard phosphodiester linkages, such as phosphoramidates, phosphorothioates, amides, positively charged linkages, etc.; nucleosides with modified and / or synthetic nucleobases, such as inosine, xanthine, hypoxanthine, etc.; and / or nucleosides with modified sugar residues, such as 2'-O-alkyl (e.g., 2'-O-methyl, 2'-O-ethyl, etc.), 2'-halo (e.g., 2'-F, 2'-Br, etc.), 2,3-dideoxy, 2'-halo-2'-deoxy, β-D-ribo-LNA; α-L-ribo-LNA (locked nucleic acid), etc. and others.

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

[0084] "Engineered," "recombinant," "non-naturally occurring," and "variant" when used with respect to cells, polynucleotides, or polypeptides refer to materials that are or correspond to native or natural forms of materials that have been modified or are identical to them in a way that is not originally found in nature but are produced or derived from synthetic materials and / or through manipulation using recombinant techniques.

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

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

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

[0088] "Reference sequence" refers to a designated sequence used as the basis for sequence comparison. A reference sequence can be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Typically, a reference sequence is a length of at least 20 nucleotides or amino acid residues, a length of at least 25 residues, a length of at least 50 residues, a length of at least 100 residues, or the full length of a nucleic acid or polypeptide. Since two polynucleotides or polypeptides can each (1) contain a sequence similar to the two sequences (i.e., a portion of the complete sequence), and (2) can also contain a sequence different from the two sequences, a sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides on a "comparison window" to identify and compare local regions of sequence similarity. In some embodiments, a "reference sequence" can be based on a primary amino acid sequence, wherein a reference sequence is a sequence that can have one or more changes in the primary sequence. For example, the phrase "the reference sequence corresponding to SEQ ID NO:2 with serine at the residue corresponding to X470" (or "the reference sequence corresponding to SEQ ID NO:2 with serine at the residue corresponding to position 470") refers to the reference sequence in which the corresponding residue at position X470 in SEQ ID NO:2 (e.g., isoleucine) has been changed to serine.

[0089] "Comparison window" refers to a conceptual segment of consecutive nucleotide positions or amino acid residues in which a sequence can be compared to a reference sequence. In some embodiments, the comparison window is at least 15 to 20 consecutive nucleotides or amino acids, and wherein the sequence portion in the comparison window may contain 20% or less additions or deletions (i.e., spaces) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. In some embodiments, the comparison window may be longer than 15-20 consecutive residues, and optionally includes 30, 40, 50, 100 or longer windows.

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

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

[0092] "Amino acid difference" and "residue difference" refer to the difference of the amino acid residue at one position of a polypeptide sequence relative to the amino acid residue at the corresponding position in the reference sequence. The position of the amino acid difference is generally referred to as "Xn" herein, where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, "residue difference at position X470 compared to SEQ ID NO:2" (or "residue difference at position 470 compared to SEQ ID NO:2") refers to the difference of the amino acid residue at the polypeptide position corresponding to SEQ ID NO:2 position 470. Therefore, if the reference polypeptide of SEQ ID NO:2 has isoleucine at position 470, "residue difference at position X470 compared to SEQ ID NO:2" refers to the amino acid substitution of any residue except isoleucine at the position of the polypeptide corresponding to SEQ ID NO:2 position 470. In some cases herein, the specific amino acid residue difference at one position is indicated as "XnY", where "Xn" specifies the corresponding residue and position of 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 different from the reference polypeptide). In some cases (e.g., in the tables in the Examples), the disclosure also provides specific amino acid differences represented by the conventional notation "AnB", where A is a single-letter identifier for a residue in a reference sequence, "n" is the number of the residue position in the reference sequence, and B is a single-letter identifier for the residue substitution in the sequence of the engineered polypeptide. In some embodiments, amino acid differences (e.g., substitutions) are represented by the abbreviation "nB" without the identifier for the residue in the reference sequence. In some embodiments, the phrase "amino acid residue nB" represents an amino acid residue present in an engineered polypeptide that may or may not be a substitution in the context of a reference sequence.

[0093] In some cases, the polypeptide of the present disclosure may include one or more amino acid residue differences relative to a reference sequence, and the amino acid residue differences are indicated by a list of designated positions where residue differences exist relative to the reference sequence. In some embodiments, when more than one amino acid can be used in a specific residue position of a polypeptide, the multiple amino acid residues that can be used are separated by " / " (e.g., X21M / X21V, X21M / V or 21M / V). The present disclosure includes engineered polypeptide sequences comprising one or more amino acid differences, and the one or more amino acid differences include one or both of conservative amino acid substitutions and non-conservative amino acid substitutions, and insertions and deletions of amino acids in the sequence.

[0094] "Amino acid substitution set" and "substitution set" refer to a set of amino acid substitutions within a polypeptide sequence. In some embodiments, the substitution set comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions. In some embodiments, the substitution set refers to 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, each substitution is separated by a semicolon (";"; for example, S36T; A241Q; K372E; I470S or abbreviated as 36T; 241Q; 372E; 470S) or a slash (" / "; for example, S36T / A241Q / K372E / I470S or abbreviated as 36T / 241Q / 372E / 470S). In some embodiments, "substitution" includes the deletion of an amino acid and can be represented by the "-" symbol.

[0095] "Conservative amino acid substitutions" refer to replacing a residue with a different residue having a similar side chain, and thus generally include replacing an amino acid in a polypeptide with an amino acid in the same or similar amino acid defined class. For example, but not limited to, an amino acid with an aliphatic side chain can be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain can be substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain can be substituted 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 substituted with another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain can be substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and a hydrophobic amino acid or a hydrophilic amino acid can be substituted with another hydrophobic amino acid or a hydrophilic amino acid, respectively.

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

[0097] "Deletion" refers to a polypeptide modification by removing one or more amino acids from a reference polypeptide. Deletion can include removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids that make up the reference enzyme, or up to 20% of the total number of amino acids that make up the reference enzyme, while retaining enzymatic activity and / or retaining improved properties of the engineered polymerase. Deletion can involve internal portions and / or terminal portions of a polypeptide. In various embodiments, deletion can include continuous segments or can be discontinuous. Deletion is represented by "-" and may be present in a substitution set.

[0098] "Insertion" refers to the modification of a polypeptide by the addition of one or more amino acids to a reference polypeptide. The insertion may be in an internal portion of the polypeptide or to the carboxyl or amino terminus. Insertions as used herein include fusion proteins as known in the art. Insertions may be continuous stretches of amino acids, or separated by one or more amino acids in a naturally occurring polypeptide.

[0099] "Functional fragment" and "biologically active fragment" are used interchangeably herein to refer to a polypeptide having an amino-terminal deletion and / or a carboxyl-terminal deletion and / or an internal deletion, but wherein the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is compared (e.g., a full-length engineered DNA polymerase of the invention) and retains substantially all of the activity of the full-length polypeptide.

[0100] "Isolated polypeptide" refers to a polypeptide that is separated from other contaminants (e.g., proteins, lipids, and polynucleotides) that naturally accompany it. The term includes polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., host cells or in vitro synthesis). Recombinant DNA polymerase polypeptides can be present in cells, in cell culture media, or prepared in various forms (such as lysates or isolated preparations). Therefore, in some embodiments, the recombinant DNA polymerase polypeptides provided herein are isolated polypeptides.

[0101] "Substantially pure polypeptide" or "pure polypeptide" refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis, it is more abundant than any other individual macromolecular species in the composition), and when the target species constitutes at least about 50% of the macromolecular species present by mole or % weight, it is generally a substantially purified composition. Typically, 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 all macromolecular species present in the composition by mole or % weight. In some embodiments, the target species is purified to basic homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods), wherein the composition is substantially composed of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ionic species are not considered macromolecular species. In some embodiments, the separated recombinant DNA polymerase polypeptide is a substantially pure polypeptide composition.

[0102] As used herein, "improved enzyme properties" refers to an engineered DNA polymerase polypeptide that exhibits improvements in any enzyme property compared to a reference DNA polymerase polypeptide (such as 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 thermal stability, increased stability, increased enzyme activity, increased substrate specificity and / or affinity, increased specific activity, increased resistance to substrate and / or end-product inhibition, increased chemical stability, improved salt tolerance, improved solvent stability, increased solubility, increased fidelity, increased processivity, increased inhibitor resistance or tolerance, and altered temperature profile.

[0103] "Increased enzymatic activity" and "enhanced catalytic activity" refer to the improved properties of engineered DNA polymerase polypeptides, which can be expressed as an increase in specific activity (e.g., product / time / weight protein produced) and / or an increase in the conversion percentage of substrate to product conversion (e.g., using a specified amount of DNA polymerase in a specified time period, the conversion percentage of a starting amount of substrate to product conversion) compared to a reference DNA polymerase (e.g., wild-type DNA polymerase and / or another engineered DNA polymerase). Exemplary methods for determining enzymatic activity are provided in the Examples. Any property associated with enzymatic activity can be affected, including typical enzyme properties Km, Vmax or kcat, whose changes can result in increased enzymatic activity. In some embodiments, the engineered DNA polymerase can be from about 1.1, 1.2, 1.3, 1.4 or 1.5 times the enzymatic activity of the corresponding wild-type enzyme, to up to 2 times, 5 times, 10 times, 20 times, 25 times, 50 times, 75 times, 100 times, 150 times, 200 times or more enzymatic activity than the enzymatic activity of another engineered DNA polymerase from which the engineered DNA polymerase polypeptide is derived. In some specific embodiments, the engineered DNA polymerase exhibits an improved enzymatic activity in the range of 1.5 to 10 times, 1.5 to 25 times, 1.5 to 50 times, 1.5 to 100 times or more compared to the enzymatic activity of the reference DNA polymerase.

[0104] "Hybridization stringency" refers to hybridization conditions in nucleic acid hybridization, such as washing conditions (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, 2001). Typically, hybridization reactions are performed under conditions of lower stringency, followed by washings of different but higher stringency. The term "moderate stringency hybridization" refers to conditions that allow target DNA to bind to complementary nucleic acids that have about 60% identity, preferably about 75% identity, about 85% identity, and greater than about 90% identity with the target polynucleotide. Exemplary moderate stringency conditions are conditions equivalent to hybridization at 42°C in 50% formamide, 5×Denhart solution, 5×SSPE, 0.2% SDS, followed by washing at 42°C in 0.2×SSPE, 0.2% SDS. "High stringency hybridization" generally refers to conditions that differ by about 10°C or less from the thermal melting temperature Tm as determined for a specified polynucleotide sequence under solution conditions. In some embodiments, high stringency conditions refer to conditions that allow only hybridization of those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65°C (i.e., if the hybrid is unstable in 0.018M NaCl at 65°C, it will be unstable under high stringency conditions as contemplated herein). For example, high stringency conditions can be provided by hybridizing under conditions equivalent to 50% formamide, 5×Denhart solution, 5×SSPE, 0.2% SDS at 42°C, followed by washing in 0.1×SSPE and 0.1% SDS at 65°C. Another high stringency condition includes hybridization under conditions equivalent to hybridizing in 5X SSC containing 0.1% (w:v) SDS at 65°C and washing in 0.1×SSC containing 0.1% SDS at 65°C. Other high stringency hybridization conditions, as well as moderate stringency conditions, are described in the references cited above.

[0105] "Codon optimization" refers to the change of the codons of the polynucleotides encoding proteins to the codons preferentially used in a specific organism, so that the encoded protein is more effectively expressed in the organism. Although the genetic code is degenerate, because most amino acids are represented by several codons referred to as "synonymous (synonym)" or "synonymous (synonymous)" codons, it is well known that the codon usage of a specific organism is non-random and biased towards specific codon triplet. Although the genetic code is degenerate, that is, most amino acids are represented by several codons referred to as "synonymous ("synonyms") or "synonymous ("synonymous") codons, it is well known that the codon usage of a specific organism is non-random and biased towards specific codon triplet. In some embodiments, the polynucleotide encoding DNA polymerase is codon optimized for optimal production from the host organism selected for expression.

[0106] "Control sequences" herein refer to all components that are necessary or advantageous for the expression of polynucleotides and / or polypeptides of the present disclosure. As used herein, "control sequences" include all components that are necessary or advantageous for the expression of polynucleotides and / or polypeptides of the present invention. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoter sequences, signal peptide sequences, start sequences, and transcription terminators. In some embodiments, at a minimum, control sequences include promoters and transcription and translation termination signals. In some embodiments, control sequences are provided with joints for the purpose of introducing specific restriction sites that facilitate the connection of control sequences to the coding region of the nucleic acid sequence encoding the polypeptide.

[0107] "Operably linked" or "operatively linked" refers to a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest so that the control sequence directs or regulates the expression of the polynucleotide of interest and / or encoded polypeptide.

[0108] "Promoter" or "promoter sequence" refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest (such as a coding sequence). The promoter sequence comprises a transcriptional control sequence that mediates the expression of the polynucleotide of interest. The promoter can be any nucleic acid sequence that shows transcriptional activity in a selected host cell, including mutants, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0109] "Suitable reaction conditions" or "suitable conditions" refer to those conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffer, cosolvent, etc.) under which the DNA polymerase polypeptide of the present disclosure is able to convert a substrate into a desired product compound. Exemplary "suitable reaction conditions" are provided herein (see Examples).

[0110] In the context of an enzymatic conversion process, "product" refers to a compound or molecule resulting from the action of a DNA polymerase polypeptide on a substrate.

[0111] "Cultivation" refers to the growth of a population of cells under suitable conditions using any suitable medium (e.g., liquid, gel or solid). Cells used for culture can be prokaryotic or eukaryotic cells, such as bacterial, fungal, insect or mammalian cells.

[0112] "Vector" is a recombinant construct for introducing a polynucleotide sequence of interest into a cell. In some embodiments, a vector is an expression vector operably linked to a suitable control sequence capable of achieving expression of a polynucleotide or polypeptide encoded in a polynucleotide sequence in a suitable host. In some embodiments, an "expression vector" has a promoter sequence operably linked to a polynucleotide sequence (e.g., a transgene) to drive expression in a host cell, and in some embodiments, further comprises a transcription terminator sequence.

[0113] "Expression" includes any step involved in the production of the polypeptide, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from the cell.

[0114] "Produce" refers to the expression or production of proteins and / or other compounds by a cell. The term is intended to encompass any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

[0115] "Heterologous" or "recombinant" refers to the relationship between two or more nucleic acid or polypeptide sequences (eg, promoter sequence, signal peptide, terminator sequence, etc.) that are derived from different sources and are not essentially related.

[0116] "Host cell" and "host strain" refer to a suitable host for an expression vector comprising a polynucleotide as provided herein (e.g., a polynucleotide sequence encoding at least one DNA polymerase variant). In some embodiments, the host cell is a prokaryotic or eukaryotic cell transformed or transfected with a vector constructed using recombinant DNA techniques as known in the art.

[0117] An "analog" refers to a polypeptide having greater than 70% sequence identity but less than 100% sequence identity (e.g., greater than 75%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 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 further comprises one or more D-amino acid residues and non-peptide linkages between two or more amino acid residues.

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

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

[0120] "Isothermal amplification" refers to nucleic acid amplification that is not limited by thermal cycling constraints, such as in PCR.

[0121] "Polymerase chain reaction" and "PCR" refer to a method for producing multiple copies of a nucleic acid template of interest by repeated cycles of denaturation, annealing, and primer extension with a polymerase in the presence of nucleic acid primers, such as described in "PCR: Methods and Protocols" Methods in Molecular Biology," Springer Protocols (2017) and "Quantitative Real-Time PCR: Methods and Protocols," Methods in Molecular Biology, Springer Protocols (2014) (incorporated herein by reference). A series of denaturation, annealing, and extension constitutes a "cycle." The steps of denaturation, primer annealing, and polymerase extension can be repeated (i.e., using multiple cycles) to amplify the nucleic acid template. In some embodiments, the annealing and extension process occurs in a single step, sometimes referred to as 2-step PCR. Therefore, as used herein, the term "PCR" includes many variations of this method, including qPCR, hot start PCR, touchdown PCR, asymmetric PCR, multiplex PCR, long or long range PCR, assembly PCR, and inverse PCR, among others.

[0122] When used in reference to methods employing DNA polymerase, "target" refers to a region of nucleic acid used to prepare complementary DNA. A "target" is selected from other nucleic acids present in a method employing DNA polymerase. In some embodiments, a "segment" is a region of nucleic acid within a target sequence.

[0123] When used in the context of DNA polymerase, "target DNA" refers to the DNA (whole or part thereof) that is the object of making complementary DNA copies. The target DNA can be the entire DNA sequence or a part thereof, such as a segment of a DNA sequence.

[0124] "Target RNA" refers to the whole or part of RNA that is the object of preparing complementary DNA copies. The target RNA can be the whole RNA sequence or a part thereof, such as a segment of the RNA sequence.

[0125] "Sample template" refers to nucleic acid derived from a sample that is used to analyze 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. The background template may be accidentally included in the sample, which may be caused by carryover, or may be caused by the presence of nucleic acid contaminants from which the target nucleic acid is purified. For example, in some embodiments, nucleic acid from an organism other than the organism to be detected may be present in the test sample as background. However, it is not intended that the present invention be limited to any specific nucleic acid sample or template.

[0126] "Amplifiable nucleic acid" is used to refer to a nucleic acid that can be amplified by any amplification method, including but not limited to PCR and isothermal amplification. In most embodiments, the amplifiable nucleic acid comprises a sample template.

[0127] "Amplification product" refers to the resulting compound (ie, product) obtained after two or more cycles of a nucleic acid amplification method (eg, isothermal amplification or PCR, or as indicated above and below).

[0128] "Amplification reagents" refer to those reagents required for amplification (e.g., deoxyribonucleotide triphosphates, buffer, etc.) other than primers, nucleic acid templates, and amplification enzymes. Typically, the amplification reagents are placed and contained in a reaction vessel (e.g., a test tube, microwell, etc.) along with other reaction components. It is not intended that the present invention be limited to any particular amplification reagent, as any suitable reagent may be used in the present invention.

[0129] "Primer" refers to an oligonucleotide (i.e., a series of nucleotides) that can serve as a starting point for nucleic acid synthesis when placed under conditions that induce the synthesis of a primer extension product complementary to a nucleic acid chain (i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase, and at a suitable temperature and pH), whether the oligonucleotide is naturally occurring or synthetically produced, recombinantly produced, or produced by amplification. In some embodiments, the primer is single-stranded, but in some embodiments, the primer is double-stranded. In some embodiments, the primer is a primer of sufficient length to initiate the synthesis of an extension product in the presence of a nucleic acid polymerase. As known to those skilled in the art, the exact primer length depends on many factors.

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

[0131] "Altered fidelity" refers to the fidelity of a polymerase variant that is different from the fidelity of a parent polymerase or a reference polymerase from which the polymerase variant is derived. In some embodiments, the altered fidelity is higher than the fidelity of the parent polymerase or the reference polymerase, while in some other embodiments, the altered fidelity is lower than the fidelity of the parent polymerase or the reference polymerase. The altered fidelity can be determined by assaying the parent polymerase and the variant polymerase using any suitable assay known in the art and comparing their activities.

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

[0133] "Altered processivity" refers to a polymerase or variant thereof that has a processivity that is different from the processivity of the parent polymerase or reference polymerase from which the variant was 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. The altered processivity can be determined by assaying the parent / reference polymerase and the variant polymerase using any suitable assay known in the art and comparing their activities.

[0134] "Subject" includes mammals, such as humans, non-human primates, livestock, pets, and laboratory animals (eg, rodents and lagomorphs). It is intended that the term include females as well as males.

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

[0136] As used herein, "sample" refers to a material or substance for reacting with nucleic acid polymerase, for example, such as for detecting the presence of target nucleic acid or for preparing a DNA copy of target nucleic acid for sequencing or producing a cDNA library. In some embodiments, sample is a "biological sample", which refers to a sample of biological tissue or fluid. Such a sample is usually from humans, but includes tissues separated from non-human primates, domestic mammals (e.g., cats, dogs, cows, sheep, etc.) or rodents (e.g., mice and rats), and includes tissue sections, such as biopsy and autopsy samples, frozen sections collected for histological purposes, blood, plasma, serum, sputum, feces, tears, mucus, hair, skin, etc.."biological sample" also refers to cells or cell groups or a certain amount of tissue or fluid from an organism. In some embodiments, biological sample has been taken out from an animal, but the term "biological sample" may also refer to cells or tissues analyzed in vivo (i.e., not taken out from an animal, including cell culture). Typically, a "biological sample" will contain cells from an animal or organism, but the term can also refer to non-cellular biological materials, such as non-cellular fractions of blood, saliva, lymph or urine. Various types of biological samples can be used with the enzymes, compositions and methods in the present disclosure, including but not limited to biopsies, blood samples, buccal scrapes, saliva samples or nipple discharge. As used herein, a "biopsy" refers to a certain amount of tissue taken out from an animal (preferably a human) for diagnostic analysis. In patients with cancer, tissue can be taken out from a tumor, allowing the cells within the tumor to be analyzed. A "biopsy" can refer to any type of biopsy, such as a needle biopsy, a fine needle biopsy, a surgical biopsy, etc. In some embodiments, the sample can be from an environmental source, such as, but not limited to, water (e.g., oceans, rivers, garbage / sewers, etc.), soil, air, vents or surfaces (e.g., floors, machines, counters, etc.).

[0137] Engineered DNA polymerase peptides

[0138] In one aspect, the disclosure provides a DNA polymerase variant that recognizes DNA and RNA as templates, and wherein the DNA polymerase has been engineered to have one or more improved properties, including enhanced activity, enhanced fidelity, enhanced processivity, enhanced stability, enhanced sensitivity under low template DNA or RNA concentrations, increased resistance to inhibitors and increased salt tolerance and other compared to a reference DNA polymerase. As described above, the engineered DNA polymerase herein is based on a large fragment of the wild-type DNA polymerase of Parageobacillus genomosp.1, wherein the large fragment includes a DNA polymerase domain, but lacks a 5' exonuclease domain. The polynucleotides encoding the large fragment of the DNA polymerase of Parageobacillus genomosp.1 (i.e., residues 285 to 876 corresponding to the full-length wild-type sequence of SEQ ID NO:540) are used as the starting point for producing and identifying a DNA polymerase variant with a desired property.

[0139] The engineered DNA polymerase variants described herein can be used for polymerase reaction, including in various methods, such as in sequencing (e.g., NGS sequencing), isothermal amplification, DNA library preparation and diagnostic methods (such as for detecting target nucleic acid), preparing target DNA or RNA template (whole or part) complementary DNA. These engineered DNA polymerase variants can be used in solution, and in fixed embodiments. In some embodiments, the engineered DNA polymerase can be prepared and used as a non-fusion polypeptide or as a fusion polypeptide.

[0140] In some embodiments, the engineered DNA polymerase or a functional fragment thereof comprises an amino acid 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 residues 12 to 604 of SEQ ID NO: 2, 10, 80, 224 or 366, or to a reference sequence corresponding to SEQ ID NO: 2, 10, 80, 224 or 366, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 2, 10, 80, 224 or 366, or to a reference sequence corresponding to SEQ ID NO: 2, 10, 80, 224 or 366.

[0141] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 amino acid residues 12 to 604 of SEQ ID NO:2, or to a reference sequence corresponding to SEQ ID NO:2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12 to 604 of SEQ ID NO:2, or to 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 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 amino acid residues 12 to 604 of SEQ ID NO: 10, 80, 224 or 366, or to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12 to 604 of SEQ ID NO: 2, or to 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 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 amino acid residues 12 to 604 of an even numbered SEQ ID NO: 4-488, or to a reference sequence corresponding to an even numbered SEQ ID NO: 4-488, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12 to 604 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0144] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at the following amino acid positions: 21, 24, 25, 25, 34, 36, 52, 58, 66, 68, 81, 84, 92, 101, 105, 114, 115, 124, 133, 133, 134, 136, 144, 152, 154, 168, 183, 184, 191, 192, 210, 212, 221, 226, 230, 241, 252 , 253, 276, 287, 290, 294, 295, 300, 304, 322, 325, 372, 373, 374, 393, 427, 432, 452, 454, 456, 462, 470, 483, 486, 495, 505, 509, 541, 541, 545, 547, 551, 552, 573, 575, 578, 584, 585 or 593 or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:2.

[0145] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution 21M / V, 24M, 25L / M, 34D, 36T, 52G, 58K, 66T, 68G, 81R, 84Q, 92G, 101A, 105S, 114S, 115Y, 124L, 133A, 133R, 134R / T, 136P, 144R, 152R, 154W, 168G, 183P, 184T, 191E / R, 192E, 210A, 212V, 221M, 226K, 230G / Q, 241E / Q, 252T, 253R, 276 578F, 584V, 585R or 593S, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0146] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution E21M / V, L24M, P25L / M, E34D, S36T, E52G, A58K, A66T, E68G, S81R, L84Q, E92G, G101A, I105S, E114S, L115Y, I124L, Q133A / R, S134R / T, E136P, T144R, S152R, E154W, E168G, R183P, A184T, H191E / R, D192E, S210A, L212V, V221M, E226K, E230G / Q, A241E / Q, Q252T, E253R, R27 6G, D287R, E290G, P294R, Q295K, E300A / G / R, H304L, V322C, R325W, K372E, P373A, D374E, I393L, R427A, Q432K / L, N452R / S, N454G, P456T, E462R, I470S, Q483K, K486R, L495F, T505H, F509R, D541G / R / T, R545K, K547A / H / V, R551K, M552L, Q573R / V, I575V, E578F, I584V, Q585R or D593S, or a combination thereof, wherein the amino acid position is relative to the amino acid corresponding to SEQ Reference sequence of ID NO:2.

[0147] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 36, 241, 372, or 470, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution 36T, 241E / Q, 372E, or 470S, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 36. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 241. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 372. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 470.

[0148] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 52, 101, 124, 212, 294, 372, 393, 452, 483, or 509, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution 52G, 101A, 124L, 212V, 294R, 372K, 393L, 452R, 483K, or 509R, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 52. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 101. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 124. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 212. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 294. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 372. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 393. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 452. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 483. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 509.

[0149] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 253, 300, 454, 505, or 573, or a combination thereof, wherein the amino acid 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 substitution 253R, 300R, 454G, 505H, or 573V, or a combination thereof, wherein the amino acid 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 substitution at amino acid position 253. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 300. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 454. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 505. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 573.

[0150] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 134, 136, 154, 505, 547, 573, or 584, or a combination thereof, wherein the amino acid 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 substitution 134R, 136P, 154W, 505H, 547H, 573V, or 583V, or a combination thereof, wherein the amino acid 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 substitution at amino acid position 134. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 136. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 154. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 505. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 547. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 573. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 584.

[0151] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 36, 52, 101, 124, 134, 136, 154, 212, 241, 253, 294, 300, 372, 393, 452, 454, 456, 470, 483, 505, 509, 547, 573 or 584, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution 36T, 52G, 101A, 124L, 134R / T, 136P, 154W, 212V, 241E / Q, 253R, 294R, 300A / G / R, 372E / K, 393L, 452R / S, 454G, 456T, 470S, 483K, 505H, 509R, 547A / H / V, 573R / V, or 584V, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:2.

[0152] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions at one or more of the following amino acid positions: 509, 300, 452, 36 / 241 / 372 / 470, 124 / 192 / 210 / 372 / 427 / 456 / 552, 124, 52, 483, 372, 393, 212, 52 / 66, 133, 454, 154, 593, 462, 541, 21, 573, 505, 152, 294, 545, 101 / 241 / 470, 584, 304, 295, 578, 456 / 470, 253, 290, 192 / 241 / 372 / 456, or 252, wherein the amino acid positions are relative to a 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 substitution or set of substitutions 509R, 300R, 452R, 36T / 241Q / 372E / 470S, 124L / 192E / 210A / 372E / 427A / 456T / 552L, 124L, 52G, 483K, 372E, 452S, 300A, 393L, 300G, 212V, 52G / 66T, 133R , 454G, 154W, 593S, 462R, 541R, 21M, 573V, 505H, 152R, 294R, 545K, 101A / 241Q / 470S, 584V, 304L, 295K, 578F, 541T, 456T / 470S, 253R, 290G, 192E / 241E / 372E / 456T or 252T, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:2.

[0154] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions F509R, E300R, N452R, S36T / A241Q / K372E / I470S, I124L / D192E / S210A / K372E / P427A / P456T / M552L, I124L, E52G, Q483K, K372E, N452S, E300A, I393L, E300G, I212V, E52G / A66T, Q133R , N454G, E154W, D593S, E462R, D541R, E21M, Q573V, T505H, S152R, P294R, R545K, G101A / A241Q / I470S, I584V, H304L, Q295K, E578F, D541T, P456T / I470S, E253R, E290G, D192E / A241E / K372E / P456T or Q252T, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:2.

[0155] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one set of substitutions at the following amino acid positions: 36 / 52 / 101 / 124 / 212 / 241 / 294 / 393 / 452 / 470 / 483 / 509, 36 / 241 / 372 / 470, 36 / 154 / 212 / 241 / 294 / 300 / 393 / 452 / 470 / 483 / 509, 36 / 52 / 101 / 154 / 241 / 294 / 300 / 393 / 452 / 470 / 483 / 509, 36 / 52 / 101 / 154 / 212 / 241 / 294 / 300 / 372 / 452 / 470 / 483 / 509. 0 / 372 / 393 / 452 / 470 / 509、36 / 52 / 101 / 212 / 241 / 294 / 300 / 372 / 393 / 452 / 470 / 483 / 509 / 593、36 / 52 / 124 / 241 / 294 / 300 / 372 / 452 / 470 / 509、36 / 124 / 241 / 300 / 372 / 393 / 452 / 470 / 483 / 509 / 593、36 / 101 / 124 / 212 / 241 / 372 / 452 / 470 / 483 / 509、36 / 52 / 393 / 241 / 372 / 452 / 470 / 509 / 593、36 / 212 / 241 / 300 / 372 / 452 / 470 / 509 / 593、36 / 52 / 241 / 372 / 452 / 470 / 509 / 593、36 / 154 / 212 / 241 / 300 / 452 / 470 / 509 / 593、36 / 52 / 124 / 212 / 241 / 294 / 372 / 393 / 452 / 470 / 593、 36 / 124 / 241 / 372 / 452 / 470 / 509、36 / 152 / 241 / 253 / 287 / 372 / 470 / 505 / 541 / 573 / 584、36 / 154 / 212 / 241 / 300 / 372 / 452 / 470、36 / 152 / 241 / 253 / 287 / 304 / 372 / 454 / 470 / 545 / 573 / 584、36 / 52 / 124 / 154 / 241 / 294 / 300 / 452 / 470 / 509、36 / 241 / 294 / 300 / 452 / 470 / 509 / 593、36 / 52 / 212 / 241 / 372 / 452 / 470、36 / 12 4 / 241 / 294 / 372 / 393 / 452 / 470、36 / 52 / 241 / 300 / 372 / 470 / 509 / 593、36 / 152 / 241 / 253 / 287 / 372 / 454 / 470 / 541 / 584、36 / 52 / 101 / 241 / 372 / 452 / 470 / 509、36 / 52 / 154 / 212 / 241 / 300 / 393 / 470 / 509、21 / 36 / 241 / 253 / 287 / 372 / 454 / 470 / 584、36 / 52 / 393 / 241 / 372 / 452 / 470、36 / 52 / 124 / 154 / 241 / 294 / 372 / 470 / 509、36 / 101 / 124 / 212 / 241 / 300 / 372 / 393 / 470、36 / 124 / 241 / 294 / 300 / 452 / 470 / 509 / 593、36 / 52 / 124 / 154 / 241 / 294 / 372 / 470 / 483 / 593、36 / 52 / 241 / 393 / 452 / 470 / 509 / 593 、36 / 154 / 241 / 300 / 372 / 393 / 452 / 470 / 593 、 36 / 52 / 101 / 241 / 294 / 393 / 452 / 470 / 509 、21 / 36 / 152 / 241 / 253 / 287 / 304 / 372 / 454 / 470 / 505 、 36 / 52 / 241 / 300 / 372 / 393 / 470 、36 / 101 / 124 / 154 / 241 / 294 / 372 / 452 / 470 / 509、 36 / 154 / 212 / 241 / 393 / 470 / 509 / 593、21 / 36 / 241 / 253 / 287 / 372 / 454 / 470 / 505 / 541 、 36 / 241 / 287 / 372 / 454 / 470 / 505 / 584、36 / 52 / 124 / 241 / 294 / 300 / 452 / 470、36 / 241 / 287 / 372 / 470 / 505 / 541 / 573、36 / 241 / 253 / 372 / 470 / 505 / 584、36 / 84 / 241 / 253 / 287 / 372 / 456 / 470 / 573、36 / 52 / 241 / 470 / 509 / 593、36 / 241 / 372 / 470 / 509 / 593、36 / 154 / 212 / 241 / 300 / 470 / 509 、 36 / 52 / 154 / 241 / 294 / 372 / 470 / 509 / 593 、36 / 152 / 241 / 295 / 372 / 470 / 505 / 545 / 573 、 36 / 241 / 253 / 372 / 470 / 541 / 584 、36 / 241 / 253 / 295 / 304 / 372 / 454 / 462 / 470 / 573 / 584、36 / 241 / 253 / 372 / 454 / 470 / 505 / 573 / 584、36 / 154 / 212 / 241 / 300 / 372 / 470、36 / 241 / 287 / 372 / 454 / 470 / 545 / 573、21 / 36 / 152 / 241 / 287 / 304 / 372 / 454 / 470、36 / 152 / 241 / 253 / 295 / 304 / 372 / 470 / 541 / 573、36 / 152 / 241 / 253 / 287 / 304 / 372 / 470 / 454、36 / 152 / 241 / 372 / 454 / 470 / 573、36 / 124 / 241 / 294 / 470 / 509 / 593、36 / 152 / 241 / 253 / 295 / 372 / 454 / 470、36 / 154 / 241 / 300 / 372 / 452 / 470、36 / 241 / 287 / 304 / 372 / 470 / 505, 36 / 152 / 241 / 253 / 372 / 454 / 470 / 505, 36 / 241 / 253 / 372 / 454 / 470 / 505, 21 / 36 / 241 / 253 / 372 / 454 / 470 / 505 / 545, 36 / 152 / 241 / 253 / 287 / 372 / 470 / 505, 36 / 241 / 372 / 470 / 505 / 541 / 584 or 36 / 152 / 241 / 253 / 372 / 454 / 470 / 584, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0156] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution set 36T / 52G / 101A / 124L / 212V / 241Q / 294R / 393L / 452R / 470S / 483K / 509R, 36T / 241Q / 372E / 470S, 36T / 154W / 212V / 241Q / 294R / 300G / 393L / 452R / 470S / 483K / 509R, 36T / 52G / 101A / 154W / 241Q / 294R / 300R / 372E / 452S / 470S / 509R / 593S , 36T / 52G / 154W / 212V / 241Q / 294R / 300G / 372E / 393L / 452R / 470S , 36T / 212V / 241Q / 300R / 372E / 393L / 452R / 470S / 509R , 36T / 52G / 101A / 212V / 241Q / 294R / 300R / 372E / 393L / 452R / 470S / 483K / 509R / 593S , 36T / 52G / 124L / 241Q / 294R / 300R / 372E / 452S / 470S / 509R , 36T / 124L / 241Q / 300R / 372E / 393L / 452R / 470S / 483K / 509R / 593S , 36T / 101A / 124L / 212V / 241Q / 372E / 452R / 470S / 483K / 509R , 36T / 52G / 393L / 241Q / 372E / 452R / 470S / 509R / 593S , 36T / 212V / 241Q / 300R / 372E / 452R / 470S / 509R / 593S ,36T / 52G / 241Q / 372E / 452R / 470S / 509R / 593S , 36T / 154W / 212V / 241Q / 300R / 452R / 470S / 509R / 593S , 36T / 52G / 124L / 212V / 241Q / 294R / 372E / 393L / 452R / 470S / 593S , 36T / 124L / 241Q / 372E / 452R / 470S / 509R , 36T / 152R / 241Q / 253R / 287R / 372E / 470S / 505H / 541R / 573V / 584V ,36T / 154W / 212V / 241Q / 300R / 372E / 452R / 470S ,36T / 152R / 241Q / 253R / 287R / 304L / 372E / 454G / 470S / 545K / 573V / 584V、36T / 52G / 124L / 154W / 241Q / 294R / 300A / 452R / 470S / 509R 、36T / 241Q / 294R / 300R / 452S / 470S / 509R / 593S、36T / 52G / 212V / 241Q / 372E / 452R / 470S、36T / 124L / 241Q / 294R / 372E / 393L / 452R / 470S、36T / 52G / 241Q / 300R / 372E / 470S / 509R / 593S、36T / 152R / 241Q / 253R / 287R / 372E / 454G / 470S / 541R / 584V 、36T / 52G / 101A / 241Q / 372E / 452S / 470S / 509R 、36T / 52G / 154W / 212V / 241Q / 300R / 393L / 470S / 509R 、21M / 36T / 241Q / 253R / 287R / 372E / 454G / 470S / 584V、36T / 52G / 393L / 241Q / 372E / 452R / 470S、36T / 52G / 124L / 154W / 241Q / 294R / 372E / 470S / 509R 、36T / 101A / 124L / 212V / 241Q / 300R / 372E / 393L / 470S 、36T / 124L / 241Q / 294R / 300G / 452S / 470S / 509R / 593S 、36T / 52G / 124L / 154W / 241Q / 294R / 372E / 470S / 483K / 593S 、36T / 52G / 241Q / 393L / 452S / 470S / 509R / 593S 、36T / 154W / 241Q / 300R / 372E / 393L / 452S / 470S / 593S 、36T / 52G / 101A / 241Q / 294R / 393L / 452S / 470S / 509R 、21M / 36T / 152R / 241Q / 253R / 287R / 304L / 372E / 454G / 470S / 505H 、36T / 52G / 241Q / 300R / 372E / 393L / 470S 、36T / 101A / 124L / 154W / 241Q / 294R / 372E / 452S / 470S / 509R 、36T / 154W / 212V / 241Q / 393L / 470S / 509R / 593S 、21M / 36T / 241Q / 253R / 287R / 372E / 454G / 470S / 505H / 541G、36T / 241Q / 287R / 372E / 454G / 470S / 505H / 584V、36T / 52G / 124L / 241Q / 294R / 300R / 452S / 470S、36T / 241Q / 287R / 372E / 470S / 505H / 541R / 573V、36T / 241Q / 253R / 372E / 470S / 505H / 584V、36T / 84Q / 241Q / 253R / 287R / 372E / 456T / 470S / 573V、36T / 52G / 241Q / 470S / 509R / 593S、36T / 241Q / 372E / 470S / 509R / 593S、 36T / 154W / 212V / 241Q / 300R / 470S / 509R、36T / 52G / 154W / 241Q / 294R / 372E / 470S / 509R / 593S 、36T / 152R / 241Q / 295K / 372E / 470S / 505H / 545K / 573V、36T / 241Q / 253R / 372E / 470S / 541R / 584V、36T / 241Q / 253R / 295K / 304L / 372E / 454G / 462R / 470S / 573V / 584V、36T / 241Q / 253R / 372E / 454G / 470S / 505H / 573V / 584V、36T / 154W / 212V / 241Q / 300R / 372E / 470S、36T / 241Q / 287R / 372E / 454G / 470S / 545K / 573V 、21M / 36T / 152R / 241Q / 287R / 304L / 372E / 454G / 470S 、36T / 152R / 241Q / 253R / 295K / 304L / 372E / 470S / 541R / 573V, 36T / 152R / 241Q / 253R / 287R / 304L / 372E / 470S / 454G, 36T / 152R / 241Q / 372E / 454G / 470S / 573V, 36T / 124L / 241Q / 294R / 470S / 509R / 593S, 36T / 152R / 241Q / 253R / 295K / 372E / 454G / 470S, 36T / 154W / 241Q / 300R / 372 E / 452S / 470S, 36T / 241Q / 287R / 304L / 372E / 470S / 505H, 36T / 152R / 241Q / 253R / 372E / 454G / 470S / 505H, 36T / 241Q / 253R / 372E / 454G / 470S / 505H, 21M / 36T / 241Q / 253R / 372E / 454G / 470S / 505H / 545K, 36T / 152R / 241Q / 253R / 287R / 372E / 470S / 505H, 36T / 241Q / 372E / 470S / 505H / 541R / 584V or 36T / 152R / 241Q / 253R / 372E / 454G / 470S / 584V, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:2.

[0157] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions provided in Table 6.1 relative to the reference sequence corresponding to SEQ ID NO:2.

[0158] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one set of substitutions at the following amino acid positions: 36 / 52 / 101 / 124 / 212 / 241 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 509 / 541 / 584, 21 / 36 / 52 / 101 / 124 / 212 / 241 / 294 / 300 / 393 / 452 / 454 / 470 / 483 / 509 / 545 / 584, 36 / 52 / 101 / 124 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 509 / 541 / 584 、36 / 52 / 101 / 124 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 573 、21 / 36 / 52 / 101 / 124 / 154 / 212 / 241 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 509 / 573 、36 / 52 / 101 / 124 / 152 / 212 / 241 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 584 / 593 、36 / 52 / 101 / 124 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 456 / 470 / 483 / 505 / 509 / 545 / 573 / 584 、36 / 52 / 101 / 124 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 573 、36 / 52 / 101 / 124 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 509 / 541 / 573 / 584 、36 / 52 / 101 / 124 / 154 / 212 / 241 / 294 / 300 / 393 / 452 / 454 / 470 / 483 / 505 / 509 、21 / 36 / 52 / 101 / 124 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 509 / 545 / 573 / 593 、36 / 52 / 101 / 124 / 168 / 212 / 241 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 509 / 545 / 573 、36 / 52 / 101 / 124 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 470 / 483 / 505 / 509 / 573、36 / 52 / 101 / 124 / 154 / 212 / 241 / 294 / 300 / 393 / 452 / 470 / 483 / 505 / 509 / 545 / 584 、36 / 52 / 101 / 124 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 456 / 470 / 483 / 509 / 541 / 573 / 584 、21 / 36 / 52 / 101 / 124 / 154 / 212 / 241 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 545 、36 / 52 / 101 / 124 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 470 / 483 / 505 / 509 、21 / 36 / 52 / 101 / 124 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 470 / 483 / 509 / 573 、36 / 52 / 101 / 124 / 212 / 241 / 294 / 300 / 393 / 452 / 454 / 470 / 483 / 505 / 509 / 541 / 584 、36 / 52 / 101 / 124 / 212 / 241 / 294 / 300 / 393 / 452 / 454 / 470 / 483 / 505 / 509 / 541 / 573 、36 / 52 / 101 / 124 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 470 / 483 / 509 、36 / 52 / 101 / 124 / 154 / 212 / 241 / 294 / 300 / 393 / 452 / 454 / 470 / 483 / 509 / 573 、36 / 52 / 101 / 124 / 212 / 241 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 509 / 545 / 573 、36 / 52 / 101 / 124 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 509 、36 / 52 / 101 / 124 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 470 / 483 / 509 / 545 / 573 、21 / 36 / 52 / 101 / 124 / 152 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 509 / 541 / 573 、36 / 52 / 101 / 124 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 509 / 573、36 / 52 / 101 / 124 / 152 / 212 / 241 / 294 / 300 / 393 / 452 / 454 / 470 / 483 / 509 、36 / 52 / 101 / 124 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 470 / 483 / 505 / 509 / 541 / 593 、21 / 36 / 52 / 101 / 124 / 154 / 212 / 241 / 294 / 300 / 393 / 452 / 454 / 470 / 483 / 509 / 573 / 584 、36 / 52 / 101 / 124 / 154 / 212 / 241 / 294 / 300 / 393 / 452 / 456 / 470 / 483 / 509 / 573 / 593 、36 / 52 / 101 / 124 / 152 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 470 / 483 / 509 / 545 / 573 、21 / 36 / 52 / 101 / 124 / 212 / 241 / 294 / 300 / 393 / 452 / 456 / 470 / 483 / 495F / 505 / 509 / 541 / 584 、36 / 52 / 101 / 124 / 154 / 212 / 241 / 294 / 300 / 393 / 452 / 454 / 470 / 483 / 509 / 541 / 584 、36 / 52 / 101 / 124 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 470 / 483 / 505 / 509 / 541 / 573 / 584 、36 / 52 / 101 / 124 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 470 / 483 / 509 / 584 、36 / 52 / 101 / 124 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 509 / 545 、21 / 36 / 52 / 101 / 124 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 470 / 483 / 509 / 573 、36 / 52 / 101 / 124 / 212 / 241 / 294 / 300 / 393 / 452 / 454 / 470 / 483 / 509 / 545 、36 / 52 / 101 / 124 / 212 / 241 / 294 / 393 / 452 / 454 / 456 / 470 / 483 / 509 / 541 / 545 / 573 / 584 、36 / 52 / 101 / 124 / 152 / 212 / 241 / 253 / 294 / 393 / 452 / 454 / 470 / 483 / 505 / 509 / 545 / 573、21 / 36 / 52 / 101 / 124 / 212 / 241 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 495 / 509 、36 / 52 / 101 / 124 / 212 / 241 / 294 / 300 / 393 / 452 / 470 / 483 / 509 / 573 、36 / 52 / 101 / 124 / 154 / 212 / 241 / 294 / 300 / 393 / 452 / 454 / 470 / 483 / 509 / 545 、36 / 52 / 101 / 124 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 470 / 483 / 509 / 545 / 584 、21 / 36 / 52 / 101 / 124 / 212 / 241 / 294 / 300 / 393 / 452 / 470 / 483 / 505 / 509 、21 / 36 / 52 / 101 / 124 / 152 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 509 / 545 、36 / 52 / 101 / 124 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 456 / 470 / 483 / 509 / 541 / 573 、36 / 52 / 101 / 124 / 154 / 212 / 241 / 294 / 300 / 393 / 452 / 454 / 470 / 483 / 509 、36 / 52 / 101 / 124 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 470 / 483 / 509 / 541 、36 / 52 / 101 / 124 / 212 / 241 / 253 / 294 / 393 / 452 / 454 / 456 / 470 / 483 / 509 / 541 / 584 、21 / 36 / 52 / 101 / 124 / 212 / 241 / 294 / 300 / 393 / 452 / 470 / 483 / 509 / 573 、36 / 52 / 101 / 124 / 212 / 241 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 509 、36 / 52 / 101 / 124 / 212 / 241 / 294 / 300 / 393 / 452 / 454 / 470 / 483 / 509 、36 / 52 / 101 / 124 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 509 、36 / 52 / 101 / 124 / 154 / 212 / 241 / 294 / 393 / 452 / 456 / 470 / 483 / 505 / 509 / 593、36 / 52 / 101 / 124 / 212 / 241 / 294 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 、36 / 52 / 101 / 124 / 212 / 241 / 253 / 294 / 393 / 452 / 454 / 456 / 470 / 483 / 509 / 584 、36 / 52 / 101 / 124 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 470 / 483 / 509 、36 / 52 / 101 / 124 / 212 / 241 / 253 / 294 / 393 / 452 / 456 / 470 / 483 / 505 / 509 / 584 、36 / 52 / 101 / 124 / 212 / 241 / 294 / 393 / 452 / 454 / 456 / 470 / 483 / 509 、36 / 52 / 101 / 124 / 154 / 212 / 241 / 294 / 393 / 452 / 454 / 470 / 483 / 509 、36 / 52 / 101 / 124 / 212 / 241 / 294 / 393 / 452 / 456 / 470 / 483 / 509 / 584 、36 / 52 / 101 / 124 / 212 / 241 / 294 / 393 / 452 / 456 / 470 / 483 / 509 / 584 , 21 / 36 / 52 / 101 / 124 / 212 / 241 / 294 / 393 / 452 / 470 / 483 / 505 / 509 / 545 / 584, 21 / 36 / 52 / 101 / 124 / 154 / 212 / 241 / 294 / 393 / 452 / 470 / 483 / 509 / 545 / 573, 36 / 52 / 101 / 124 / 212 / 241 / 294 / 393 / 452 / 454 / 470 / 483 / 509 / 584 or 36 / 52 / 101 / 124 / 154 / 212 / 241 / 253 / 294 / 393 / 452 / 470 / 483 / 505 / 509 Reference sequence of ID NO:2.

[0159] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution set 36T / 52G / 101A / 124L / 212V / 241Q / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 509R / 541R / 584V, 21M / 36T / 52G / 101A / 124L / 212V / 241Q / 294R / 300R / 393L / 452R / 454G / 470S / 483K / 509R / 541R / 584V, R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 509R / 584V, 36T / 52G / 101A / 124L / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 573V, 21M / 36T / 52G / 101A / 124L / 154W / 212V / 241Q / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 509R / 57 3V, 36T / 52G / 101A / 124L / 152R / 212V / 241Q / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 584V / 593S, 36T / 52G / 101A / 124L / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 456T / 470S / 483K / 505H / 509R / 545K / 573V / 584V, 36T / 52G / 101A / 124L / 212V / 241Q / 253R / 29 4R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 573V, 36T / 52G / 101A / 124L / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454 G / 456T / 470S / 483K / 509R / 541G / 573V / 584V, 36T / 52G / 101A / 124L / 154W / 212V / 241Q / 294R / 300R / 393L / 452R / 454G / 470S / 483K / 505H / 509R,21M / 36T / 52G / 101A / 124L / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 509R / 545K / 573V / 593S、36T / 52G / 101A / 124L / 168G / 212V / 241Q / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 509R / 545K / 573V、36T / 52G / 101A / 124L / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 470S / 483K / 505H / 509R / 573V、36T / 52G / 101A / 124L / 154W / 212V / 241Q / 294R / 300R / 393L / 452R / 470S / 483K / 505H / 509R / 545K / 584V、36T / 52G / 101A / 124L / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 456T / 470S / 483K / 509R / 541R / 573V / 584V、21M / 36T / 52G / 101A / 124L / 154W / 212V / 241Q / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 545K、36T / 52G / 101A / 124L / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 470S / 483K / 505H / 509R、21M / 36T / 52G / 101A / 124L / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 470S / 483K / 509R / 573V、36T / 52G / 101A / 124L / 212V / 241Q / 294R / 300R / 393L / 452R / 454G / 470S / 483K / 505H / 509R / 541R / 584V、36T / 52G / 101A / 124L / 212V / 241Q / 294R / 300R / 393L / 452R / 454G / 470S / 483K / 505H / 509R / 541R / 573V、36T / 52G / 101A / 124L / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 470S / 483K / 509R、36T / 52G / 101A / 124L / 154W / 212V / 241Q / 294R / 300R / 393L / 452R / 454G / 470S / 483K / 509R / 573V、36T / 52G / 101A / 124L / 212V / 241Q / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 509R / 545K / 573V、36T / 52G / 101A / 124L / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 509R、36T / 52G / 101A / 124L / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 470S / 483K / 509R / 545K / 573V、21M / 36T / 52G / 101A / 124L / S152R / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 509R / 541R / 573V、36T / 52G / 101A / 124L / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 509R / 573V、36T / 52G / 101A / 124L / S152R / 212V / 241Q / 294R / 300R / 393L / 452R / 454G / 470S / 483K / 509R、36T / 52G / 101A / 124L / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 470S / 483K / 505H / 509R / 541R / 593S、21M / 36T / 52G / 101A / 124L / 154W / 212V / 241Q / 294R / 300R / 393L / 452R / 454G / 470S / 483K / 509R / 573V / 584V、36T / 52G / 101A / 124L / 154W / 212V / 241Q / 294R / 300R / 393L / 452R / 456T / 470S / 483K / 509R / 573V / 593S、36T / 52G / 101A / 124L / S152R / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 470S / 483K / 509R / 545K / 573V、21M / 36T / 52G / 101A / 124L / 212V / 241Q / 294R / 300R / 393L / 452R / 456T / 470S / 483K / L495F / 505H / 509R / 541R / 584V、36T / 52G / 101A / 124L / 154W / 212V / 241Q / 294R / 300R / 393L / 452R / 454G / 470S / 483K / 509R / 541G / 584V、36T / 52G / 101A / 124L / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 470S / 483K / 505H / 509R / 541R / 573V / 584V、36T / 52G / 101A / 124L / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 470S / 483K / 509R / 584V、36T / 52G / 101A / 124L / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 509R / 545K、21M / 36T / 52G / 101A / 124L / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 470S / 483K / 509R / 573V、36T / 52G / 101A / 124L / 212V / 241Q / 294R / 300R / 393L / 452R / 454G / 470S / 483K / 509R / 545K、36T / 52G / 101A / 124L / 212V / 241Q / 294R / 393L / 452R / 454G / 456T / 470S / 483K / 509R / 541G / 545K / 573V / 584V、36T / 52G / 101A / 124L / S152R / 212V / 241Q / 253R / 294R / 393L / 452R / 454G / 470S / 483K / 505H / 509R / 545K / 573V、21M / 36T / 52G / 101A / 124L / 212V / 241Q / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / L495F / 509R、36T / 52G / 101A / 124L / 212V / 241Q / 294R / 300R / 393L / 452R / 470S / 483K / 509R / 573V、36T / 52G / 101A / 124L / 154W / 212V / 241Q / 294R / 300R / 393L / 452R / 454G / 470S / 483K / 509R / 545K、36T / 52G / 101A / 124L / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 470S / 483K / 509R / 545K / 584V、21M / 36T / 52G / 101A / 124L / 212V / 241Q / 294R / 300R / 393L / 452R / 470S / 483K / 505H / 509R、21M / 36T / 52G / 101A / 124L / S152R / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 509R / 545K、36T / 52G / 101A / 124L / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 456T / 470S / 483K / 509R / 541R / 573V、36T / 52G / 101A / 124L / 154W / 212V / 241Q / 294R / 300R / 393L / 452R / 454G / 470S / 483K / 509R、36T / 52G / 101A / 124L / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 470S / 483K / 509R / 541R、36T / 52G / 101A / 124L / 212V / 241Q / 253R / 294R / 393L / 452R / 454G / 456T / 470S / 483K / 509R / 541G / 584V、21M / 36T / 52G / 101A / 124L / 212V / 241Q / 294R / 300R / 393L / 452R / 470S / 483K / 509R / 573V、36T / 52G / 101A / 124L / 212V / 241Q / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 509R 、36T / 52G / 101A / 124L / 212V / 241Q / 294R / 300R / 393L / 452R / 454G / 470S / 483K / 509R 、36T / 52G / 101A / 124L / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 509R、36T / 52G / 101A / 124L / 154W / 212V / 241Q / 294R / 393L / 452R / 456T / 470S / 483K / 505H / 509R / 593S、36T / 52G / 101A / 124L / 212V / 241Q / 294R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R, 36T / 52G / 101A / 124L / 2 12V / 241Q / 253R / 294R / 393L / 452R / 454G / 456T / 470S / 483K / 509R / 584V, 36T / 52G / 101A / 124L / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 470S / 483K / 509R, 36T / 52G / 101A / 124L / 212V / 241Q / 253R / 294R / 393L / 452R / 456T / 470S / 483K / 505H / 509R / 584V, 36T / 52G / 101A / 124L / 212V / 241Q / 294R / 393L / 452R / 454G / 456T / 470S / 483K / 509R ,36T / 52G / 101A / 124L / 154W / 212V / 241Q / 294R / 393L / 452R / 454G / 470S / 483K / 509R ,36T / 52G / 101A / 124L / 212V / 241Q / 294R / 393L / 452R / 470S / 483K / 509R / 584V ,36T / 52G / 101A / 124L / 212V / 241Q / 294R / 393L / 452R / 470S / 483K / 509R / 584V ,36T / 52G / 101A / 124L / 212V / 241Q / 294R / 393L / 452R / 456T / 470S / 483K / 509R / 584V ,21M / 36T / 52G / 101A / 124L / 212V / 241Q / 294R / 393L / 452R / 470S / 483K / 505H / 509R / 545K / 584V, 21V / 36T / 52G / 101A / 124L / 154W / 212V / 241Q / 294R / 393L / 452R / 470S / 483K / 509R / 545K / 573V, 36 T / 52G / 101A / 124L / 212V / 241Q / 294R / 393L / 452R / 454G / 470S / 483K / 509R / 584V or 36T / 52G / 101A / 124L / 154W / 212V / 241Q / 253R / 294R / 393L / 452R / 470S / 483K / 505H / 509R, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0160] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions provided in Table 7.1 relative to the reference sequence corresponding to SEQ ID NO:2.

[0161] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one set of substitutions at the following amino acid positions: 36 / 52 / 101 / 124 / 144 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573, 36 / 52 / 101 / 124 / 154 / 191 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 573, 36 / 52 / 101 / 124 / 154 / 191 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 573, 294 / 300 / 373 / 374 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 573、36 / 52 / 101 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、36 / 52 / 81 / 101 / 124 / 144 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、36 / 52 / 101 / 124 / 154 / 2 12 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 573、36 / 52 / 101 / 124 / 144 / 154 / 191 / 212 / 230 / 241 / 253 / 294 / 300 / 322 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 573、36 / 52 / 68 / 101 / 124 / 144 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 573、36 / 52 / 101 / 124 / 14 4 / 154 / 212 / 226 / 230 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 50 5 / 509 / 573, 36 / 52 / 101 / 124 / 144 / 154 / 212 / 241 / 253 / 294 / 300 / 374 / 393 / 4 52 / 454 / 456 / 470 / 483 / 325 / 505 / 509 / 573、36 / 52 / 81 / 101 / 114S / 124 / 144 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 573、36 / 52 / 101 / 124 / 144 / 154 / 212 / 241 / 253 / 276 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 573、36 / 52 / 101 / 124 / 134 / 144 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 573、36 / 52 / 101 / 124 / 144 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 5 09 / 573、36 / 52 / 81 / 101 / 124 / 144 / 154 / 183 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、36 / 52 / 68 / 81 / 101 / 124 / 133 / 134 / 144 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、36 / 52 / 101 / 124 / 144 / 154 / 212 / 241 / 253 / 294 / 300 / 3 74 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 573、36 / 52 / 92 / 101 / 124 / 133 / 144 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 573、36 / 52 / 101 / 124 / 144 / 154 / 212 / 241 / 253 / 294 / 300 / 373 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 573、36 / 52 / 101 / 114 / 124 / 144 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 573, 36 / 52 / 101 / 124 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 551 / 573 / 585 or 36 / 52 / 81 / 101 / 124 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:2.

[0162] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution set 36T / 52G / 101A / 124L / 144R / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V, 36T / 52G / 101A / 124L / 154W / 191E / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V 2G / 101A / 124L / 144R / 154W / 212V / 241Q / 253R / 294R / 300R / 373A / 374E / 393 L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 573V, 36T / 52G / 101A / 124L / S 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 4 70S / 483K / 505H / 509R / 547H / 573V, 36T / 52G / 81R / 101A / 124L / 144R / 154W / 2 12V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 50 9R / 547H / 573V, 36T / 52G / 101A / 124L / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 573V, 36T / 52G / 101A / 124 L / 144R / 154W / 191R / 212V / 230G / 241Q / 253R / 294R / 300R / 322C / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 573V, 36T / 52G / 68G / 101A / 124L / 144R / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 573V, 36T / 52G / 101A / 124L / 144R / 154W / 212V / 226K / 230Q / 241 Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 573V、36T / 52G / 101A / 124L / 144R / 154W / 212V / 241Q / 253R / 294R / 300R / 374E / 393L / 452R / 454G / 456T / 470S / 483K / 486R / 505H / 509R / 573V、36T / 52G / 81R / 101A / 114S / 124L / 144R / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 573V、36T / 52G / 101A / 124L / 144R / 154W / 212V / 241Q / 253R / 276G / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 573V、36T / 52G / 101A / 124L / S134T / 144R / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 573V、36T / 52G / 101A / 124L / 144R / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 573V、36T / 52G / 81R / 101A / 124L / 144R / 154W / 183P / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547A / 573V、36T / 52G / 68G / 81R / 101A / 124L / 133A / S134R / 144R / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V、36T / 52G / 101A / 124L / 144R / 154W / 212V / 241Q / 253R / 294R / 300R / 374E / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 573V、36T / 52G / 92G / 101A / 124L / 133A / 144R / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 573V、36T / 52G / 101A / 124L / 144R / 154W / 212V / 241Q / 253R / 294R / 300R / 373A / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 573V, 36T / 52G / 101A / 114S / 124L / 144R / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 573V, 36T / 52G / 1 01A / 124L / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 551K / 573R / 585R or 36T / 52G / 81R / 101A / 124L / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 551K / 573R / 585R or 36T / 52G / 81R / 101A / 124L / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547V / 573V

[0163] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions provided in Table 8.1 relative to the reference sequence corresponding to SEQ ID NO:2.

[0164] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one set of substitutions at the following amino acid positions: 24 / 36 / 52 / 58 / 101 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573, 36 / 52 / 58 / 101 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573 75、36 / 52 / 58 / 101 / 115 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573 / 575、24 / 36 / 52 / 101 / 115 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573 / 575、24 / 25 / 36 / 52 / 101 / 124 / 134 / 136 / 154 / 212 / 221 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、25 / 36 / 52 / 58 / 101 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573 / 575、24 / 25 / 36 / 52 / 58 / 101 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 43 2 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573 / 575、36 / 52 / 101 / 124 / 134 / 136 / 154 / 212 / 221 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573 / 575、36 / 52 / 101 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573 / 575、24 / 36 / 52 / 101 / 124 / 134 / 136 / 154 / 212 / 221 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、24 / 36 / 52 / 101 / 115 / 124 / 134 / 136 / 154 / 212 / 221 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、24 / 36 / 52 / 101 / 115 / 124 / 134 / 136 / 154 / 212 / 221 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573 / 575、36 / 52 / 101 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、24 / 34 / 36 / 52 / 101 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、24 / 36 / 52 / 101 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573 / 575、36 / 52 / 101 / 124 / 134 / 136 / 154 / 184 / 212 / 221 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573 / 575、24 / 36 / 52 / 101 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、24 / 25 / 36 / 52 / 58 / 101 / 115 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573 / 575、25 / 36 / 52 / 101 / 124 / 134 / 136 / 154 / 212 / 221 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、25 / 36 / 52 / 58 / 101 / 115 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、36 / 52 / 101 / 124 / 134 / 136 / 154 / 212 / 221 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573 / 575、36 / 52 / 101 / 124 / 134 / 136 / 154 / 212 / 221 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、24 / 36 / 52 / 58 / 101 / 115 / 124 / 134 / 136 / 154 / 212 / 221 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573 / 575、24 / 25 / 36 / 52 / 101 / 124 / 134 / 136 / 154 / 212 / 221 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573 / 575、24 / 25 / 36 / 52 / 58 / 101 / 124 / 134 / 136 / 154 / 212 / 221 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、34 / 36 / 52 / 58 / 101 / 105 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、24 / 25 / 36 / 52 / 101 / 115 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、24 / 36 / 52 / 101 / 124 / 134 / 136 / 154 / 212 / 221 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573 / 575、36 / 52 / 101 / 115 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、36 / 52 / 58 / 101 / 124 / 134 / 136 / 154 / 212 / 221 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、36 / 52 / 58 / 101 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573 / 575、25 / 36 / 52 / 101 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573 / 575、25 / 36 / 52 / 101 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、24 / 36 / 52 / 101 / 124 / 134 / 136 / 154 / 212 / 221 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573 / 575、24 / 36 / 52 / 101 / 115 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、24 / 25 / 36 / 52 / 101 / 105 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、25 / 34 / 36 / 52 / 101 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、25 / 36 / 52 / 101 / 105 / 124 / 134 / 136 / 154 / 212 / 221 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、24 / 25 / 36 / 52 / 101 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573、24 / 36 / 52 / 58 / 101 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573 / 575 or 24 / 25 / 36 / 52 / 101 / 105 / 124 / 134 / 136 / 154 / 212 / 241 / 253 / 294 / 300 / 393 / 432 / 452 / 454 / 456 / 470 / 483 / 505 / 509 / 547 / 573 / 575, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0165] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution set 24M / 36T / 52G / 58K / 101A / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V, 36T / 52G / 58K / 101A / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V 2R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V / 575V, 36T / 52G / 58K / 101A / 115Y / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V / 575V、24M / 36T / 52G / 101A / 115Y / 124L / 134R / 136P / 154W / 212V / 241Q / 2 53R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V / 575V, 24M / 25L / 36T / 52G / 101A / 124L / 134R / 136P / 154W / 212V / 221M / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V、25L / 36T / 52G / 58K / 101A / 124L / 13 4R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V / 575V、24M / 25M / 36T / 52G / 58K / 101A / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V / 575V,36T / 52G / 101A / 124L / 134R / 136P / 154W / 212V / 221M / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V / 575V、36T / 52G / 101A / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V / 575V、24M / 36T / 52G / 101A / 124L / 134R / 136P / 154W / 212V / 221M / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V、24M / 36T / 52G / 101A / 115Y / 124L / 134R / 136P / 154W / 212V / 221M / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V、24M / 25M / 36T / 52G / 101A / 124L / 134R / 136P / 154W / 212V / 221M / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V、24M / 36T / 52G / 101A / 115Y / 124L / 134R / 136P / 154W / 212V / 221M / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V / 575V、36T / 52G / 101A / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V、24M / 34D / 36T / 52G / 101A / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V、24M / 36T / 52G / 101A / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V / 575V、36T / 52G / 101A / 124L / 134R / 136P / 154W / 184T / 212V / 221M / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V / 575V、24M / 36T / 52G / 101A / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V、24M / 25L / 36T / 52G / 58K / 101A / 115Y / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V / 575V、25L / 36T / 52G / 101A / 124L / 134R / 136P / 154W / 212V / 221M / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V、25M / 36T / 52G / 58K / 101A / 115Y / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V、36T / 52G / 101A / 124L / 134R / 136P / 154W / 212V / 221M / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V / 575V、36T / 52G / 101A / 124L / 134R / 136P / 154W / 212V / 221M / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V、24M / 36T / 52G / 58K / 101A / 115Y / 124L / 134R / 136P / 154W / 212V / 221M / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V / 575V、24M / 25M / 36T / 52G / 101A / 124L / 134R / 136P / 154W / 212V / 221M / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V / 575V、24M / 25M / 36T / 52G / 58K / 101A / 124L / 134R / 136P / 154W / 212V / 221M / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V、34D / 36T / 52G / 58K / 101A / 105S / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V、24M / 25M / 36T / 52G / 101A / 115Y / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V、24M / 36T / 52G / 101A / 124L / 134R / 136P / 154W / 212V / 221M / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V / 575V、36T / 52G / 101A / 115Y / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V、36T / 52G / 58K / 101A / 124L / 134R / 136P / 154W / 212V / 221M / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V、36T / 52G / 58K / 101A / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V / 575V、25L / 36T / 52G / 101A / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V / 575V、25L / 36T / 52G / 101A / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V、24M / 36T / 52G / 101A / 124L / 134R / 136P / 154W / 212V / 221M / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V / 575V、24M / 36T / 52G / 101A / 115Y / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V、24M / 25M / 36T / 52G / 101A / 105S / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V、25M / 34D / 36T / 52G / 101A / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V、24M / 25L / 36T / 52G / 101A / 105S / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V、25M / 36T / 52G / 101A / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 45 4G / 456T / 470S / 483K / 505H / 509R / 547H / 573V, 25M / 36T / 52G / 101A / 105S / 124L / 134R / 136P / 154W / 212V / 221M / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V、24M / 25L / 36T / 52G / 101A / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432 L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V, 24M / 36T / 52G / 58K / 101A / 124L / 134R / 1 36P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 5 73V / 575V or 24M / 25M / 36T / 52G / 101A / 105S / 124L / 134R / 136P / 154W / 212V / 241Q / 253R / 294R / 300R / 393L / 432L / 452R / 454G / 456T / 470S / 483K / 505H / 509R / 547H / 573V / 575V, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0166] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions provided in Table 9.1 relative to the reference sequence corresponding to SEQ ID NO:2.

[0167] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of SEQ ID NO: 10, 80, 224 or 366. In some embodiments, the engineered DNA polymerase comprises the following restriction: a DNA polymerase having an amino acid sequence corresponding to residues 12 to 604 of SEQ ID NO: 2 is excluded.

[0168] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of an even numbered SEQ ID NO:4-488, or to a reference sequence corresponding to an even numbered SEQ ID NO:4-488.

[0169] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of SEQ ID NO: 10, 80, 224 or 366, or to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 10, 80, 224 or 366, or to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366.

[0170] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of an even numbered SEQ ID NO: 4-488, or to a reference sequence corresponding to an even numbered SEQ ID NO: 4-488, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 10, 80, 224, or 366, or relative to the reference sequence corresponding to SEQ ID NO: 10, 80, 224, or 366.

[0171] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at the following amino acid positions: 21, 24, 25, 25, 34, 36, 52, 58, 66, 68, 81, 84, 92, 101, 105, 114, 115, 124, 133, 133, 134, 136, 144, 152, 154, 168, 183, 184, 191, 192, 210, 212, 221, 226, 230, 241, 252 , 253, 276, 287, 290, 294, 295, 300, 304, 322, 325, 372, 373, 374, 393, 427, 432, 452, 454, 456, 462, 470, 483, 486, 495, 505, 509, 541, 541, 545, 547, 551, 552, 573, 575, 578, 584, 585 or 593 or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366.

[0172] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or amino acid residue 21M / V, 24M, 25L / M, 34D, 36T, 52G, 58K, 66T, 68G, 81R, 84Q, 92G, 101A, 105S, 114S, 115Y, 124L, 133A, 133R, 134R / T, 136P, 144R, 152R, 154W, 168G, 183P, 184T, 191E / R, 192E, 210A, 212V, 221M, 226K, 230G / Q, 241E / Q, 252T, 253R, 2 76G, 287R, 290G, 294R, 295K, 300A / G / R, 304L, 322C, 325W, 372E / K, 373A, 374E, 393L, 427A, 432K / L, 452R / S, 454G, 456T, 462R, 470S, 483K, 486R, 495F, 505H, 509R, 541G / R / T, 545K, 547A / H / V, 551K, 552L, 573R / V, 575V, 578F, 584V / I, 585R or 593S, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366.

[0173] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 36, 241, 372, or 470, or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to SEQ ID NO: 10, 80, 224, or 366. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or amino acid residues 36T, 241Q, 372K / E, or 470S, or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to SEQ ID NO: 10, 80, 224, or 366.

[0174] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at the following amino acid positions: 36, 52, 101, 124, 134, 136, 154, 212, 241, 253, 294, 300, 372, 393, 452, 454, 456, 470, 483, 505, 509, 547, 573 or 584 or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366. In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or amino acid residue 36T, 52G, 101A, 124L, 134R, 136P, 154W, 212V, 241Q, 253R, 294R, 300R, 372K / E, 393L, 452R, 454G, 456T, 470S, 483K, 505H, 509R, 547H, 573V, or 584V / I, or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to SEQ ID NO: 10, 80, 224, or 366.

[0175] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of SEQ ID NO: 10, or to a reference sequence corresponding to SEQ ID NO: 10, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 10, or to a reference sequence corresponding to SEQ ID NO: 10.

[0176] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of SEQ ID NOs: 10-218, or to a reference sequence corresponding to an even numbered SEQ ID NOs: 10-218, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 10, or relative to the reference sequence corresponding to SEQ ID NO: 10.

[0177] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one set of substitutions at one or more of the following amino acid positions: 52 / 101 / 124 / 212 / 294 / 372 / 393 / 452 / 483 / 509, 52 / 124 / 300 / 393 / 452, 154 / 212 / 294 / 300 / 372 / 393 / 452 / 483 / 509, 52 / 101 / 154 / 294 / 300 / 452 / 509 / 593, 52 / 154 / 212 / 294 / 300 / 393 / 452, 212 / 300 / 393 / 452 / 509, 52 / 101 / 212 / 294 / 300 / 393 / 452 / 483 / 509 / 593、52 / 124 / 294 / 300 / 452 / 509、124 / 300 / 393 / 452 / 483 / 509 / 593、101 / 124 / 212 / 452 / 483 / 509、52 / 393 / 452 / 509 / 593、212 / 300 / 452 / 509 / 59 3. 52 / 452 / 509 / 593, 154 / 212 / 300 / 372 / 452 / 509 / 593, 52 / 124 / 212 / 294 / 393 / 452 / 593, 124 / 452 / 509, 152 / 253 / 287 / 505 / 541 / 573 / 584, 154 / 212 / 300 / 4 52, 152 / 253 / 287 / 304 / 454 / 545 / 573 / 584, 52 / 124 / 154 / 294 / 300 / 372 / 452 / 509, 294 / 300 / 372 / 452 / 509 / 593, 52 / 212 / 452, 124 / 294 / 393 / 452, 52 / 300 / 509 / 593、152 / 253 / 287 / 454 / 541 / 584、52 / 101 / 452 / 509、52 / 154 / 212 / 300 / 372 / 393 / 509、21 / 253 / 287 / 454 / 584、52 / 393 / 452、52 / 124 / 154 / 294 / 509、1 01 / 124 / 212 / 300 / 393、124 / 294 / 300 / 372 / 452 / 509 / 593、52 / 124 / 154 / 294 / 483 / 593、52 / 372 / 393 / 452 / 509 / 593、154 / 300 / 393 / 452 / 593、52 / 101 / 294 / 372 / 393 / 452 / 509、21 / 152 / 253 / 287 / 304 / 454 / 505、52 / 300 / 393、101 / 124 / 154 / 294 / 452 / 509、154 / 212 / 372 / 393 / 509 / 593、21 / 253 / 287 / 454 / 505 / 541、287 / 454 / 505 / 584、52 / 124 / 294 / 300 / 372 / 452、287 / 505 / 541 / 573、253 / 505 / 584、84 / 253 / 287 / 456 / 573、52 / 372 / 509 / 593、509 / 593、154 / 212 / 300 / 372 / 509、52 / 154 / 294 / 509 / 593、152 / 295 / 505 / 545 / 573、253 / 541 / 584、253 / 295 / 304 / 454 / 462 / 573 / 584、253 / 454 / 505 / 573 / 584、154 / 212 / 300、287 / 454 / 54 or 152 / 253 / 454 / 584, wherein the amino acid positions are relative to the amino acid positions corresponding to SEQ ID NO: 5 / 573, 21 / 152 / 287 / 304 / 454, 152 / 253 / 295 / 304 / 541 / 573, 152 / 253 / 287 / 304 / 454, 152 / 454 / 573, 124 / 294 / 372 / 509 / 593, 152 / 253 / 295 / 454, 154 / 300 / 452, 287 / 304 / 505, 152 / 253 / 454 / 505, 253 / 454 / 505, 21 / 253 / 454 / 505 / 545, 152 / 253 / 287 / 505, 505 / 541 / 584 or 152 / 253 / 454 / 584, wherein the amino acid positions are relative to the amino acid positions corresponding to SEQ ID NO: 5 / 573, 21 / 152 / 287 / 304 / 454, 152 / 253 / 295 / 304 / 541 Reference sequence of ID NO:10.

[0178] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution set 52G / 101A / 124L / 212V / 294R / 372K / 393L / 452R / 483K / 509R, 52G / 124L / 300A / 393L / 452R, 154W / 212V / 294R / 300G / 372K / 393L / 452R / 483K / 509R, 52G / 101A / 154W / 294R / 300R / 452S / 509R / 593S, 52G / 154W / 212V / 294R / 300G / 393L / 452R, 212V / 300R / 393L / 452R / 509R, 52G / 101A / 212V / 294R / 300R / 393L / 452R / 483K / 509R / 593S, 52G / 124L / 294R / 300R / 452S / 509R, 124L / 300R / 393L / 45 2R / 483K / 509R / 593S, 101A / 124L / 212V / 452R / 483K / 509R, 52G / 393L / 452R / 509R / 593S, 212V / 300R / 452R / 509R / 593S, 52G / 452R / 509R / 593S , 154W / 212V / 300R / 372K / 452R / 509R / 593S, 52G / 124L / 212V / 294R / 393L / 452R / 593S, 124L / 452R / 509R , 152R / 253R / 287R / 505H / 541R / 573V / 584V, 154W / 212V / 300R / 452R ,152R / 253R / 287R / 304L / 454G / 545K / 573V / 584V, 52G / 124L / 154W / 294R / 300A / 372K / 452R / 5 09R、294R / 300R / 372K / 452S / 509R / 593S、52G / 212V / 452R、124L / 294R / 393L / 452R、52G / 300R / 509R / 593S, 152R / 253R / 287R / 454G / 541R / 584V, 52G / 101A / 452S / 509R, 52G / 154W / 212V / 300R / 372K / 393L / 509R, 21M / 253R / 287R / 454G / 584V, 52G / 393L / 452R, 52G / 124L / 154W / 294R / 509R , 101A / 124L / 212V / 300R / 393L, 124L / 294R / 300G / 372K / 452S / 509R / 593S,52G / 124L / 154W / 294R / 483K / 593S, 52G / 372K / 393L / 452S / 509R / 593S, 154W / 300R / 393L / 452S / 593S , 52G / 101A / 294R / 372K / 393L / 452S / 509R, 21M / 152R / 253R / 287R / 304L / 454G / 505H, 52G / 300R / 393L, 101A / 124L / 154W / 2 94R / 452S / 509R, 154W / 212V / 372K / 393L / 509R / 593S, 21M / 253R / 287R / 454G / 505H / 541G, 287R / 454G / 505H / 584V, 52G / 12 4L / 294R / 300R / 372K / 452S, 287R / 505H / 541R / 573V, 253R / 505H / 584V, 84Q / 253R / 287R / 456T / 573V, 52G / 372K / 509R / 593 S, 509R / 593S, 154W / 212V / 300R / 372K / 509R, 52G / 154W / 294R / 509R / 593S, 152R / 295K / 505H / 545K / 573V, 253R / 541R / 584V , 253R / 295K / 304L / 454G / 462R / 573V / 584V, 253R / 454G / 505H / 573V / 584V, 154W / 212V / 300R, 287R / 454G / 545K / 573V, 21M / 152R / 287R / 304L / 454G, 152R / 253R / 295K / 304L / 541R / 573V, 152R / 253R / 287R / 304L / 454G, 152R / 454G / 573V, 124L / 294R / 372K / 509R / 593S, 152R / 253R / 295K / 454G, 154W / 300R / 452S, 287R / 304L / 505H, 152R / 253R / 454G / 505H, 253R / 454G / 505H, 21M / 253R / 454G / 505H / 545K, 152R / 253R / 287R / 505H, 505H / 541R / 584V or 152R / 253R / 454G / 584V, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:10.

[0179] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution set E52G / G101A / I124L / I212V / P294R / E372K / I393L / N452R / Q483K / F509R, E52G / I124L / E300A / I393L / N452R, E154W / I212V / P294R / E300G / E372K / I393L / N452R / Q483K / F509R, E52G / G101A / E154W / P294R / E300R / N452S / F509R / D593S , E52G / E154W / I212V / P294R / E300G / I393L / N452R, I212V / E300R / I393L / N452R / F509R, E52 G / G101A / I212V / P294R / E300R / I393L / N452R / Q483K / F509R / D593S, E52G / I124L / P294R / E30 0R / N452S / F509R, I124L / E300R / I393L / N452R / Q483K / F509R / D593S, G101A / I124L / I212V / N452R / Q483K / F509R, E52G / I393L / N452R / F509R / D593S, I212V / E300R / N452R / F509R / D593S , E52G / N452R / F509R / D593S, E154W / I212V / E300R / E372K / N452R / F509R / D593S, E52G / I124L / I212V / P294R / I393L / N452R / D593S, I124L / N452R / F509R , S152R / E253R / D287R / T505H / D541R / Q573V / I584V, E154W / I212V / E300R / N452R, S152R / E253R / D287R / H304L / N454G / R545K / Q573V / I584V , E52G / I124L / E154W / P294R / E300A / E372K / N452R / F509R、P294R / E300R / E372K / N452S / F509R / D593S、E52G / I212V / N452R、I124L / P294R / I393L / N452R、E52G / E300R / F509R / D593S、S152R / E253R / D287R / N454G / D541R / I584V、E52G / G101A / N452S / F509R、E52G / E154W / I212V / E300R / E372K / I393L / F509R、E21M / E253R / D287R / N454G / I584V、E52G / I393L / N452R、E52G / I124L / E154W / P294R / F509R、G101A / I124L / I212V / E300R / I393L、I124L / P294R / E300G / E372K / N452S / F509R / D593S、E52G / I124L / E154W / P294R / Q483K / D593S、E52G / E372K / I393L / N452S / F509R / D593S、E154W / E300R / I393L / N452S / D593S、E52G / G101A / P294R / E372K / I393L / N452S / F509R、E21M / S152R / E253R / D287R / H304L / N454G / T505H、E52G / E300R / I393L、G101A / I124L / E154W / P294R / N452S / F509R、E154W / I212V / E372K / I393L / F509R / D593S、E21M / E253R / D287R / N454G / T505H / D541G、 D287R / N454G / T505H / I584V、E52G / I124L / P294R / E300R / E372K / N452S 、D287R / T505H / D541R / Q573V, E253R / T505H / I584V, L84Q / E253R / D287R / P456T / Q573V, E52G / E372K / F509R / D593S, F509R / D593S , E154W / I212V / E300R / E372K / F509R, E52G / E154W / P294R / F509R / D593S, S152R / Q295K / T505H / R545K / Q573V, E253R / D541R / I584V, E253R / Q295K / H304L / N454G / E462R / Q573V / I584V, E253R / N454G / T505 H / Q573V / I584V, E154W / I212V / E300R, D287R / N454G / R545K / Q573V, E21M / S152R / D287R / H304L / N454 G、S152R / E253R / Q295K / H304L / D541R / Q573V、S152R / E253R / D287R / H304L / N454G、S152R / N454G / Q5 73V, I124L / P294R / E372K / F509R / D593S, S152R / E253R / Q295K / N454G, E154W / E300R / N452S, D287R / H 304L / T505H, S152R / E253R / N454G / T505H, E253R / N454G / T505H, E21M / E253R / N454G / T505H / R545K, S152R / E253R / D287R / T505H, T505H / D541R / I584V or S152R / E253R / N454G / I584, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:10.

[0180] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of SEQ ID NO:80, or to a reference sequence corresponding to SEQ ID NO:80, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO:80, or to a reference sequence corresponding to SEQ ID NO:80.

[0181] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of even numbered SEQ ID NOs: 220-258 or a reference sequence corresponding to even numbered SEQ ID NOs: 220-258, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 80 or relative to the reference sequence corresponding to SEQ ID NO: 80.

[0182] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions at one or more of the following amino acid positions: 300 / 454 / 456 / 541 / 584, 21 / 300 / 454 / 545 / 584, 253 / 300 / 454 / 456 / 584, 154 / 253 / 300 / 454 / 456 / 505 / 573, 21 / 154 / 300 / 454 / 456 / 573, 152 / 300 / 454 / 456 / 505 / 584 / 593, 154 / 253 / 300 / 456 / 505 / 541 / 584, 21 / 300 / 454 / 545 / 584, 253 / 300 / 454 / 456 / 505 / 573, 154 / 253 / 300 / 454 / 456 / 541 / 573 / 584、154 / 300 / 454 / 505、21 / 253 / 300 / 454 / 456 / 545 / 573 / 593、168 / 300 / 454 / 456 / 545 / 573、253 / 300 / 454 / 505 / 573、 154 / 300 / 505 / 545 / 584、154 / 253 / 300 / 456 / 541 / 573 / 584、21 / 154 / 300 / 454 / 456 / 505 / 545、154 / 253 / 300 / 454 / 505、21 / 154 / 253 / 300 / 454 / 573、300 / 45 4 / 505 / 541 / 584、300 / 454 / 505 / 541 / 573、154 / 253 / 300 / 454、154 / 300 / 454 / 573、300 / 454 / 456 / 545 / 573、253 / 300 / 454 / 456、154 / 253 / 300 / 454 / 545 / 5 73、21 / 152 / 253 / 300 / 454 / 456 / 541 / 573、154 / 253 / 300 / 454 / 456 / 573、152 / 300 / 454、154 / 253 / 300 / 454 / 505 / 541 / 593、21 / 154 / 300 / 454 / 573 / 584、15 4 / 300 / 456 / 573 / 593、152 / 253 / 300 / 454 / 545 / 573、21 / 300 / 456 / 495 / 505 / 541 / 584、154 / 300 / 454 / 541 / 584、253 / 300 / 505 / 541 / 573 / 584、253 / 300 / 58 4. 253 / 300 / 454 / 456 / 545, 21 / 253 / 300 / 573, 300 / 454 / 545, 454 / 456 / 541 / 545 / 573 / 584, 152 / 253 / 454 / 505 / 545 / 573, 21 / 300 / 454 / 456 / 495, 300 / 573,154 / 300 / 454 / 545, 154 / 253 / 300 / 545 / 584, 21 / 300 / 505, 21 / 152 / 253 / 300 / 454 / 456 / 545, 154 / 253 / 300 / 456 / 541 / 573, 154 / 300 / 454, 253 / 300 / 454 / 541, 253 / 454 / 456 / 541 / 584, 21 / 300 / 573, 300 / 454 / 456, 300 / 454, 154 / 253 / 300 / 454 4 / 456, 154 / 456 / 505 / 593, 454 / 456 / 505, 253 / 454 / 456 / 584, 154 / 253 / 300, 253 / 456 / 505 / 584, 454 / 456, 154 / 454, 584, 432 / 505 / 573, 456 / 584, 21 / 505 / 545 / 584, 21 / 154 / 545 / 573, 454 / 584, 154 / 253 / 505 or 505, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 80.

[0183] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions 300R / 454G / 456T / 541R / 584V, 21M / 300R / 454G / 545K / 584V, 253R / 300R / 454G / 456T / 584V, 154W / 253R / 300R / 454G / 456T / 505H / 573V, 21M / 154W / 300R / 454G / 456T / 505H / 573V 573V, 152R / 300R / 454G / 456T / 505H / 584V / 593S, 154W / 253R / 300R / 456T / 505H / 545K / 573V / 584V, 253R / 300R / 454G / 456T / 505H / 573V、154W / 253R / 300R / 454G / 456T / 541G / 573V / 584V、154W / 300R / 454G / 505H , 21M / 253R / 300R / 454G / 456T / 545K / 573V / 593S, 168G / 300R / 454G / 456T / 545K / 573V, 253R / 300R / 454G / 505H / 573V , 154W / 300R / 505H / 545K / 584V, 154W / 253R / 300R / 456T / 541R / 573V / 584V, 21M / 154W / 300R / 454G / 456T / 505H / 545K, 154W / 253R / 300R / 454G / 505H, 21M / 154W / 253R / 300R / 454G / 573V, 300R / 454G / 505H / 541R / 584V , 300R / 454G / 505H / 541R / 573V, 154W / 253R / 300R / 454G, 154W / 300R / 454G / 573V, 300R / 454G / 456T / 545K / 573V, 253R / 300R / 454G / 456T, 1 54W / 253R / 300R / 454G / 545K / 573V, 21M / 152R / 253R / 300R / 454G / 456T / 541R / 573V, 154W / 253R / 300R / 454G / 456T / 573V, 152R / 300R / 454G , 154W / 253R / 300R / 454G / 505H / 541R / 593S, 21M / 154W / 300R / 454G / 573V / 584V, 154W / 300R / 456T / 573V / 593S, 152R / 253R / 300R / 454G / 545K / 573V,21M / 300R / 456T / 495F / 505H / 541R / 584V, 154W / 300R / 454G / 541G / 584V, 253R / 300R / 505H / 541R / 573V / 584V, 253R / 30 0R / 584V, 253R / 300R / 454G / 456T / 545K, 21M / 253R / 300R / 573V, 300R / 454G / 545K, 454G / 456T / 541G / 545K / 573V / 584V ,152R / 253R / 454G / 505H / 545K / 573V , 21M / 300R / 454G / 456T / 495F, 300R / 573V, 154W / 300R / 454G / 545K, 154W / 253R / 300R / 545K / 584V, 21M / 300R / 505H, 21M / 152R / 253R / 300R / 454G / 456T / 545K、154W / 253R / 300R / 456T / 541R / 573V, 154W / 300R / 454G, 253R / 300R / 454G / 541R, 253R / 454G / 456T / 541G / 584V、21M / 300R / 573V、300R / 454G / 456T、300R / 454G、154W / 253R / 300R / 454G / 456T / 505H / 593S, 454G / 456T / 505H, 253R / 454G / 456T / 584V, 154W / 253R / 300R, 253R / 456T / 505H / 584V, 454G / 456T, 154W / 454G, 584V, 432K / 505H / 573V, 456T / 584V, 21M / 505H / 545K / 584V, 21V / 154W / 545K / 573V, 454G / 584V, 154W / 253R / 505H or 505H, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:80.

[0184] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions E300R / N454G / P456T / D541R / I584V, E21M / E300R / N454G / R545K / I584V, E253R / E300R / N454G / P456T / I584V, E154W / E253R / E300R / N454G / P456T / T505H / Q573V, E21M / E154W / E300R / N454G / P456T / Q573V, S152R / E300R / N454G / P456T / T505H / I584V / D593S , E154W / E253R / E300R / P456T / T505H / R545K / Q573V / I584V , E253R / E300R / N454G / P456T / T505H / Q573V , E154W / E253R / E300R / N454G / P456T / D541G / Q573V / I584V, E154W / E300R / N454G / T505H, E21M / E253R / E300R / N454G / P456T / R54 5K / Q573V / D593S, E168G / E300R / N454G / P456T / R545K / Q573V, E253R / E300R / N454G / T505H / Q573V, E154W / E300R / T505H / R545K / I 584V, E154W / E253R / E300R / P456T / D541R / Q573V / I584V, E21M / E154W / E300R / N454G / P456T / T505H / R545K, E154W / E253R / E300R / N454G / T505H, E21M / E154W / E253R / E300R / N454G / Q573V, E300R / N454G / T505H / D541R / I584V, E300R / N454G / T505H / D541R / Q573V , E154W / E253R / E300R / N454G, E154W / E300R / N454G / Q573V, E300R / N454G / P456T / R545K / Q573V , E253R / E300R / N454G / P456T, E154W / E253R / E300R / N454G / R545K / Q573V, E21M / S152R / E253R / E300R / N454G / P456T / D541R / Q573V , E154W / E253R / E300R / N454G / P456T / Q573V,S152R / E300R / N454G 、E154W / E253R / E300R / N454G / T505H / D541R / D593S 、E21M / E154W / E300R / N454G / Q573V / I584V、E154W / E300R / P456T / Q573V / D593S、S152R / E253R / E300R / N454G / R545K / Q573V、E21M / E300R / P456T / L495F / T505H / D541R / I584V、E154W / E300R / N454G / D541G / I584V、E253R / E300R / T505H / D541R / Q573V / I584V、E253R / E300R / I584V、E253R / E300R / N454G / P456T / R545K、E21M / E253R / E300R / Q573V、E300R / N454G / R545K、N454G / P456T / D541G / R545K / Q573V / I584V、S152R / E253R / N454G / T505H / R545K / Q573V、E21M / E300R / N454G / P456T / L495F、E300R / Q573V、E154W / E300R / N454G / R545K、E154W / E253R / E300R / R545K / I584V 、 E21M / E300R / T505H 、E21M / S152R / E253R / E300R / N454G / P456T / R545K 、E154W / E253R / E300R / P456T / D541R / Q573V 、E154W / E300R / N454G, E253R / E300R / N454G / D541R, E253R / N454G / P456T / D541G / I584V, E21M / E300R / Q573V, E300R / N454G / P456T, E300R / N454 G. E154W / E253R / E300R / N454G / P456T, E154W / P456T / T505H / D593S, N454G / P456T / T505H, E253R / N454G / P456T / I584V, E1 54W / E253R / E300R, E253R / P456T / T505H / I584V, N454G / P456T, E154W / N454G, I584V, Q432K / T505H / Q573V, P456T / I584V, E21M / T505H / R545K / I584V, E21V / E154W / R545K / Q573V, N454G / I584V, E154W / E253R / T505H or T505H, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:80.

[0185] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of SEQ ID NO: 224, or to a reference sequence corresponding to SEQ ID NO: 224, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 224, or to a reference sequence corresponding to SEQ ID NO: 224.

[0186] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of even numbered SEQ ID NOs in SEQ ID NOs: 360-400, or a reference sequence corresponding to even numbered SEQ ID NOs in SEQ ID NOs: 360-400, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 224, or relative to the reference sequence corresponding to SEQ ID NO: 224.

[0187] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one set of substitutions at one or more of the following amino acid positions: 144 / 154 / 505 / 547 / 573 / 584, 154 / 191 / 325 / 505 / 573 / 584, 144 / 154 / 373 / 374 / 505 / 573 / 584, 134 / 136 / 154 / 505 / 547 / 573 / 584, 81 / 144 / 154 / 505 / 547 / 573 / 584、154 / 505 / 573 / 584、144 / 154 / 191 / 230 / 322 / 505 / 573 / 584、68 / 144 / 154 / 505 / 573 / 584、144 / 154 / 226 / 230 / 505 / 573 / 584、144 / 154 / 374 / 486 / 505 / 573 / 584、81 / 114 / 144 / 154 / 505 / 573 / 584、144 / 154 / 276 / 505 / 573 / 584、134 / 144 / 154 / 505 / 573 / 584、144 / 154 / 505 / 573 / 584、81 / 144 / 154 / 183 / 505 / 547 / 573 / 584、68 / 81 / 133 / 134 / 144 / 154 / 505 / 547 / 573 / 584、144 / 15 or 81 / 154 / 505 / 547 / 573 / 584, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:224.

[0188] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution set 144R / 154W / 505H / 547H / 573V / 584I, 154W / 191E / 325W / 505H / 573V / 584I, 144R / 154W / 373A / 374E / 505H / 573V / 584I, 134R / 136P / 154W / 505H / 547H / 573V / 584I, 81R / 144R / 154W / 505H / 547H / 573V / 584 I、154W / 505H / 573V / 584I、144R / 154W / 191R / 230G / 322C / 505H / 573V / 584I、68G / 144R / 154W / 505H / 573V / 584I、144R / 1 54W / 226K / 230Q / 505H / 573V / 584I, 144R / 154W / 374E / 486R / 505H / 573V / 584I, 81R / 114S / 144R / 154W / 505H / 573V / 584I, 144R / 154W / 276G / 505H / 573V / 584I, 134T / 144R / 154W / 505H / 573V / 584I, 144R / 154W / 505H / 573V / 584I ,81R / 144R / 154W / 183P / 505H / 547A / 573V / 584I , 68G / 81R / 133A / 134R / 144R / 154W / 505H / 547H / 573V / 584I, 144R / 154W / 374E / 505H / 573V / 584I, 92G / 133A / 144R / 154W / 505H / 573V / 584I, 144R / 154W / 373A / 505H / 573V / 584I, 114S / 144R / 154W / 505H / 573V / 584I, 154W / 505H / 551K / 573R / 584I / 585R or 81R / 154W / 505H / 547V / 573V / 584I, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:224.

[0189] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution set T144R / E154W / T505H / K547H / Q573V / V584I, E154W / H191E / R325W / T505H / Q573V / V584I, T144R / E154W / P373A / D374E / T505H / Q573V / V584I, S134R / E136P / E154W / T505H / K547H / Q573V / V584I , S81R / T144R / E154W / T505H / K547H / Q573V / V584I, E154W / T505H / Q573V / V584I, T144R / E154W / H191R / E230G / V322C / T505H / Q573V / V584I , E68G / T144R / E154W / T505H / Q573V / V584I, T144R / E154W / E226K / E230Q / T505H / Q573V / V584I , T144R / E154W / D374E / K486R / T505H / Q573V / V584I ,S81R / E114S / T144R / E154W / T505H / Q573V / V584I , T144R / E154W / K276G / T505H / Q573V / V584I, S134T / T144R / E154W / T505H / Q573V / V584I, T144R / E154W / T505H / Q573V / V584I ,S81R / T144R / E154W / R183P / T505H / K547A / Q573V / V584I , E68G / S81R / Q133A / S134R / T144R / E154W / T505H / K547H / Q573V / V584I, T144R / E154W / D374E / T505H / Q573V / V584I , E92G / Q133A / T144R / E154W / T505H / Q573V / V584I , T144R / E154W / P373A / T505H / Q573V / V584I, E114S / T144R / E154W / T505H / Q573V / V584I, E154W / T505H / R551K / Q573R / V584I / Q585R or S81R / E154W / T505H / K547V / Q573V / V584I, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:224.

[0190] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of SEQ ID NO: 366, or to a reference sequence corresponding to SEQ ID NO: 366, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 366, or to a reference sequence corresponding to SEQ ID NO: 366.

[0191] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of even numbered SEQ ID NOs: 402-488 or a reference sequence corresponding to even numbered SEQ ID NOs: 402-488, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 366 or relative to the reference sequence corresponding to SEQ ID NO: 366.

[0192] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions at one or more of the following amino acid positions: 24 / 58 / 432, 58 / 432 / 575, 58 / 115 / 432 / 575, 24 / 115 / 432 / 575, 24 / 25 / 221 / 432, 25 / 58 / 432 / 575, 24 / 25 / 58 / 432 / 575, 221 / 432 / 575, 432 / 575, 24 / 221 / 432, 24 / 115 / 221 / 432, 24 / 115 / 221 / 432 / 575, 432, 24 / 34 / 432, 24 / 432 / 575, 184 / 221 / 432 / 575, 24 / 432, 24 / 25 / 58 / 115 / 432 / 575, 25 / 221 / 432, 25 / 58 / 115 / 432, 221 / 575, 221 / 432, 24 / 58 / 115 / 221 / 575, 24 / 25 / 221 / 432 / 575, 24 / 25 / 58 / 221 / 432, 34 / 58 / 105 / 432, 24 / 25 / 115 / 432, 24 / 221 / 575, 115 / 432, 58 / 221, 58 / 575, 25 / 432 / 575, 25 / 432, 24 / 221 / 432 / 575, 24 / 115 / 432, 24 / 25 / 105 / 432, 25 / 34 / 432, 25 / 105 / 221 / 432, 24 / 25 / 432, 24 / 58 / 575 or 24 / 25 / 105 / 432 / 575, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:366.

[0193] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions 24M / 58K / 432L, 58K / 432L / 575V, 58K / 115Y / 432L / 575V, 24M / 115Y / 432L / 575V, 24M / 25L / 221M / 432L, 25L / 58K / 432L / 575V, 24M / 25M / 58K / 432L / 575V, 221M / 432L / 575V, 432L / 575V, 24M / 25L / 221M / 432L M / 221M / 432L, 24M / 115Y / 221M / 432L, 24M / 25M / 221M / 432L, 24M / 115Y / 221M / 432L / 575V, 432L, 24M / 34D / 432L, 24 M / 432L / 575V, 184T / 221M / 432L / 575V, 24M / 432L, 24M / 25L / 58K / 115Y / 432L / 575V, 25L / 221M / 432L, 25M / 58K / 115Y / 432L、221M / 575V、221M / 432L、24M / 58K / 115Y / 221M / 575V、24M / 25M / 221M / 432L / 575V、24M / 25M / 58K / 221M / 432L , 34D / 58K / 105S / 432L, 24M / 25M / 115Y / 432L, 24M / 221M / 575V, 115Y / 432L, 58K / 221M, 58K / 575V, 25L / 432L / 575V, or 24M / 25M / 105S / 432L / 575V, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:3 66.

[0194] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions L24M / A58K / Q432L, A58K / Q432L / I575V, A58K / L115Y / Q432L / I575V, L24M / L115Y / Q432L / I575V, L24M / P25L / V221M / Q432L, P25L / A58K / Q432L / I575V, L24M / P25M / A58K / Q432L / I575V, V221M / Q432L / I575V, Q432L / I575V, L24M / V22 1M / Q432L, L24M / L115Y / V221M / Q432L, L24M / P25M / V221M / Q432L, L24M / L115Y / V221M / Q432L / I575V, Q432L, L24M / E34D / Q432L, L24M / Q4 32L / I575V, A184T / V221M / Q432L / I575V, L24M / Q432L, L24M / P25L / A58K / L115Y / Q432L / I575V, P25L / V221M / Q432L, P25M / A58K / L115Y / Q 432L, V221M / I575V, V221M / Q432L, L24M / A58K / L115Y / V221M / I575V, L24M / P25M / V221M / Q432L / I575V, L24M / P25M / A58K / V221M / Q432L, E34D / A58K / I105S / Q432L, L24M / P25M / L115Y / Q432L, L24M / V221M / I575V, L115Y / Q432L, A58K / V221M, A58K / I575V, P25L / Q432L / I575V, P25L / Q432L, L24M / V221M / Q432L / I575V, L24M / L115Y / Q432L, L24M / P25M / I105S / Q432L, P25M / E34D / Q432L, L24M / P25L / I105S / Q432L, P25M / Q432L, P25M / I105S / V221M / Q432L, L24M / P25L / Q432L, L24M / A58K / I575V or L24M / P25M / I105S / Q432L / I575V, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:366.

[0195] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at an amino acid position provided in Tables 5.1, 6.1, 7.1, 8.1, and 9.1, wherein the substitution is relative to a reference sequence corresponding to SEQ ID NO: 2, 10, 80, 244, or 366, as provided in each table.

[0196] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution provided in Tables 5.1, 6.1, 7.1, 8.1, and 9.1, wherein the substitution is relative to a reference sequence corresponding to SEQ ID NO: 2, 10, 80, 244, or 366, as provided in each table.

[0197] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions at one or more amino acid positions provided in Tables 5.1, 6.1, 7.1, 8.1, and 9.1, wherein the substitution or set of substitutions is relative to a reference sequence corresponding to SEQ ID NO: 2, 10, 80, 244, or 366, as provided in each table.

[0198] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions of the DNA polymerase variants provided in Tables 5.1, 6.1, 7.1, 8.1, and 9.1, wherein the substitution or set of substitutions is relative to a reference sequence corresponding to SEQ ID NO: 2, 10, 80, 244, or 366, as provided in each table.

[0199] In some embodiments, the engineered DNA polymerase comprises an amino acid 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 amino acid sequence comprising a substitution or set of substitutions of the engineered DNA polymerase variants listed in Tables 5.1, 6.1, 7.1, 8.1, and 9.1.

[0200] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises residues 12 to 604 of the even-numbered SEQ ID NOs in SEQ ID NOs: 4-488 or comprises the even-numbered SEQ ID NOs in SEQ ID NOs: 4-488. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally has 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 has 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 has 1, 2, 3, 4, or 5 substitutions, insertions and / or deletions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally has 1, 2, 3, 4, or 5 substitutions.

[0201] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises SEQ ID NO:4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80 ,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,11 4, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 1 44, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 26 0, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, or 488 of residues 12 to 604.In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally has 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 has 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 has 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 has 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally has 1, 2, 3, 4 or 5 substitutions.

[0202] In some embodiments, the amino acid sequence of the engineered DNA polymerase comprises SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142 , 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 257 8, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 3 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, or 488.In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally has 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 has 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 has 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 has 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally has 1, 2, 3, 4 or 5 substitutions.

[0203] In some embodiments, the engineered DNA polymerase polypeptide has 1, 2, 3, 4 or up to 5 substitutions in the amino acid sequence. In some embodiments, the engineered DNA polymerase polypeptide has 1, 2, 3 or 4 substitutions in the amino acid sequence. In some embodiments, the substitutions include conservative substitutions. In some embodiments, the substitutions include non-conservative substitutions. In some embodiments, the substitutions include conservative and non-conservative substitutions. In some embodiments, the variants disclosed herein provide guidance on non-conservative and conservative substitutions.

[0204] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence comprising residues 12 to 604 of SEQ ID NO: 10, 80, 224 or 366 or an amino acid sequence comprising SEQ ID NO: 10, 80, 224 or 366. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally has 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 has 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 has 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally has 1, 2, 3, 4 or 5 substitutions.

[0205] It will be apparent that the description herein, including the embodiments, provides a table showing sequence structure information that associates a specific amino acid sequence feature with the functional activity of an engineered DNA polymerase polypeptide. This structure-function related information is provided in the form of specific amino acid residue differences with respect to a reference engineered DNA polymerase polypeptide of SEQ ID NO:2, 10, 80, 224 or 366 and experimentally determined correlated activity data for an exemplary engineered DNA polymerase polypeptide. Such information provides guidance and information about the replacement implemented in the preparation of an engineered DNA polymerase variant.

[0206] In some embodiments, the engineered DNA polymerase of the present disclosure has DNA polymerase activity. In some embodiments, the engineered DNA polymerase of the present disclosure has reverse transcriptase activity. In some embodiments, the engineered DNA polymerase of the present disclosure has DNA polymerase activity using DNA and RNA as templates.

[0207] In some embodiments, compared with a reference DNA polymerase, the engineered DNA polymerase has DNA polymerase and / or reverse transcriptase activity and at least one or more improved properties. In some embodiments, compared with a reference DNA polymerase, the engineered DNA polymerase has increased activity. In some embodiments, compared with a reference DNA polymerase, the engineered DNA polymerase has increased stability. In some embodiments, compared with a reference DNA polymerase, the engineered DNA polymerase has increased thermal stability. In some embodiments, compared with a reference DNA polymerase, the engineered DNA polymerase has increased continuous synthesis ability. In some embodiments, compared with a reference DNA polymerase, the engineered DNA polymerase has increased fidelity. In some embodiments, compared with a reference DNA polymerase, the engineered DNA polymerase has increased input DNA or RNA (e.g., target) sensitivity. In some embodiments, compared with a reference DNA polymerase, the engineered DNA polymerase has increased product yield in an isothermal amplification reaction. In some embodiments, compared with a reference DNA polymerase, the engineered DNA polymerase has increased salt tolerance. In some embodiments, the reference DNA polymerase has a sequence corresponding to residues 12 to 604 of SEQ ID NO: 2, 10, 80, 224, or 366, or a sequence corresponding to SEQ ID NO: 2, 10, 80, 224, or 366. In some embodiments, the reference DNA polymerase has a sequence corresponding to residues 12 to 604 of SEQ ID NO: 2, or a sequence corresponding to SEQ ID NO: 2.

[0208] In some embodiments, the engineered DNA polymerase has increased resistance to inhibitors. In some embodiments, compared with the reference DNA polymerase, the engineered DNA polymerase has increased resistance to inhibitors. In some embodiments, the engineered DNA polymerase has increased resistance to ethanol, guanidine thiocyanate, viral transport medium (VTM), heparin, heme, RNA or genomic DNA. The exemplary inhibitor concentration for assessing increased resistance includes 3% (v / v) ethanol, 25mM guanidine thiocyanate, 20% (v / v) VTM (see, Preparation of Viral Transport Medium, Centers for Disease Control and Prevention, SOP#:DSR-052-05), 6.25 units / mL heparin, 10 μM heme, 50ng total RNA or 100ng human genomic DNA. In some embodiments, the reference DNA polymerase has a sequence corresponding to residues 12 to 604 of SEQ ID NO:2 or a sequence corresponding to SEQ ID NO:2. In some embodiments, the reference DNA polymerase is Bst 3.0 (New England Biolabs, catalog number M0374). In some embodiments, the reference DNA polymerase is a LavaLAMP TM DNA Master Mix (LGC Biosearch Technologies) is commercially available.

[0209] In some embodiments, the engineered DNA polymerase has increased sensitivity to input DNA or RNA (e.g., target) compared to a reference DNA polymerase, wherein the input is about 1250 copies, about 1000 copies, about 750 copies, about 500 copies, about 200 copies, about 100 copies, or about 75 copies of a target RNA or DNA in a 5uL volume, e.g., as provided in the Examples. In some embodiments, the engineered DNA polymerase has increased product yield in an isothermal amplification reaction compared to a reference DNA polymerase, wherein the input target is about 1250 copies, about 1000 copies, about 750 copies, about 500 copies, about 200 copies, or about 100 copies of a target RNA or DNA in a 5uL volume, as provided in the Examples.

[0210] In some embodiments, the improved property of the engineered DNA polymerase compared to a reference DNA polymerase is selected from i) increased activity, ii) increased stability, iii) increased thermostability, iv) increased processivity, v) increased fidelity, vi) increased sensitivity to input target RNA or DNA, vii) increased product yield in isothermal amplification reactions, viii) increased salt tolerance, and ix) increased resistance to inhibitors, or any combination of i), ii), iii), iv), v), vii), vii), viii), and ix). In some embodiments, the reference DNA polymerase has a sequence corresponding to residues 12 to 604 of SEQ ID NO: 2, 10, 80, 224, or 366 or a sequence corresponding to SEQ ID NO: 2, 10, 80, 224, or 366. In some embodiments, the reference DNA polymerase has a sequence corresponding to residues 12 to 604 of SEQ ID NO: 2 or a sequence corresponding to SEQ ID NO: 2.

[0211] In some embodiments, the DNA polymerase described herein does not have significant or measurable 3'-exonuclease activity. In some embodiments, the DNA polymerase described herein does not have significant or measurable 5'-exonuclease activity. In some embodiments, the DNA polymerase described herein does not have significant or measurable 3'-exonuclease and 5'-exonuclease activities.

[0212] Although the engineered DNA polymerase described herein relates to a large fragment lacking a 5'-exonuclease domain, it should be understood that in some embodiments, the engineered DNA polymerase comprises a 5'-exonuclease domain. In some embodiments, the replacement or replacement set of the variant described herein can be incorporated into a full-length DNA polymerase containing a DNA polymerase function and a 5'-exonuclease domain. In some embodiments, the engineered DNA polymerase described herein can be expressed as a fusion protein with the 5'-exonuclease domain of the DNA polymerase of Parageobacillus genomosp 1. In some embodiments, the engineered DNA polymerase comprises a sequence corresponding to residues 1 to 284 of SEQ IDNO:540 encoded by the polynucleotide sequence of SEQ ID NO:539 or is fused to the sequence. Therefore, for each and every engineered DNA polymerase described herein as "large fragments", the present disclosure also includes an engineered DNA polymerase comprising a 5'-exonuclease domain.

[0213] In some embodiments, the disclosure also provides large fragments of DNA polymerases as provided in Table 10.2. In some embodiments, the engineered DNA polymerase comprises an amino acid sequence comprising:

[0214] Amino acid residues 12-603 of SEQ ID NO:490;

[0215] Amino acid residues 12 to 605 of SEQ ID NO:492;

[0216] Amino acid residues 12-603 of SEQ ID NO:494;

[0217] Amino acid residues 12 to 603 of SEQ ID NO:496;

[0218] Amino acid residues 12-600 of SEQ ID NO:498;

[0219] Amino acid residues 12 to 603 of SEQ ID NO:500;

[0220] Amino acid residues 12-603 of SEQ ID NO:502;

[0221] Amino acid residues 12 to 605 of SEQ ID NO:504;

[0222] Amino acid residues 12-606 of SEQ ID NO:506;

[0223] Amino acid residues 12 to 503 of SEQ ID NO:508;

[0224] Amino acid residues 12-607 of SEQ ID NO:510;

[0225] Amino acid residues 12 to 604 of SEQ ID NO:512;

[0226] Amino acid residues 12-605 of SEQ ID NO:514;

[0227] Amino acid residues 12 to 600 of SEQ ID NO:516;

[0228] Amino acid residues 12-605 of SEQ ID NO:518;

[0229] Amino acid residues 12 to 601 of SEQ ID NO:520;

[0230] Amino acid residues 12-599 of SEQ ID NO:522;

[0231] Amino acid residues 12 to 599 of SEQ ID NO:524;

[0232] Amino acid residues 12-605 of SEQ ID NO:526;

[0233] amino acid residues 12 to 600 of SEQ ID NO:528;

[0234] Amino acid residues 12-600 of SEQ ID NO:530; or

[0235] Amino acid residues 12 to 602 of SEQ ID NO:532.

[0236] In some embodiments, the engineered DNA polymerase comprises an amino acid sequence comprising SEQ ID NO: 2, 490, 492, 494, 496, 498, 500, 503, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, or 532.

[0237] In some embodiments, the present disclosure also provides isolated DNA polymerases of the full-length DNA polymerases provided in Table 10.2. In some embodiments, the DNA polymerase comprises the amino acid sequence of UniProt ID No. A0A023CMU9, P52026, A0A167UH07, M8D3Y0, A0A0A2SK72, Q08IE4, A0A4R1QH44, Q45458, A0A0B4SB30, A0A0D1JLC4, A0A0N0I8N0, A0A3R9UCK4, A0A084GX94, A0A176JAP1, A0A1W1II73, A0A2S0U8D5, A0A1I5VYY5, D5DMV6, E6U0L1, G8PDR9, K1KNJ5, L5N8Z2, or Q03RJ7, wherein each UniProt ID The sequence of No. is the sequence as of September 12, 2022.

[0238] In some embodiments, the engineered DNA polymerase is in the form of a fusion protein. In some embodiments, the engineered DNA polymerase described herein can be fused with a variety of polypeptide sequences, such as, for example, but not limited to, a polypeptide tag that can be used for detection and / or purification. In some embodiments, the fusion protein of the engineered DNA polymerase comprises a glycine-histidine tag or a histidine tag (His-tag). In some embodiments, the fusion protein of the engineered DNA polymerase comprises an epitope tag, such as c-myc, FLAG, V5 or hemagglutinin (HA). In some embodiments, the fusion protein of the engineered DNA polymerase comprises a GST, SUMO, Strep, MBP or GFP tag. In some embodiments, the fusion protein is fused to the amino (N-) end of the engineered DNA polymerase polypeptide. In some embodiments, the fusion protein is fused to the carboxyl (C-) end of the engineered DNA polymerase polypeptide.

[0239] In some embodiments, the disclosure also provides functional fragments or bioactive fragments of the engineered DNA polymerase polypeptide described herein.Therefore, for each and every embodiment of the engineered DNA polymerase, functional fragments or bioactive fragments of the engineered DNA polymerase are provided herein.In some embodiments, the functional fragments or bioactive fragments of the engineered DNA polymerase comprise at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% activity of the DNA polymerase polypeptide (i.e., parent DNA polymerase) derived therefrom.In some embodiments, the functional fragments or bioactive fragments comprise at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the parent sequence of the DNA polymerase.In some embodiments, the functional fragments are truncated less than 5, less than 10, less than 15, less than 20, less than 25, less than 30, less than 35, less than 40, less than 45 and less than 50 amino acids.

[0240] In some embodiments, the functional fragment of the engineered DNA polymerase herein comprises at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the parent sequence of the engineered DNA polymerase. In some embodiments, the functional fragment is truncated to less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, less than 50, less than 55, less than 60, less than 65 or less than 70 amino acids.

[0241] In some embodiments, the functional fragments 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 the engineered DNA polymerase described herein. Therefore, in some embodiments, the functional fragments or biologically active fragments of the engineered DNA polymerase exhibit enhanced or improved properties associated with the mutation or set of mutations in the parent DNA polymerase.

[0242] Polynucleotides encoding engineered polypeptides, expression vectors and host cells

[0243] On the other hand, the disclosure provides the recombinant polynucleotides encoding the engineered DNA polymerase polypeptides described herein. In some embodiments, the recombinant polynucleotides are operably connected to one or more heterologous regulatory sequences controlling gene expression to produce a recombinant polynucleotide construct capable of expressing a DNA polymerase. In some embodiments, an expression construct comprising at least one recombinant polynucleotide encoding one or more engineered DNA polymerase polypeptides is introduced into a suitable host cell to express one or more corresponding DNA polymerase polypeptides.

[0244] As will be apparent to the skilled artisan, the availability of protein sequences and knowledge of codons corresponding to a variety of amino acids provide a description of all polynucleotides capable of encoding a subject polypeptide. The degeneracy of the genetic code (wherein the same amino acid is encoded by an optional or synonymous codon) allows the preparation of a very large number of nucleic acids, all of which encode the engineered DNA polymerase polypeptides of the present disclosure. Therefore, the present disclosure provides methods and compositions for producing each and every possible variant of an engineered DNA polymerase polynucleotide encoding an engineered DNA polymerase polypeptide described herein, which can be prepared by selecting a combination based on possible codon selections, and for any DNA polymerase polypeptide described herein, including the amino acid sequences presented in the embodiments (e.g., Tables 5.1, 6.1, 7.1, 8.1, 9.1, and 10.2) and the sequence table, all such polynucleotide variants are considered to be specifically disclosed.

[0245] In some embodiments, codons are preferably optimized for use in the selected host cells for protein production. For example, preferred codons used in bacteria are generally used for expression in bacteria, and preferred codons used in mammalian cells are generally used for expression in mammalian cells. Therefore, codon-optimized polynucleotides encoding engineered DNA polymerase polypeptides comprise preferred codons at about 40%, 50%, 60%, 70%, 80%, 90% or greater than 90% of the codon positions in the full-length coding region.

[0246] Thus, in some embodiments, the recombinant polynucleotides of the present disclosure comprise a polynucleotide sequence encoding an engineered DNA polymerase described herein. In some embodiments, the polynucleotide sequence of the recombinant polynucleotide is codon optimized.

[0247] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase or a functional fragment thereof, the engineered DNA polymerase comprising an amino acid 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 amino acid residues 12 to 604 of SEQ ID NO: 2, 10, 80, 224 or 366, or to a reference sequence corresponding to SEQ ID NO: 2, 10, 80, 224 or 366, wherein the amino acid sequence is relative to the reference sequence corresponding to amino acid residues 12 to 604 of SEQ ID NO: 2, 10, 80, 224 or 366 as described herein, or to a reference sequence corresponding to SEQ ID NO: The reference sequence of NO: 2, 10, 80, 224 or 366 comprises one or more substitutions.

[0248] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid 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 amino acid residues 12 to 604 of SEQ ID NO:2, or to a reference sequence corresponding to SEQ ID NO:2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO:2 or to the reference sequence corresponding to SEQ ID NO:2.

[0249] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid 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 amino acid residues 12 to 604 of SEQ ID NO: 10, 80, 224 or 366, or to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12 to 604 of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2.

[0250] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid 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 amino acid residues 12 to 604 of SEQ ID NO: 4-488 or to a reference sequence corresponding to an even numbered SEQ ID NO: 4-488, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12 to 604 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0251] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least one substitution at the following amino acid positions: 21, 24, 25, 25, 34, 36, 52, 58, 66, 68, 81, 84, 92, 101, 105, 114, 115, 124, 133, 133, 134, 136, 144, 152, 154, 168, 183, 184, 191, 192, 210, 212, 221 , 226, 230, 241, 252, 253, 276, 287, 290, 294, 295, 300, 304, 322, 325, 372, 373, 374, 393, 427, 432, 452, 454, 456, 462, 470, 483, 486, 495, 505, 509, 541, 541, 545, 547, 551, 552, 573, 575, 578, 584, 585 or 593 or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:2.

[0252] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence containing at least one substitution at amino acid position 36, 241, 372, or 470, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:2.

[0253] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence containing at least one substitution at the following amino acid positions: 36, 52, 101, 124, 134, 136, 154, 212, 241, 253, 294, 300, 372, 393, 452, 454, 456, 470, 483, 505, 509, 547, 573 or 584 or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:2.

[0254] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least one substitution or set of substitutions at one or more of the following amino acid positions: 509, 300, 452, 36 / 241 / 372 / 470, 124 / 192 / 210 / 372 / 427 / 456 / 552, 124, 52, 483, 372, 393, 212, 52 / 66, 133, 454, 154, 593, 462, 541, 21, 573, 505, 152, 294, 545, 101 / 241 / 470, 584, 304, 295, 578, 456 / 470, 253, 290, 192 / 241 / 372 / 456, or 252, wherein the amino acid positions are relative to the amino acid corresponding to SEQ ID Reference sequence of NO:2.

[0255] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence containing at least one substitution at the amino acid positions listed in Tables 5.1, 6.1, 7.1, 8.1, and 9.1, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:2.

[0256] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least one set of substitutions listed in Tables 5.1, 6.1, 7.1, 8.1, and 9.1, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:2.

[0257] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence containing at least one substitution or set of substitutions at one or more amino acid positions listed in Tables 5.1, 6.1, 7.1, 8.1, and 9.1, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[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 substitution or set of substitutions listed for the variants listed in Tables 5.1, 6.1, 7.1, 8.1, and 9.1, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:2.

[0259] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid 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 residues 12 to 604 of SEQ ID NO: 10, 80, 224 or 366, or to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366.

[0260] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid 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 residues 12 to 604 of an even numbered SEQ ID NO:4-488 or to a reference sequence corresponding to an even numbered SEQ ID NO:4-488.

[0261] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid 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 residues 12 to 604 of SEQ ID NO: 10, 80, 224 or 366, or to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 10, 80, 224 or 366, or to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366.

[0262] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid 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 residues 12 to 604 of SEQ ID NOs: 10, 80, 224, or 366, or to a reference sequence corresponding to an even numbered SEQ ID NO: 10, 80, 224, or 366.

[0263] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least one substitution at the following amino acid positions: 21, 24, 25, 25, 34, 36, 52, 58, 66, 68, 81, 84, 92, 101, 105, 114, 115, 124, 133, 133, 134, 136, 144, 152, 154, 168, 183, 184, 191, 192, 210, 212, 221 , 226, 230, 241, 252, 253, 276, 287, 290, 294, 295, 300, 304, 322, 325, 372, 373, 374, 393, 427, 432, 452, 454, 456, 462, 470, 483, 486, 495, 505, 509, 541, 541, 545, 547, 551, 552, 573, 575, 578, 584, 585 or 593 or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366.

[0264] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence containing at least one substitution at amino acid position 36, 241, 372 or 470, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366.

[0265] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence containing at least one substitution at the following amino acid positions: 36, 52, 101, 124, 134, 136, 154, 212, 241, 253, 294, 300, 372, 393, 452, 454, 456, 470, 483, 505, 509, 547, 573 or 584 or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366.

[0266] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid 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 residues 12 to 604 of SEQ ID NO: 10, or to a reference sequence corresponding to SEQ ID NO: 10, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 10, or to a reference sequence corresponding to SEQ ID NO: 10.

[0267] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid 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 residues 12 to 604 of SEQ ID NO: 10 or to a reference sequence corresponding to an even numbered SEQ ID NO: 10-218, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 10 or to the reference sequence corresponding to SEQ ID NO: 10.

[0268] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least one set of substitutions at one or more of the following amino acid positions: 52 / 101 / 124 / 212 / 294 / 372 / 393 / 452 / 483 / 509, 52 / 124 / 300 / 393 / 452, 154 / 212 / 294 / 300 / 372 / 393 / 452 / 483 / 509, 52 / 101 / 154 / 294 / 300 / 452 / 509 / 593, 52 / 154 / 212 / 294 / 300 / 393 / 452, 212 / 300 / 393 / 452 452 / 509、52 / 101 / 212 / 294 / 300 / 393 / 452 / 483 / 509 / 593、52 / 124 / 294 / 300 / 452 / 509、124 / 300 / 393 / 452 / 483 / 509 / 593、101 / 124 / 212 / 452 / 483 / 509、 52 / 393 / 452 / 509 / 593、212 / 300 / 452 / 509 / 593、52 / 452 / 509 / 593、154 / 212 / 300 / 372 / 452 / 509 / 593、52 / 124 / 212 / 294 / 393 / 452 / 593、124 / 452 / 509、15 2 / 253 / 287 / 505 / 541 / 573 / 584、154 / 212 / 300 / 452、152 / 253 / 287 / 304 / 454 / 545 / 573 / 584、52 / 124 / 154 / 294 / 300 / 372 / 452 / 509、294 / 300 / 372 / 452 / 509 / 593、52 / 212 / 452、124 / 294 / 393 / 452、52 / 300 / 509 / 593、152 / 253 / 287 / 454 / 541 / 584、52 / 101 / 452 / 509、52 / 154 / 212 / 300 / 372 / 393 / 509、21 / 253 / 287 / 454 / 584、52 / 393 / 452、52 / 124 / 154 / 294 / 509、101 / 124 / 212 / 300 / 393、124 / 294 / 300 / 372 / 452 / 509 / 593、52 / 124 / 154 / 294 / 483 / 593、52 / 372 / 39 3 / 452 / 509 / 593、154 / 300 / 393 / 452 / 593、52 / 101 / 294 / 372 / 393 / 452 / 509、21 / 152 / 253 / 287 / 304 / 454 / 505、52 / 300 / 393、101 / 124 / 154 / 294 / 452 / 509、154 / 212 / 372 / 393 / 509 / 593、21 / 253 / 287 / 454 / 505 / 541、287 / 454 / 505 / 584、52 / 124 / 294 / 300 / 372 / 452、287 / 505 / 541 / 573、253 / 505 / 584、84 / 253 / 287 / 456 / 573、52 / 3 72 / 509 / 593、509 / 593、154 / 212 / 300 / 372 / 509、52 / 154 / 294 / 509 / 593、152 / 295 / 505 / 545 / 573、253 / 541 / 584、253 / 295 / 304 / 454 / 462 / 573 / 584、253 / 454 / 505 / 573 / 584 、154 / 212 / 300、287 / 454 / 545 / 573、21 / 152 / 287 / 304 / 454、152 / 253 / 295 / 304 / 541 / 573、152 / 253 / 287 / 304 / 454、152 / 454 / 573、124 / 294 / 372 / 509 / 593、152 / 253 / 295 / 4 54, 154 / 300 / 452, 287 / 304 / 505, 152 / 253 / 454 / 505, 253 / 454 / 505, 21 / 253 / 454 / 505 / 545, 152 / 253 / 287 / 505, 505 / 541 / 584 or 152 / 253 / 454 / 584, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 10.

[0269] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid 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 residues 12 to 604 of SEQ ID NO: 80, or to a reference sequence corresponding to SEQ ID NO: 80, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 80, or to a reference sequence corresponding to SEQ ID NO: 80.

[0270] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid 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 residues 12 to 604 of SEQ ID NOs: 220-258 or to a reference sequence corresponding to even numbered SEQ ID NOs: 220-258, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 80 or to the reference sequence corresponding to SEQ ID NO: 80.

[0271] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least one substitution and set of substitutions at one or more of the following amino acid positions: 300 / 454 / 456 / 541 / 584, 21 / 300 / 454 / 545 / 584, 253 / 300 / 454 / 456 / 584, 154 / 253 / 300 / 454 / 456 / 505 / 573, 21 / 154 / 300 / 454 / 456 / 573, 152 / 300 / 454 / 456 / 505 / 584 / 593, 154 / 253 / 300 / 456 / 505 / 545 / 574. 3 / 584、253 / 300 / 454 / 456 / 505 / 573、154 / 253 / 300 / 454 / 456 / 541 / 573 / 584、154 / 300 / 454 / 505、21 / 253 / 300 / 454 / 456 / 545 / 573 / 593、168 / 300 / 454 / 456 / 545 / 573、253 / 300 / 454 / 505 / 573、154 / 300 / 505 / 545 / 584、154 / 253 / 300 / 456 / 541 / 573 / 584、21 / 154 / 300 / 454 / 456 / 505 / 545、154 / 253 / 300 / 454 / 505、 21 / 154 / 253 / 300 / 454 / 573、300 / 454 / 505 / 541 / 584、300 / 454 / 505 / 541 / 573、154 / 253 / 300 / 454、154 / 300 / 454 / 573、300 / 454 / 456 / 545 / 573、253 / 300 / 454 / 456、154 / 253 / 300 / 454 / 545 / 573、21 / 152 / 253 / 300 / 454 / 456 / 541 / 573、154 / 253 / 300 / 454 / 456 / 573、152 / 300 / 454、154 / 253 / 300 / 454 / 505 / 541 / 5 93, 21 / 154 / 300 / 454 / 573 / 584, 154 / 300 / 456 / 573 / 593, 152 / 253 / 300 / 454 / 545 / 573, 21 / 300 / 456 / 495 / 505 / 541 / 584, 154 / 300 / 454 / 541 / 584, 253 / 300 / 505 / 541 / 573 / 584、253 / 300 / 584、253 / 300 / 454 / 456 / 545、21 / 253 / 300 / 573、300 / 454 / 545、454 / 456 / 541 / 545 / 573 / 584、152 / 253 / 454 / 505 / 545 / 573、21 / 300 / 454 / 456 / 495、300 / 573、154 / 300 / 454 / 545、154 / 253 / 300 / 545 / 584、21 / 300 / 505、21 / 152 / 253 / 300 / 454 / 456 / 545、154 / 253 / 300 / 456 / 541 / 573、154 / 300 / 454、253 / 300 / 454 / 541、253 / 454 / 456 / 541 / 584、21 / 300 / 573、300 / 454 / 456、300 / 454、 154 / 253 / 300 / 454 / 456, 154 / 456 / 505 / 593, 454 / 456 / 505, 253 / 454 / 456 / 584, 154 / 253 / 300, 253 / 456 / 505 / 584, 454 / 456, 154 / 454, 584, 432 / 505 / 573, 456 / 584, 21 / 505 / 545 / 584, 21 / 154 / 545 / 573, 454 / 584, 154 / 253 / 505 or 505, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 80.

[0272] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid 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 residues 12 to 604 of SEQ ID NO: 224 or to a reference sequence corresponding to SEQ ID NO: 224, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 224 or to the reference sequence corresponding to SEQ ID NO: 224.

[0273] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid 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 residues 12 to 604 of SEQ ID NO: 360-400 or to a reference sequence corresponding to even numbered SEQ ID NOs: 360-400, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 224 or to the reference sequence corresponding to SEQ ID NO: 224.

[0274] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least one set of substitutions at the following amino acid positions: 144 / 154 / 505 / 547 / 573 / 584, 154 / 191 / 325 / 505 / 573 / 584, 144 / 154 / 373 / 374 / 505 / 573 / 584, 134 / 136 / 154 / 505 / 547 / 573 / 584、81 / 144 / 154 / 505 / 547 / 573 / 584、154 / 505 / 573 / 584、144 / 154 / 191 / 230 / 322 / 505 / 573 / 584、68 / 144 / 154 / 505 / 573 / 584、144 / 154 / 226 / 230 / 505 / 573 / 584、144 / 154 / 374 / 486 / 505 / 573 / 58 4. 81 / 114 / 144 / 154 / 505 / 573 / 584, 144 / 154 / 276 / 505 / 573 / 584, 134 / 144 / 154 / 505 / 573 / 584, 144 / 154 / 505 / 573 / 584, 81 / 144 / 154 / 183 / 505 / 547 / 573 / 584, 68 / 81 / 133 / 134 / 144 / 154 / 505 / 547 / 573 / 584 , 144 / 154 / 374 / 505 / 573 / 584, 92 / 133 / 144 / 154 / 505 / 573 / 584, 144 / 154 / 373 / 505 / 573 / 584, 114 / 144 / 154 / 505 / 573 / 584, 154 / 505 / 551 / 573 / 584 / 585 or 81 / 154 / 505 / 547 / 573 / 584, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:224.

[0275] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid 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 residues 12 to 604 of SEQ ID NO: 366 or to a reference sequence corresponding to SEQ ID NO: 366, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 366 or to the reference sequence corresponding to SEQ ID NO: 366.

[0276] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid 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 residues 12 to 604 of SEQ ID NOs: 402-488 or to a reference sequence corresponding to even numbered SEQ ID NOs: 402-488, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 366 or to the reference sequence corresponding to SEQ ID NO: 366.

[0277] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least one substitution or set of substitutions at one or more of the following amino acid positions: 24 / 58 / 432, 58 / 432 / 575, 58 / 115 / 432 / 575, 24 / 115 / 432 / 575, 24 / 25 / 221 / 432, 25 / 5 8 / 432 / 575, 24 / 25 / 58 / 432 / 575, 221 / 432 / 575, 432 / 575, 24 / 221 / 432, 24 / 115 / 221 / 432, 24 / 115 / 221 / 432 / 575, 432, 24 / 34 / 432, 24 / 432 / 575, 184 / 221 / 432 / 575, 24 / 432, 24 / 25 / 58 / 115 / 432 / 575, 25 / 221 / 432, 25 / 58 / 115 / 432, 221 / 575, 221 / 432, 24 / 58 / 115 / 221 / 575, 24 / 25 / 221 / 432 / 575, 24 / 25 / 58 / 221 / 432, 34 / 58 / 105 / 432, 24 / 25 / 115 / 432, 24 / 221 / 575, 115 / 432, 58 / 221, 58 / 575, 25 / 432 / 575, 25 / 432, 24 / 221 / 432 / 575, 24 / 115 / 432, 24 / 25 / 105 / 432, 25 / 34 / 432, 25 / 105 / 221 / 432, 24 / 25 / 432, 24 / 58 / 575 or 24 / 25 / 105 / 432 / 575, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:366.

[0278] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence containing at least one substitution at an amino acid position provided in Tables 6.1, 7.1, 8.1, and 9.1, wherein the positions are relative to a reference sequence corresponding to SEQ ID NO: 2, 10, 80, 224, or 366, as provided in each table.

[0279] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence containing at least one substitution provided in Tables 6.1, 7.1, 8.1, and 9.1, wherein the substituted amino acid position is relative to a reference sequence corresponding to SEQ ID NO: 2, 10, 80, 224, or 366, as provided in each table.

[0280] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence containing at least one substitution or set of substitutions at one or more amino acid positions provided in Tables 6.1, 7.1, 8.1, and 9.1, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 2, 10, 80, 224, or 366, as provided in each table.

[0281] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least one substitution or set of substitutions of a variant listed in Tables 6.1, 7.1, 8.1, and 9.1, wherein the amino acid positions of the substitutions or sets of substitutions are relative to a reference sequence corresponding to SEQ ID NO: 2, 10, 80, 224, or 366, as provided in each table.

[0282] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid 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 amino acid sequence comprising a substitution or set of substitutions of the engineered DNA polymerase variants listed in Tables 5.1, 6.1, 7.1, 8.1, and 9.1.

[0283] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence containing residues 12 to 604 of an even-numbered SEQ ID NO. in SEQ ID NO: 4-488 or comprising an even-numbered SEQ ID NO. in SEQ ID NO: 4-488.

[0284] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising residues 12 to 604 comprising SEQ ID NO: 10, 80, 224, or 366 or an amino acid sequence comprising SEQ ID NO: 10, 80, 224, or 366.

[0285] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase,The engineered DNA polymerase comprises an amino acid sequence comprising residues 12 to 604 of the following sequence: NO:4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80 ,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,11 4, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144、146、148、150、152、154、156、158、160、162、164、166、168、170、172、174、176、178、180、182、184、186、188、190、192、194、196、198、200、202、204、206、208、210、212、214、216、218、220、222、224、226、228、230、232、234、236、238、240、242、244、246、248、250、252、254、256、258、 260、262、264、266、268、270、272、274、276、278、280、282、284、286、288、290、292、294、296、298、300、302、304、306、308、310、312、314、316、318、320、322、324、326、328、330、332、334、336、338、340、342、344、346、348、350、352、354、356、358、360、362、364、366、368、370、372、374、 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 440, 442, 444, 446, 448, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486 or 488, optionally,wherein the engineered DNA polymerase has 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions in the amino acid sequence.

[0286] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polypeptide comprising an amino acid sequence comprising: SEQ ID NO:4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80 ,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,11 4, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144、146、148、150、152、154、156、158、160、162、164、166、168、170、172、174、176、178、180、182、184、186、188、190、192、194、196、198、200、202、204、206、208、210、212、214、216、218、220、222、224、226、228、230、232、234、236、238、240、242、244、246、248、250、252、254、256、258、 260、262、264、266、268、270、272、274、276、278、280、282、284、286、288、290、292、294、296、298、300、302、304、306、308、310、312、314、316、318、320、322、324、326、328、330、332、334、336、338、340、342、344、346、348、350、352、354、356、358、360、362、364、366、368、370、372、374、 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 440, 442, 444, 446, 448, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486 or 488, optionally,wherein the engineered DNA polymerase has 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions in the amino acid sequence.

[0287] In some embodiments, the encoded engineered DNA polymerase polypeptide has 1, 2, 3, 4, up to 5 substitutions in the amino acid sequence. In some embodiments, the engineered DNA polymerase polypeptide has 1, 2, 3 or 4 substitutions in the amino acid sequence. In some embodiments, the substitutions include conservative substitutions. In some embodiments, the substitutions include non-conservative substitutions. In some embodiments, the substitutions include non-conservative substitutions and conservative substitutions.

[0288] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising residues 12 to 604 of SEQ ID NO: 10, 80, 224, or 366 or an amino acid sequence comprising SEQ ID NO: 10, 80, 224, or 366, optionally wherein the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions in the polypeptide sequence. In some embodiments, the encoded DNA polymerase has 1, 2, 3, 4, up to 5 substitutions in the amino acid sequence. In some embodiments, the encoded DNA polymerase has 1, 2, 3, or 4 substitutions in the amino acid sequence.

[0289] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 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 1812 of SEQ ID NO: 1, 9, 79, 223 or 365, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 1, 9, 79, 223 or 365, wherein the recombinant polynucleotide encodes an engineered DNA polymerase.

[0290] In some embodiments, the recombinant polynucleotide comprises 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 polynucleotide sequence corresponding to nucleotide residues 34 to 1812 of an odd-numbered SEQ ID NO. in SEQ ID NOs: 3-487, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. in SEQ ID NOs: 3-487, wherein the recombinant polynucleotide encodes an engineered DNA polymerase.

[0291] In some embodiments, the recombinant polynucleotide encoding the engineered DNA polymerase comprises 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 polynucleotide sequence corresponding to nucleotide residues 34 to 1812 of the following sequence:NO:3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79 ,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257 , 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 3 73, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, or 487.

[0292] In some embodiments, the recombinant polynucleotide encoding the engineered DNA polymerase comprises 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 the polynucleotide sequence corresponding to the following sequence: SEQ ID NO:NO:3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79 ,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257 , 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 3 73, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, or 487.

[0293] In some embodiments, the recombinant polynucleotide encoding the engineered DNA polymerase comprises a polynucleotide sequence comprising nucleotide residues 34 to 1812 of SEQ ID NO: NO:3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79 ,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257 , 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 3 73, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, or 487.

[0294] In some embodiments, the recombinant polynucleotide encoding the engineered DNA polymerase comprises a polynucleotide sequence comprising: SEQ ID NO:3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79 ,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257 , 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 3 73, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, or 487.

[0295] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 1812 of SEQ ID NO: 9, 79, 223, or 365 or a polynucleotide sequence comprising SEQ ID NO: 9, 79, 223, or 365.

[0296] In some embodiments, the recombinant polynucleotide encodes a DNA polymerase comprising an amino acid sequence comprising:

[0297] Amino acid residues 12 to 603 of SEQ ID NO:490;

[0298] Amino acid residues 12 to 605 of SEQ ID NO:492;

[0299] Amino acid residues 12 to 603 of SEQ ID NO:494;

[0300] Amino acid residues 12 to 603 of SEQ ID NO:496;

[0301] amino acid residues 12 to 600 of SEQ ID NO:498;

[0302] Amino acid residues 12 to 603 of SEQ ID NO:500;

[0303] Amino acid residues 12 to 603 of SEQ ID NO:502;

[0304] Amino acid residues 12 to 605 of SEQ ID NO:504;

[0305] Amino acid residues 12 to 606 of SEQ ID NO:506;

[0306] Amino acid residues 12 to 503 of SEQ ID NO:508;

[0307] Amino acid residues 12 to 607 of SEQ ID NO:510;

[0308] Amino acid residues 12 to 604 of SEQ ID NO:512;

[0309] Amino acid residues 12 to 605 of SEQ ID NO:514;

[0310] Amino acid residues 12 to 600 of SEQ ID NO:516;

[0311] Amino acid residues 12 to 605 of SEQ ID NO:518;

[0312] Amino acid residues 12 to 601 of SEQ ID NO:520;

[0313] Amino acid residues 12 to 599 of SEQ ID NO:522;

[0314] Amino acid residues 12 to 599 of SEQ ID NO:524;

[0315] Amino acid residues 12 to 605 of SEQ ID NO:526;

[0316] amino acid residues 12 to 600 of SEQ ID NO:528;

[0317] Amino acid residues 12 to 600 of SEQ ID NO:530; or

[0318] Amino acid residues 12 to 602 of SEQ ID NO:532.

[0319] In some embodiments, the recombinant polynucleotide encodes an engineered DNA polymerase comprising an amino acid sequence comprising SEQ ID NO: 2, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530 or 532.

[0320] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 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, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529 or 531, wherein the recombinant polynucleotide encodes a DNA polymerase as described herein. In some embodiments, the recombinant polynucleotide encoding the engineered DNA polymerase comprises a polynucleotide sequence comprising SEQ ID NO: 1, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529 or 531.

[0321] In some embodiments, the recombinant polynucleotide is hybridized under highly stringent conditions with a reference polynucleotide sequence encoding an engineered DNA polymerase described herein, or its reverse complement. In some embodiments, the reference polynucleotide sequence corresponds to residues 34 to 1812 of SEQ ID NO: 1, 9, 79, 223, or 365, or corresponds to a sequence of SEQ ID NO: 1, 9, 79, 223, or 365, or its reverse complement, or a polynucleotide sequence encoding any other engineered DNA polymerase provided herein, or its reverse complement. In some embodiments, the recombinant polynucleotide is hybridized under highly stringent conditions with a reference polynucleotide sequence corresponding to residues 34 to 1812 of an odd-numbered SEQ ID NO: 1-487, or with a reference polynucleotide comprising a sequence corresponding to an odd-numbered SEQ ID NO: 1-487, or with its reverse complement.

[0322] In some embodiments, a recombinant polynucleotide that hybridizes under highly stringent conditions comprises a sequence that has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one reference polynucleotide sequence corresponding to residues 34 to 1812 of a polynucleotide sequence listed in Table 5.1, 6.1, 7.1, 8.1 or 9.1, or a polynucleotide sequence listed in Table 5.1, 6.1, 7.1, 8.1 or 9.1.

[0323] In some embodiments, the recombinant polynucleotide encoding any one of the DNA polymerases herein is manipulated in various ways to promote the 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 the expression of the DNA polymerase polynucleotide and / or polypeptide. According to the expression vector used, the manipulation of the isolated polynucleotide before the isolated polynucleotide is inserted into the vector may be desirable or necessary. The technology utilizing recombinant DNA methods to modify polynucleotides and nucleotide sequences is 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.

[0324] In some embodiments, a suitable promoter is selected based on the host cell used. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure include, but are not limited to, promoters obtained from the following: the Escherichia coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis α-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens α-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic β-lactamase genes (see, e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA, 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, but are not limited to, promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-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. oxysporum) trypsin-like protease (see, e.g., WO96 / 00787), and the NA2-tpi promoter (a hybrid of the promoters of the Aspergillus niger neutral α-amylase gene and the Aspergillus oryzae triosephosphate isomerase gene), and mutants, truncations, and hybrid promoters thereof. Exemplary yeast cell promoters can be from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.Other useful promoters for yeast host cells are known in the art (see, e.g., Romanos et al., Yeast, 1992, 8: 423-488). Exemplary promoters for use in mammalian cells include, but are not limited to, promoters from cytomegalovirus (CMV), chicken β-actin promoter fused to CMV enhancer, simian vacuolating virus 40 (SV40), promoters from Homo sapiens phosphoglycerate kinase, promoters from β-actin, elongation factor-1a or glyceraldehyde-3-phosphate dehydrogenase genes, or promoters from Gallus β-actin.

[0325] In some embodiments, the control sequence is a suitable transcription terminator sequence (i.e., a sequence recognized by the 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 selected host cell can be used in the present invention. For bacterial expression, the transcription terminator can be a Rho-dependent terminator that depends on the Rho transcription factor, or a Rho-independent or intrinsic terminator that does not require a transcription factor. Exemplary bacterial transcription terminators are described in Peters et al., J Mol Biol., 2011, 412 (5): 793-813. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the following genes: Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger α-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the following genes: 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). Exemplary terminators for mammalian cells include, but are not limited to, those from cytomegalovirus (CMV), Simian virus 40 (SV40), from the Homo sapiens growth hormone gene hGH, bovine growth hormone BGH, and human or rabbit beta globulin.

[0326] In some embodiments, the control sequence is a suitable leader sequence (non-translated region of 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 selected host cell can be used in the present invention. Exemplary leaders for filamentous fungal host cells are obtained from the following genes: Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the following genes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP). Suitable leaders for mammalian host cells include, but are not limited to, 5'-UTR elements present in Orthopoxvirus mRNA or SV40 mRNA.

[0327] In some embodiments, the control sequence is a polyadenylation sequence (i.e., a sequence operably linked to the 3' end of the nucleic acid sequence, and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to the transcribed mRNA). Any suitable polyadenylation sequence that is functional in the selected host cell can be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, the following genes: 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). Useful polyadenylation and 3'UTR sequences for mammalian host cells include, but are not limited to, 3-UTRs of α- and β-globin mRNAs, which contain several sequence elements that increase mRNA stability and translation, and those from mammalian viruses such as SV40.

[0328] In some embodiments, the control sequence is also a signal peptide (i.e., a coding region encoding an amino acid sequence that is linked to the amino terminus of a polypeptide and directs the encoded polypeptide to the secretory pathway of the cell). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence contains a signal peptide coding region that is naturally linked to the segment of the coding region encoding the secreted polypeptide in accordance with the translation reading frame. Any suitable signal peptide coding region that directs the expressed polypeptide to the secretory pathway of the selected host cell can be used for expression of one or more engineered polypeptides. Effective signal peptide coding regions for bacterial host cells include, but are not limited to, those obtained from the following genes: Bacillus NClB 11837 maltogenic amylase, Bacillus stearothermophilus α-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM) and Bacillus subtilis prsA. Additional signal peptides are known in the art (see, e.g., Simonen and Palva, Microbiol. Rev., 1993, 57: 109-137). In some embodiments, the signal peptide coding region effective for filamentous fungal host cells includes, but is not limited to, the signal peptide coding region obtained from the following genes: 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 from the following genes: Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Useful signal peptides for mammalian host cells include, but are not limited to, those from the gene for immunoglobulin gamma (IgG) and signal peptides in human secretory proteins (such as human β-galactosidase polypeptide).

[0329] In some embodiments, the control sequence is a propeptide coding region encoding an amino acid sequence positioned at the amino terminus of a polypeptide. The polypeptide produced is referred to as a "proenzyme", "propolypeptide" or "zymogen". The propolypeptide can be converted into a mature active polypeptide by catalyzing or autocatalyzing the cleavage of the propeptide from the propolypeptide. The propeptide coding region can be obtained from any suitable source including but not limited to the following genes: Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae α-factor, Rhizomucor miehei aspartic protease and Myceliophthora thermophila lactase (see, e.g., WO 95 / 33836). When both the signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned adjacent to the amino terminus of the polypeptide and the signal peptide region is positioned adjacent to the amino terminus of the propeptide region.

[0330] In some embodiments, control sequences are regulatory sequences that promote the regulation of expression of recombinant polynucleotides and / or encoded polypeptides. Examples of regulatory systems are those that cause the expression of genes to be turned on or off in response to chemical or physical stimuli (including the presence of regulatory compounds). In prokaryotic host cells, suitable regulatory sequences include but are not limited to lac, tac and trp operator systems. In yeast host cells, suitable regulatory systems include but are not limited to ADH2 systems or GAL1 systems. In filamentous fungi, suitable regulatory sequences include but are not limited to TAKA α-amylase promoters, Aspergillus niger glucoamylase promoters and Aspergillus oryzae glucoamylase promoters.

[0331] On the other hand, the disclosure provides a kind of recombinant expression vector, it comprises the recombinant polynucleotide of coding engineering DNA polymerase polypeptide, wherein the recombinant polynucleotide is operably connected to one or more control sequences, such as promoter and terminator, replication origin etc., this depends on the type of host into which they will be introduced, for example, for expressing polynucleotide and / or encoded polypeptide.In some embodiments, various nucleic acids described herein and control sequences are linked together (that is, operably connected) with the recombinant polynucleotide to produce recombinant expression vector, and the recombinant expression vector comprises one or more convenient restriction sites, to allow the nucleic acid sequence of encoding DNA polymerase polypeptide to be inserted or replaced at such site.In some embodiments, the recombinant expression vector can be any suitable vector (for example, plasmid or virus), it can easily stand the recombinant DNA procedure and cause the expression of DNA polymerase polynucleotide and / or encoded polypeptide.The selection of carrier usually depends on the compatibility of carrier with the host cell to be introduced into the carrier.Carrier can be a linear plasmid or a closed circular plasmid.

[0332] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome). The vector may include any means for ensuring self-replication. In some selectable embodiments, the vector is a vector that, when introduced into a host cell, is integrated into the genome and replicated with one or more chromosomes into which it is integrated. In addition, in some embodiments, a single vector or plasmid is utilized, or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, and / or a transposon.

[0333] In some embodiments, the recombinant polynucleotide can be provided on a non-replicating expression vector or plasmid. In some embodiments, the non-replicating expression vector or plasmid can be based on a replication-deficient viral vector (see, e.g., Travieso et al., npj Vaccines, 2022, vol. 7, Article 75).

[0334] In some embodiments, the expression vector comprises one or more selectable markers that allow easy selection of transformed cells. "Selectable markers" are genes whose products provide antimicrobial or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc. Examples of selectable markers for bacteria include, but are not limited to, 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.

[0335] On the other hand, the disclosure provides a host cell comprising a polynucleotide encoding at least one engineered DNA polymerase polypeptide of the disclosure, the polynucleotide being operably linked to one or more control sequences for expressing one or more engineered DNA polymerases in the host cell. In some embodiments, the host cell comprises an expression vector comprising a recombinant polynucleotide encoding an engineered DNA polymerase polypeptide described herein. In some embodiments, the host cell suitable for expressing a polypeptide encoded by the expression vector is a prokaryotic cell or a eukaryotic cell. Host cells known in the art include, but are not limited to, bacterial cells such as Escherichia coli, Vibrio fluvialis, Streptomyces, and Salmonella typhimurium cells; fungal cells such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal (e.g., mammalian) cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Exemplary host cells also include a variety of Escherichia coli strains (e.g., W3110 (ΔfhuA) and BL21).

[0336] On the other hand, present disclosure provides a kind of method producing engineered DNA polymerase polypeptide, wherein said method comprises cultivating the host cell of the polynucleotide that can express the coding engineered DNA polymerase polypeptide under the condition that is suitable for expressing polypeptide, so that engineered DNA polymerase is produced.In some embodiments, said method also comprises separating engineered DNA polymerase from culture medium and / or host cell.In some embodiments, said method also comprises purifying DNA polymerase polypeptide, such as by method described herein.

[0337] Suitable culture media and growth conditions for host cells are known in the art. It is contemplated that any suitable method for introducing a polynucleotide expressing a DNA polymerase polypeptide into a host cell can be used in the present invention. Suitable techniques include, but are not limited to, electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0338] In some embodiments, recombinant polypeptide (for example, DNA polymerase variant) can be produced using any suitable method known in the art. For example, there are many different mutagenesis techniques well known to those skilled in the art. In addition, mutagenesis kits can also be obtained from many commercial molecular biology suppliers. The method can be used to make specific substitutions at a determined amino acid (site-directed), specificity (region-specificity) or random mutations in a local region of a gene, or random mutagenesis (for example, saturation mutagenesis) in a whole gene. Many suitable methods for producing enzyme variants known to those skilled in the art include, but are not limited to, using PCR to single-stranded DNA or double-stranded DNA site-directed mutagenesis, cassette mutagenesis, gene synthesis, fallible PCR, reorganization and chemical saturation mutagenesis, or any other suitable method known in the art. Non-limiting examples of methods for DNA and protein engineering are provided in the following patents: U.S. Pat. No. 6,117,679; U.S. Pat. No. 6,420,175; U.S. Pat. No. 6,376,246; U.S. Pat. No. 6,586,182; U.S. Pat. No. 7,747,391; U.S. Pat. No. 7,747,393; U.S. Pat. No. 7,783,428; and U.S. Pat. No. 8,383,346. After the variants are generated, they can be screened for any desired properties (e.g., high or increased activity, or low or decreased activity, increased thermal activity, increased stability, increased processivity, increased fidelity, increased inhibitor resistance or tolerance, increased salt tolerance and / or pH stability, etc.).

[0339] In some embodiments, the engineered DNA polymerase polypeptides with properties disclosed herein can be obtained by, for example, subjecting the polynucleotides encoding naturally occurring or engineered DNA polymerase polypeptides to suitable mutagenesis and / or directed evolution methods known in the art as described herein. Exemplary directed evolution techniques are mutagenesis and / or DNA shuffling (see, for example, Stemmer, Proc. Natl. Acad. Sci. USA, 1994, 91: 10747-10751; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767 and U.S. Patent No. 6,537,746). Other directed evolution procedures that can be used include the staggered extension process (StEP), in vitro recombination (see, e.g., Zhao et al., Nat. Biotechnol., 1998, 16:258-261), mutagenic PCR (see, e.g., Caldwell et al., PCR Methods Appl., 1994, 3:S136-S140), and cassette mutagenesis (see, e.g., Black et al., Proc. Natl. Acad. Sci. USA, 1996, 93:3525-3529), among others.

[0340] Methods of mutagenesis and directed evolution can be applied to polynucleotides encoding DNA polymerases to generate libraries of variants that can be expressed, screened, and assayed. Any suitable mutagenesis and directed evolution methods may be used in the context of the present disclosure (see, 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, 6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,9 10、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、6,57 6,467、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、7,7,747,391,7,747,393,7,751,986,7,776,598,7,783,428,7,795,030,7,853,410,7,868,138,7,783,428,7,873,477,7,873,499,7,904,249,7,957,912,7,981,614,8,014,961,8,029,988,8,048,674,8,058 ,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 related PCT and non-U.S. counterpart applications; Ling et al., Anal. Biochem., 1997, 254(2):157-78; Dale et al., Meth. Mol. Biol., 1996, 57:369-74; Sm ith, 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, 1 7:259-264; 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, 366:389-391; Stemmer, Proc. Nat. Acad. Sci. USA, 1994,91: 10747-10751; EP 3 049 973; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767; WO 2009 / 152336; and WO 2015 / 048573, all of which are incorporated herein by reference).

[0341] In some embodiments, clones obtained after mutagenesis treatment are screened by subjecting the enzyme preparation to defined treatment conditions or assay conditions (e.g., temperature, pH conditions, template type (e.g., DNA, RNA, GC content, secondary structure, etc.), input template concentration, nucleotides, etc.) and measuring enzyme activity after treatment or other suitable assay conditions. Clones containing polynucleotides encoding polypeptides of interest are then isolated from the gene, sequenced to identify changes in the nucleotide sequence (if any), and used to express the enzyme in a host cell. Measuring enzyme activity from an expression library can be performed using any suitable method known in the art and as described in the Examples.

[0342] For engineered polypeptides of known sequences, polynucleotides encoding enzymes can be prepared by standard solid phase methods according to known synthetic methods. In some embodiments, nucleic acid fragments of up to about 100 bases can be synthesized individually, and then connected (for example, by enzymatic or chemical connection methods or polymerase-mediated methods) to form any desired continuous sequence. For example, polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using classical phosphoramidite methods (see, for example, Beaucage et al., Tet. Lett., 22: 1859-69

[1981] ; and Matthes et al., EMBO J., 3: 801-05

[1984] ), as usually practiced in automatic synthesis methods. Lett., 1981, 22: 1859-69; and Matthes et al., EMBO J., 1984, 3: 801-05), as usually practiced in automatic synthesis methods, prepared by chemical synthesis.

[0343] In some embodiments, the method for preparing an engineered DNA polymerase polypeptide may include: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from any variant described herein, and (b) expressing a DNA polymerase polypeptide encoded by the polynucleotide. In some embodiments of the method, the amino acid sequence encoded by the polynucleotide may optionally have one or several (e.g., up to 3, 4, 5 or up to 10) amino acid residues missing, inserted and / or replaced. In some embodiments, the amino acid sequence optionally has 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45 or 1-50 amino acid residues missing, inserted and / or replaced. In some embodiments, the amino acid sequence optionally has 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, 30, 30, 35, 40, 45 or 50 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24 or 25 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the substitutions are conservative or non-conservative substitutions.

[0344] In some embodiments, any of the engineered DNA polymerase polypeptides expressed in host cells is recovered and / or purified from the cells and / or culture medium using any one or more of the known techniques for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography.

[0345] Chromatographic techniques for separating and purifying DNA polymerase polypeptides include, among others, reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, gel electrophoresis and affinity chromatography. The conditions for purifying a specific enzyme may depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, and will be apparent to those skilled in the art. In some embodiments, affinity techniques may be used to separate improved DNA polymerases. For affinity chromatography purification, any antibody specifically binding to the DNA polymerase polypeptide of interest may be used. In order to produce antibodies, various host animals are immunized by injection of DNA polymerase polypeptides or fragments thereof, including but not limited to rabbits, mice, rats, etc. In some embodiments, DNA polymerase polypeptides or fragments are attached to suitable carriers, such as BSA, by means of side chain functional groups or joints attached to side chain functional groups. In the case where engineered DNA polymerases include fusion polypeptides such as His tags that allow for affinity purification, standard affinity methods for specific fusion proteins may be used.

[0346] DNA polymerase composition

[0347] On the other hand, the present disclosure provides compositions of DNA polymerase disclosed herein. In some embodiments, compositions include at least one engineered DNA polymerase polypeptide described herein. In some embodiments, the engineered DNA polymerase polypeptide in the compositions is separated or purified. In some embodiments, DNA polymerase is combined with other components and compounds to provide compositions and preparations comprising engineered DNA polymerase polypeptides suitable for different applications and purposes (e.g., diagnostic methods, molecular biology tools and compositions).

[0348] In some embodiments, the composition comprises an amino acid sequence comprising residues 12 to 604 of SEQ ID NO: 2 or a DNA polymerase comprising a sequence of SEQ ID NO: 2 and / or at least one engineered DNA polymerase described herein. In some embodiments, the composition comprises an amino acid sequence comprising residues 12 to 604 of an even-numbered SEQ ID NO. in SEQ ID NO: 4-488 or an engineered DNA polymerase comprising an amino acid sequence of an even-numbered SEQ ID NO. in SEQ ID NO: 4-488.

[0349] In some embodiments, the composition comprises a large fragment of the DNA polymerase provided in Table 10.2. In some embodiments, the composition comprises a DNA polymerase comprising the following amino acid sequence:

[0350] Amino acid residues 12-603 of SEQ ID NO:490;

[0351] Amino acid residues 12-605 of SEQ ID NO:492;

[0352] Amino acid residues 12-603 of SEQ ID NO:494;

[0353] Amino acid residues 12-603 of SEQ ID NO:496;

[0354] Amino acid residues 12-600 of SEQ ID NO:498;

[0355] Amino acid residues 12-603 of SEQ ID NO:500;

[0356] Amino acid residues 12-603 of SEQ ID NO:502;

[0357] Amino acid residues 12-605 of SEQ ID NO:504;

[0358] Amino acid residues 12-606 of SEQ ID NO:506;

[0359] Amino acid residues 12-503 of SEQ ID NO:508;

[0360] Amino acid residues 12-607 of SEQ ID NO:510;

[0361] Amino acid residues 12 to 604 of SEQ ID NO:512;

[0362] Amino acid residues 12-605 of SEQ ID NO:514;

[0363] Amino acid residues 12-600 of SEQ ID NO:516;

[0364] Amino acid residues 12-605 of SEQ ID NO:518;

[0365] Amino acid residues 12-601 of SEQ ID NO:520;

[0366] Amino acid residues 12-599 of SEQ ID NO:522;

[0367] Amino acid residues 12-599 of SEQ ID NO:524;

[0368] Amino acid residues 12-605 of SEQ ID NO:526;

[0369] Amino acid residues 12-600 of SEQ ID NO:528;

[0370] Amino acid residues 12-600 of SEQ ID NO:530; or

[0371] Amino acid residues 12-602 of SEQ ID NO:532.

[0372] In some embodiments, the composition comprises a DNA polymerase comprising the amino acid sequence of SEQ ID NO: 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, or 532.

[0373] In some embodiments, the composition comprises a full-length DNA polymerase provided in Table 10.2. In some embodiments, the composition comprises a DNA polymerase having an amino acid sequence of UniProt ID No. A0A023CMU9, P52026, A0A167UH07, M8D3Y0, A0A0A2SK72, Q08IE4, A0A4R1QH44, Q45458, A0A0B4SB30, A0A0D1JLC4, A0A0N0I8N0, A0A3R9UCK4, A0A084GX94, A0A176JAP1, A0A1W1II73, A0A2S0U8D5, A0A1I5VYY5, D5DMV6, E6U0L1, G8PDR9, K1KNJ5, L5N8Z2, or Q03RJ7.

[0374] In some embodiments, the composition further comprises one or more of a buffer, a nucleotide substrate (e.g., dNTP, dNTP analogs and / or modified dNTP) and / or at least one primer (e.g., a synthetic primer complementary to the target nucleic acid). In some embodiments, the composition further comprises a template polynucleotide, particularly a template DNA or RNA. In some embodiments, the template polynucleotide comprises a heterologous template DNA or RNA. In some embodiments, the template polynucleotide comprises a mixture of DNA and RNA.

[0375] In some embodiments, the composition can also include a DNA polymerase (e.g., a second DNA polymerase) other than the engineered DNA polymerase described herein. In some embodiments, the second DNA polymerase is a second thermostable DNA polymerase, such as Taq or Pfu polymerase, or a reverse transcriptase, such as those useful in RT-PCR coupled reactions. In some embodiments, the composition includes a probe or indicator, such as a nucleic acid binding dye (e.g., Green, etc.), for detecting and / or quantifying the amount of product formed, for example, in a qRT-PCR reaction.

[0376] In some embodiments, the engineered DNA polymerase described herein is provided in solution or is fixed on a substrate. In some embodiments, the substrate is a solid substrate or a film or a particle. The enzyme can be embedded in a matrix or a film. In some embodiments, the matrix includes a polymer material such as calcium alginate, agar, k-carrageenan, polyacrylamide and collagen, or a solid matrix such as activated carbon, porous ceramics and diatomaceous earth. In some embodiments, the matrix is ​​a particle, a film or a fiber. The type of film includes nylon, cellulose, polysulfone or polyacrylate and other.

[0377] In some embodiments, enzyme is fixed on the surface of support material. In some embodiments, enzyme is adsorbed on support material. In some embodiments, enzyme is fixed on support material by covalent attachment. Support material comprises inorganic material, such as aluminum oxide, silicon dioxide, porous glass, ceramic, diatomite, clay and bentonite or organic material, such as cellulose (CMC, DEAE-cellulose), starch, activated carbon, polyacrylamide, polystyrene and ion exchange resin, such as Amberlite, Sephadex and Dowex and other.

[0378] Uses of engineered DNA polymerase polypeptides and kits

[0379] In another aspect, the present disclosure provides the use of engineered DNA polymerases for diagnostic and molecular biology purposes, such as for detecting the presence of a target nucleic acid, nucleic acid sequencing, and direct / indirect amplification of nucleic acids.

[0380] In some embodiments, engineered DNA polymerases are used to prepare complementary DNA of target nucleic acid / polynucleotides. In some embodiments, the method for preparing complementary DNA of target nucleic acid / polynucleotides includes contacting target nucleic acid / polynucleotides with a sequence comprising residues 12 to 604 of SEQ ID NO:2 or an amino acid sequence comprising SEQ ID NO:2 under reaction conditions suitable for producing complementary DNA complementary to all or a portion (i.e., whole or in part) of target nucleic acid / polynucleotides in the presence of a substrate sufficient to produce complementary DNA; an engineered DNA polymerase described herein; or a DNA polymerase described in Table 10.2. In some embodiments, target nucleic acid / polynucleotides are DNA. In some embodiments, target nucleic acid / polynucleotides are RNA. In some embodiments, target nucleic acid / polynucleotides comprise a mixture of DNA and RNA.

[0381] In some embodiments, the DNA polymerase for use in the methods comprises an amino acid sequence comprising:

[0382] Amino acid residues 12 to 603 of SEQ ID NO:490;

[0383] Amino acid residues 12 to 605 of SEQ ID NO:492;

[0384] Amino acid residues 12 to 603 of SEQ ID NO:494;

[0385] Amino acid residues 12 to 603 of SEQ ID NO:496;

[0386] amino acid residues 12 to 600 of SEQ ID NO:498;

[0387] Amino acid residues 12 to 603 of SEQ ID NO:500;

[0388] Amino acid residues 12 to 603 of SEQ ID NO:502;

[0389] Amino acid residues 12 to 605 of SEQ ID NO:504;

[0390] Amino acid residues 12 to 606 of SEQ ID NO:506;

[0391] Amino acid residues 12 to 503 of SEQ ID NO:508;

[0392] Amino acid residues 12 to 607 of SEQ ID NO:510;

[0393] Amino acid residues 12 to 604 of SEQ ID NO:512;

[0394] Amino acid residues 12 to 605 of SEQ ID NO:514;

[0395] Amino acid residues 12 to 600 of SEQ ID NO:516;

[0396] Amino acid residues 12 to 605 of SEQ ID NO:518;

[0397] Amino acid residues 12 to 601 of SEQ ID NO:520;

[0398] Amino acid residues 12 to 599 of SEQ ID NO:522;

[0399] Amino acid residues 12 to 599 of SEQ ID NO:524;

[0400] Amino acid residues 12 to 605 of SEQ ID NO:526;

[0401] amino acid residues 12 to 600 of SEQ ID NO:528;

[0402] Amino acid residues 12 to 600 of SEQ ID NO:530; or

[0403] Amino acid residues 12 to 602 of SEQ ID NO:532.

[0404] In some embodiments, the DNA polymerase for use in the method comprises an amino acid sequence comprising SEQ ID NO:490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530 or 532.

[0405] In some embodiments, the DNA polymerase for use in the methods herein comprises a full-length DNA polymerase provided in Table 10.2. In some embodiments, the DNA polymerase for use in the method comprises the amino acid sequence of UniProt ID No. A0A023CMU9, P52026, A0A167UH07, M8D3Y0, A0A0A2SK72, Q08IE4, A0A4R1QH44, Q45458, A0A0B4SB30, A0A0D1JLC4, A0A0N0I8N0, A0A3R9UCK4, A0A084GX94, A0A176JAP1, A0A1W1II73, A0A2S0U8D5, A0A1I5VYY5, D5DMV6, E6U0L1, G8PDR9, K1KNJ5, L5N8Z2, or Q03RJ7.

[0406] As discussed herein and known in the art, substrates include nucleotides (e.g., dNTPs or dNTP analogs) and / or oligonucleotide primers for DNA polymerase activity. Primers can be complementary to a specific sequence of a target nucleic acid, or can be random primers, such as for generating DNA libraries. In some embodiments, the reaction conditions include components required for DNA polymerase activity, such as a divalent metal, e.g., Mg. +2 , and / or a buffer at an appropriate pH.

[0407] In some embodiments, the target nucleic acid / polynucleotide is any DNA or RNA suitable as a template for an engineered DNA polymerase, including but not limited to genomic DNA or mRNA, mitochondrial DNA or RNA, cell-free DNA or cell-free RNA (e.g., obtained from blood / serum), bacterial DNA or RNA, fungal DNA or RNA, plant DNA or RNA, mammalian DNA or RNA, or viral DNA or RNA.

[0408] In some embodiments, the DNA polymerase of SEQ ID NO:2, the engineered DNA polymerase described herein, or other DNA polymerases in Table 10.2 can be used for diagnostic applications, for example, for detecting the presence of target nucleic acids / polynucleotides (including DNA and RNA). In some embodiments, the method for detecting the presence of target nucleic acids / polynucleotides includes reacting a sample suspected of containing target nucleic acids / polynucleotides with a DNA polymerase in the presence of a substrate, under conditions suitable for DNA polymerase-mediated production of DNA complementary to all or part (i.e., the whole or part) of the target nucleic acids / polynucleotides, and detecting the presence of complementary DNA, wherein the DNA polymerase comprises an amino acid sequence containing residues 12 to 604 of SEQ ID NO:2 or an amino acid sequence containing SEQ ID NO:2; an engineered DNA polymerase described herein; or a DNA polymerase shown in Table 10.2. In some embodiments, the target nucleic acid / polynucleotide is DNA. In some embodiments, the target nucleic acid / polynucleotide is RNA. In some embodiments, the target nucleic acid / polynucleotide comprises a mixture of DNA and RNA.

[0409] In some embodiments, suitable reaction conditions, particularly for engineered DNA polymerases, include reaction temperatures of 15° C. to 80° C., 15° C. to 75° C., 20° C. to 70° C., 25° C. to 65° C., 30° C. to 60° C., or 35° C. to 55° C. In some embodiments, the reaction temperature is about 15° C., 20° C., 25° C., 35° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., or 80° C.

[0410] In some embodiments, sample can be any material or substance suspected of containing target nucleic acid.In some embodiments, sample is biological sample, such as biopsy and autopsy sample, frozen section, blood, plasma, serum, sputum, feces, tears, mucus, hair, skin etc. obtained for histological purposes.In some embodiments, biological sample is cell or virus, such as cell or virus from bacterial culture, viral culture or cell culture.In some embodiments, sample is environmental sample, including sample from water (including ocean, river, garbage / sewer etc.), soil, air, vent or surface (such as floor, machine, counter etc.) and other.

[0411] In some embodiments, the detection of the complementary DNA product produced by the archaeal dna polymerase can be realized by methods known in the art.In some embodiments, complementary DNA is detected by increasing this complementary DNA, such as by PCR or isothermal amplification.In some embodiments, the isothermal amplification method used for use with the engineered DNA polymerase includes LAMP, whole genome amplification (WGA) and multiple displacement amplification and other.In some embodiments, one or more of the nucleotide substrates can be marked, and depending on the mark, can be directly (for example, fluorescently labeled nucleotides) or indirectly (for example, biotin-labeled nucleotides) detected.In some embodiments, one or more of the primers used in the archaeal dna polymerase reaction can be marked.

[0412] In some embodiments, engineered DNA polymerase is used to detect target RNA, wherein the reverse transcriptase activity of engineered DNA polymerase described herein is used to prepare a DNA copy of target RNA. In some embodiments, DNA polymerase is also used to amplify a DNA copy of target RNA. In some embodiments, reverse transcriptase reaction is carried out separately from the amplification reaction using DNA polymerase. In some embodiments, reverse transcriptase reaction and the DNA amplification reaction using DNA polymerase are carried out successively. In some embodiments, reverse transcriptase reaction and the DNA amplification reaction using DNA polymerase are carried out simultaneously or in parallel. In some embodiments, reverse transcriptase reaction and DNA amplification reaction are "one pot" reactions. In some embodiments, reverse transcriptase reaction and DNA amplification reaction are carried out in separate reactions.

[0413] In some embodiments, the method of amplifying a target DNA or RNA comprises contacting the target DNA or RNA with a DNA polymerase comprising an amino acid sequence comprising residues 12 to 604 of SEQ ID NO: 2 or an amino acid sequence comprising SEQ ID NO: 2; an engineered DNA polymerase described herein; or a DNA polymerase provided in Table 10.2 in the presence of a substrate under conditions suitable for amplifying the target DNA or RNA. In some embodiments, amplifying the DNA or RNA is by isothermal amplification, such as by LAMP.

[0414] In some embodiments, engineered DNA polymerases are used to sequence nucleic acids. Various methods for DNA sequencing, particularly NGS sequencing methods, are well known in the art. In some embodiments, the method for sequencing a target DNA comprises contacting the target DNA with a DNA polymerase containing an amino acid sequence of residues 12 to 604 of SEQ ID NO: 2 or an amino acid sequence of SEQ ID NO: 2, in the presence of a substrate suitable for sequencing, under conditions suitable for DNA polymerase-mediated extension of a complementary DNA of the target DNA; or an engineered DNA polymerase described herein; or a DNA polymerase provided in Table 10.2, and determining the sequence of the target DNA.

[0415] On the other hand, the disclosure provides a kind of test kit, the test kit comprises an amino acid sequence containing residues 12 to 604 of SEQ ID NO:2 or an amino acid sequence having SEQ ID NO:2; or at least one engineered DNA polymerase disclosed herein; or at least one DNA polymerase provided in Table 10.2 as described herein. In some embodiments, the test kit also comprises one or more of a buffer, a nucleotide substrate and / or an oligonucleotide primer. In some embodiments, the test kit may comprise a plurality of (e.g., two or more) oligonucleotide primers, for example, for different parts of a target nucleic acid. In some embodiments, the test kit also comprises a template DNA or RNA, for example, a control template DNA or RNA for a limited sequence and / or amount used as a positive control for detecting a target DNA or RNA. In some embodiments, the test kit comprises a second DNA polymerase, such as Taq or Pfu DNA polymerase. Example

[0416] The following examples, including the experiments and results obtained, are provided for illustrative purposes only and should not be construed as limiting the present invention.

[0417] In the experiments disclosed below, the following abbreviations are used where relevant: ppm (parts per million); M (mole / liter); mM (millimolar), uM and μM (micromolar); nM (nanomolar); mol (mole); gm and g (gram); mg (milligram); ug and μg (microgram); L and l (liter); ml and mL (milliliter); μl and μL (microliter); cm (centimeter); mm (millimeter); um and μm (micrometer); sec. (second); min (minute); h and hr (hour); U (unit); MW (molecular weight); rpm (revolutions per minute); rcf (relative centrifugal force); psi and PSI (pounds per square inch); °C (degrees Celsius); RT and rt (room temperature); NGS (next generation sequencing); ds (double stranded); ss (single stranded); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); E. coli W3110 (a commonly used laboratory strain of Escherichia coli, available from the Coli Genetic Stock Center [CGSC]); HTP (high throughput); HPLC (high pressure liquid chromatography); ddH 2 O (double distilled water); PBS (phosphate buffered saline); BSA (bovine serum albumin); DTT (dithiothreitol); CAM (chloramphenicol); CAT (chloramphenicol acetyltransferase); IPTG (isopropyl β-D-1-thiogalactopyranoside); FIOPC (fold improvement over positive control) or FIOP (fold improvement over parent); LB (Luria-Bertani); TB (Terrific-Broth); SPRI (solid phase reversible immobilization); GITC (guanidine thiocyanate); CDC (Centers for Disease Control).

[0418] Example 1

[0419] E. coli expression host containing recombinant DNA polymerase gene

[0420] The initial DNA polymerase for producing variants disclosed herein is SEQ ID NO:2, which is a large fragment (i.e., amino acid residues 285 to 876) of the wild-type DNA polymerase of Parageobacillus genomosp.1, cloned into expression vector pCK110900 (see, Fig. 3 of U.S. Patent Application Publication No. 2006 / 0195947) operably connected to the lac promoter under the control of the lac1 repressor. The expression vector also comprises a P15a origin of replication and a chloramphenicol resistance gene. Using standard methods known in the art, Escherichia coli W3110 is transformed with the resulting plasmid. Transformants are isolated by making cells undergo chloramphenicol selection, as known in the art (see, e.g., U.S. Patent No. 8,383,346 and WO2010 / 144103).

[0421] Example 2

[0422] Preparation of wet cell pellet containing HTP reverse transcriptase (RT)

[0423] E. coli cells containing the recombinant DNA polymerase encoding gene from a single clonal colony were inoculated into 180 μL LB containing 1% glucose and 30 μg / mL chloramphenicol (CAM) in a well of a 96-well shallow microtiter plate. The plate was inoculated with O 2 The plates were sealed with permeable seals and the cultures were grown overnight at 30°C, 200 rpm and 85% humidity. 10 μL of each cell culture was then transferred to a well of a 96-well deep-well plate containing 390 mL TB and 30 μg / mL CAM. 2 The deep well plates were sealed with a permeable seal and incubated at 30°C, 250 rpm and 85% humidity until the OD 600 0.6-0.8. The cell culture was then induced with IPTG at a final concentration of 1 mM and incubated overnight under the same conditions used initially. The cells were then pelleted by centrifugation at 4,000 rpm for 10 min. The supernatant was discarded and the pellet was frozen at -80°C before lysis.

[0424] Example 3

[0425] Preparation of cell lysate containing HTP DNA polymerase

[0426] To prepare cell lysates, 200 μL of buffer containing 50 mM triethanolamine-HCl (pH 7.5) was added to the cell paste produced as described in Example 2 in each well. The plate was shaken on a benchtop shaker to resuspend the cells. The suspension (100 μL) was transferred to a 96-well hard shell PCR plate and lysed in a thermal cycler at a set lysis temperature ranging from 68°C to 80°C, specified in the specific examples, for 60 min. The plate was then centrifuged at 4,000 rpm and 4°C for 15 min. The clarified supernatant was optionally diluted and used for biocatalytic reactions to determine their activity, RNA sensitivity and thermal stability levels.

[0427] Example 4

[0428] Use of heat-treated lysates for RT-LAMP reactions for SARS-CoV-2 detection

[0429] RT-LAMP assay was performed using a primer set targeting CoV-Orf1a, which consisted of six oligonucleotides at different concentrations (final concentrations FIP: 1.6uM, BIP: 1.6uM, F3: 0.2uM, B3: 0.2uM, loop F: 0.4uM, loop B: 0.5uM). Each variant was screened in a 5 uL reaction containing: SARS-CoV-2 synthetic RNA control 1 (GenBank / GISAID ID MT007544.1, Twist Biosciences part number 102019, 50-1250 copies per reaction as specified in the Examples), 0.5 uL 10× Isothermal Amplification Buffer (NEB catalog number B0537S), 0.5 uL 10× Orf1a LAMP primer mix (FIP: 16 uM, BIP: 16 uM, F3: 2 uM, B3: 2 uM, loop F: 4 uM, loop B: 4 uM), dNTP mix (final concentration of each dNTP 1.1 mM), 0.3 uL 100 mM MgSO 4 (final concentration 6 mM; total 8 mM, including contribution from 10× isothermal amplification buffer), 0.25 uL (20× water stock, 1.25uM final concentration), 2uL of heat-treated E. coli lysate containing DNA polymerase variants (lysate was diluted before adding to the reaction as specified in the examples below). Finally, the synthetic RNA control was added to initiate the reaction. The 384-well PCR plate was briefly vortexed and centrifuged to mix, then inserted into the CFX Touch 384-well Real-Time PCR Detection System (Bio-Rad). The isothermal amplification reaction was performed at 65°C for 120 cycles of 30 seconds, and the fluorescence of the reaction was analyzed in the FAM channel after each cycle.

[0430]

[0431] Example 5

[0432] Improvement in thermal stability relative to SEQ ID NO: 2

[0433] In the absence of a separate reverse transcriptase, after screening the reverse transcriptase and LAMP activities of large fragments of various wild-type enzymes (see Table 10.2) in the RT-LAMP reaction, the large fragment of the DNA polymerase of SEQ ID NO: 2 described in Example 1 was selected as the parent enzyme. Libraries of engineered genes were generated using established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were generated with HTP as described in Example 2, and soluble lysates were generated as described in Example 3, with a lysis temperature of 68°C. The clarified lysate was diluted fourfold, and then the RT-LAMP reaction was established with 1250 copies of SARS-CoV-2 RNA as described in Example 4.

[0434] The residual activity (active FIOP) after heat treatment relative to SEQ ID NO: 2 was calculated as the inverse of the quantitative cycle (Cq) value and is shown in Table 5.1. Samples that did not have a detectable signal above the threshold after 120 cycles (including positive and negative controls with no detectable signal) were set to a CQ (maximum cycle measured) of 120 instead of not detected (nd) to achieve fold improvement calculations.

[0435]

[0436]

[0437] Example 6

[0438] Improvement in thermal stability relative to SEQ ID NO: 10

[0439] Select SEQ ID NO:10 as the parent enzyme for this round of directed evolution. Use established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations) to generate a library of engineered genes. The polypeptides encoded by each gene were generated with HTP as described in Example 2, and soluble lysates were generated as described in Example 3, with replicate plates lysed at 71°C, 72°C, 73.5°C, and 75°C lysis temperatures. The clarified lysate was diluted 8-fold, and then RT-LAMP reactions were established with 1250 copies of SARS-CoV-2 RNA as described in Example 4.

[0440] The residual activity (active FIOP) after heat treatment relative to SEQ ID NO: 10 was calculated as the inverse of the quantification cycle (Cq) value and is shown in Table 6.1. Samples that did not have a detectable signal above the threshold after 120 cycles (including positive and negative controls with no detectable signal) were set to a CQ (maximum cycle measured) of 120 instead of not detected (nd) to achieve fold improvement calculations.

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448] Example 7

[0449] Improvement in thermal stability relative to SEQ ID NO: 80

[0450] Select SEQ ID NO:80 as the parent enzyme for this round of directed evolution. Use well-established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations) to generate a library of engineered genes. The polypeptides encoded by each gene were generated with HTP as described in Example 2, and soluble lysates were generated as described in Example 3, with replicate plates lysed at 77°C, 78°C, and 79°C lysis temperatures. The clarified lysate was diluted 8-fold, and then RT-LAMP reactions were established with 1000 copies of SARS-CoV-2 RNA as described in Example 4.

[0451] The residual activity (active FIOP) after heat treatment relative to SEQ ID NO: 80 was calculated as the inverse of the quantitative cycle (Cq) value and is shown in Table 7.1. Samples that did not have a detectable signal above the threshold after 120 cycles (including positive and negative controls with no detectable signal) were set to a CQ (maximum cycle measured) of 120 instead of not detected (nd) to achieve fold improvement calculations.

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463] Example 8

[0464] Improvement in isothermal polymerase activity relative to SEQ ID NO: 224

[0465] Select SEQ ID NO:224 as the parent enzyme for this round of directed evolution. Use established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations) to generate a library of engineered genes. The polypeptides encoded by each gene were generated with HTP as described in Example 2, and soluble lysates were generated as described in Example 3, with replicate plates lysed at a lysis temperature of 75°C. The clarified lysate was diluted 8-fold, and then RT-LAMP reactions were established with 100 copies of SARS-CoV-2 RNA as described in Example 4.

[0466] The residual activity (active FIOP) after heat treatment relative to SEQ ID NO: 224 was calculated as the inverse of the quantitative cycle (Cq) value and is shown in Table 8.1. Samples that did not have a detectable signal above the threshold after 120 cycles (including positive and negative controls with no detectable signal) were set to a CQ (maximum cycle measured) of 120 instead of not detected (nd) to achieve fold improvement calculations.

[0467]

[0468]

[0469]

[0470] Example 9

[0471] Improvement in isothermal polymerase activity relative to SEQ ID NO: 366

[0472] SEQ ID NO: 366 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were generated using well-established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). Polypeptides encoded by each gene were generated with HTP as described in Example 2, and soluble lysates were generated as described in Example 3, with replicate plates lysed at a lysis temperature of 80°C. The clarified lysate was diluted 8-fold, and then RT-LAMP reactions were established with 500 copies of SARS-CoV-2 RNA as described in Example 4.

[0473] The residual activity (active FIOP) after heat treatment relative to SEQ ID NO: 366 was calculated as the inverse of the quantitative cycle (Cq) value and is shown in Table 9.1. Samples that did not have a detectable signal above the threshold after 120 cycles (including positive and negative controls with no detectable signal) were set to a CQ (maximum cycle measured) of 120 instead of not detected (nd) to achieve fold improvement calculations.

[0474]

[0475]

[0476]

[0477]

[0478] Example 10

[0479] DNA polymerases used for initial activity screening:

[0480] Purified DNA polymerase activity in RT-LAMP reactions for SARS-CoV-2 detection

[0481] Shake flask expression

[0482] Selected cultures grown as described above were plated onto LB agar plates containing 1% glucose and 30 μg / ml chloramphenicol and grown overnight at 30° C. A single colony from each culture was transferred to 5 ml LB broth containing 1% glucose and 30 μg / ml chloramphenicol. The cultures were grown at 30° C., 250 rpm for 20 h and subcultured at approximately 1:50 dilution into 250 ml Terrific broth with 30 μg / ml chloramphenicol to a final OD of 1. 600 The culture was incubated at 30°C, 250 rpm for about 195 min to an OD of about 0.6. 600, and then add IPTG at a final concentration of 1mM for induction. The induced culture was incubated at 30°C, 250rpm for 20h. After the incubation period, the culture was centrifuged at 4000rpm×10min. The culture supernatant was discarded, and the precipitate was resuspended in 30mL of 50mM Tris-HCl, pH 8. The cell suspension was cooled in an ice bath and lysed using a microfluidized cell disruptor (Microfluidics M-110L). The crude lysate was precipitated by centrifugation (11,000rpm, at 4°C, 60min), and the supernatant was then filtered through a 0.2μm PES membrane to further clarify the lysate.

[0483] Purification of DNA polymerase from shake flask lysates

[0484] DNA polymerase lysate was purified using the AKTAPure purification system and a 5 ml HisTrap FF column (GE Healthcare); the run parameters are provided in Table 10.1. The shake flask wash buffer contained 50 mM Tris-HCl, pH 8, 500 mM NaCl, 20 mM imidazole, 0.02% v / v Triton X-100 reagent, and the shake flask elution buffer contained 50 mM Tris-HCl, pH 8, 500 mM NaCl, 250 mM imidazole, 0.02% v / v Triton X-100 reagent.

[0485]

[0486] The four most concentrated 1.5 mL fractions based on UV absorbance (A280) were combined and analyzed on a 3.5K Slide-A-Lyzer TM Buffer exchange was performed by dialyzing overnight in dialysis buffer (40 mM Tris-HCl, pH 8, 100 mM KCl, 0.1 mM EDTA, and 50% glycerol) in dialysis cassettes (Thermo Fisher). The DNA polymerase concentration in the preparation was measured by absorbance at 280 nm and normalized to 2 mg mL -1 .

[0487] RT-LAMP reaction using purified large fragment of DNA polymerase

[0488] RT-LAMP assay of the purified large fragment using DNA polymerase was performed using a primer set targeting CoV-Orf1a consisting of six oligonucleotides at different concentrations (final concentrations FIP: 1.6uM, BIP: 1.6uM, F3: 0.2uM, B3: 0.2uM, loop F: 0.4uM, loop B: 0.4uM). Each variant was screened in a 5 uL reaction containing: SARS-CoV-2 synthetic RNA control 1 (0.5 uL, GenBank / GISAID ID MT007544.1, Twist Biosciences part number 102019, 5000 copies per reaction), 0.5 uL 10× Isothermal Amplification Buffer (NEB catalog number B0537S), 0.5 uL 10× Orf1a LAMP primer mix (FIP: 16 uM, BIP: 16 uM, F3: 2 uM, B3: 2 uM, loop F: 4 uM, loop B: 4 uM), dNTP mix (final concentration of each dNTP 1.1 mM), 0.3 uL 100 mM MgSO 4 (final concentration 6 mM; total 8 mM, including contribution from 10× isothermal amplification buffer), 0.25 uL Evagreen (20× water stock solution, 1.25 uM final concentration), 1 uL purified DNA polymerase (final concentration 10 ng uL -1 ). The synthetic RNA control was added last to start the reaction. The no template control (NTC) had nuclease-free water added in place of the synthetic RNA control solution. The 384-well PCR plate was briefly vortexed and centrifuged to mix before being inserted into the CFX Touch 384-well Real-Time PCR Detection System (Bio-Rad). The isothermal amplification reaction was performed at 55°C for 120 cycles of 30 sec, and the fluorescence of the reaction was analyzed in the FAM channel after each cycle.

[0489] The RT-LAMP activity of DNA polymerase in the absence of a separate reverse transcriptase was calculated as the ratio of the quantification cycle (Cq) value of NTC to the Cq of the sample containing 5000 copies of synthetic RNA control and is shown in Table 10.2. Samples that did not have a detectable signal above the threshold after 120 cycles (including positive and negative controls that did not have a detectable signal) were set to a Cq of 120 (maximum cycle measured) instead of not detected (nd) to enable calculation of fold improvement.

[0490]

[0491]

[0492] Embodiment 11

[0493] Determining the resistance of engineered DNA polymerases to inhibitors

[0494] The engineered DNA polymerase was tested for resistance to inhibitors by reverse transcriptase loop-mediated amplification (RT-LAMP). The reaction of fluorescent dye was carried out in 20 μ L volume (n=3), wherein each of the following inhibitors was added at the following final concentrations: 3% (v / v) ethanol, 25mM guanidine thiocyanate (GTC), 20% (v / v) virus transport medium (VTM), 6.25U / mL heparin, 10μM heme, 50ng of human total RNA or 100ng of human genomic DNA (hgDNA). The other reaction of polymerase without inhibitor was run in parallel for comparison. The reaction was carried out for 72 minutes at 65°C (or 68°C for LavaLAMP) using Bio-Rad CFX Opus thermal cycler (Bio Rad, CA). The result time (time to result, TTR) of each repetition was calculated by multiplying the measured Cq value by the time of each cycle. The inhibitor resistance was compared with the engineered DNA polymerase without inhibitor, with DNA polymerase Bst 3.0 (New England Biolabs, catalog #MO374) and with LGC Lava AMP (LGC Biosearch Technologies).

[0495] Although the present invention has been described with reference to specific embodiments, various changes may be made and equivalents may be substituted to adapt a particular situation, material, composition of matter, process, process step or steps so as to achieve the benefits of the present invention without departing from the scope of what is claimed.

[0496] Each publication and patent document cited in this disclosure is incorporated herein by reference for all purposes to the same extent as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not to be construed as an indication that any such document is relevant prior art nor constitutes an admission as to its contents or date.

Claims

1. An engineered DNA polymerase, or a functional fragment thereof, comprising an amino acid 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 amino acid residues 12 to 604 of SEQ ID NO: 2, 10, 80, 224 or 366, or to a reference sequence corresponding to SEQ ID NO: 2, 10, 80, 224 or 366, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12 to 604 of SEQ ID NO: 2, 10, 80, 224 or 366, or to a reference sequence corresponding to SEQ ID NO: 2, 10, 80, 224 or 366.

2. The engineered DNA polymerase of claim 1, comprising an amino acid 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 amino acid residues 12 to 604 of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12 to 604 of SEQ ID NO: 2 or to the reference sequence corresponding to SEQ ID NO:

2.

3. The engineered DNA polymerase of claim 1, comprising an amino acid 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 amino acid residues 12 to 604 of SEQ ID NO: 10, 80, 224 or 366, or to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12 to 604 of SEQ ID NO: 2 or to a reference sequence corresponding to SEQ ID NO:

2.

4. The engineered DNA polymerase of claim 1, comprising an amino acid 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 amino acid residues 12 to 604 of an even numbered SEQ ID NO: 4-488 or a reference sequence corresponding to an even numbered SEQ ID NO: 4-488, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12 to 604 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO:

2.

5. The engineered DNA polymerase of any one of claims 2-4, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at the following amino acid positions: 21, 24, 25, 25, 34, 36, 52, 58, 66, 68, 81, 84, 92, 101, 105, 114, 115, 124, 133, 133, 134, 136, 144, 152, 154, 168, 183, 184, 191, 192, 210, 212, 221, 226 , 230, 241, 252, 253, 276, 287, 290, 294, 295, 300, 304, 322, 325, 372, 373, 374, 393, 427, 432, 452, 454, 456, 462, 470, 483, 486, 495, 505, 509, 541, 541, 545, 547, 551, 552, 573, 575, 578, 584, 585 or 593 or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

2.

6. according to the engineered DNA polymerase described in any one of claims 2-5, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or amino acid residue 21M / V, 24M, 25L / M, 34D, 36T, 52G, 58K, 66T, 68G, 81R, 84Q, 92G, 101A, 105S, 114S, 115Y, 124L, 133A, 133R, 134R / T, 136P, 144R, 152R, 154W, 168G, 183P, 184T, 191E / R, 192E, 210A, 212V, 221M, 226K, 230G / Q, 241E / Q, 578F, 584V, 585R, or 593S, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

2.

7. The engineered DNA polymerase according to any one of claims 2-4, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 36, 241, 372 or 470 or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to SEQ ID NO:

2.

8. The engineered DNA polymerase according to any one of claims 2-4 and 7, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one substitution 36T, 241E / Q, 372E or 470S or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to SEQ ID NO:

2.

9. The engineered DNA polymerase of any one of claims 2-4, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at the following amino acid positions: 36, 52, 101, 124, 134, 136, 154, 212, 241, 253, 294, 300, 372, 393, 452, 454, 456, 470, 483, 505, 509, 547, 573 or 584 or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

2.

10. The engineered DNA polymerase of any one of claims 2-4 and 9, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one substitution 36T, 52G, 101A, 124L, 134R / T, 136P, 154W, 212V, 241E / Q, 253R, 294R, 300A / G / R, 372E / K, 393L, 452R / S, 454G, 456T, 470S, 483K, 505H, 509R, 547A / H / V, 573R / V or 584V or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

2.

11. The engineered DNA polymerase of any one of claims 2-4, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions at one or more of the following amino acid positions: 509, 300, 452, 36 / 241 / 372 / 470, 124 / 192 / 210 / 372 / 427 / 456 / 552, 124, 52, 483, 372, 393, 212, 52 / 66, 133, 454, 154, 593, 462, 541, 21, 573, 505, 152, 294, 545, 101 / 241 / 470, 584, 304, 295, 578, 456 / 470, 253, 290, 192 / 241 / 372 / 456, or 252, wherein the amino acid positions are relative to the amino acid corresponding to SEQ ID Reference sequence of NO:

2.

12. The engineered DNA polymerase according to any one of claims 2-4 and 11, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions 509R, 300R, 452R, 36T / 241Q / 372E / 470S, 124L / 192E / 210A / 372E / 427A / 456T / 552L, 124L, 52G, 483K, 372E, 452S, 300A, 393L, 300G, 21 2V, 52G / 66T, 133R, 454G, 154W, 593S, 462R, 541R, 21M, 573V, 505H, 152R, 294R, 545K, 101A / 241Q / 470S, 584V, 304L, 295K, 578F, 541T, 456T / 470S, 253R, 290G, 192E / 241E / 372E / 456T or 252T, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

2.

13. The engineered DNA polymerase of claim 1, comprising an amino acid 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 residues 12 to 604 of SEQ ID NO: 10, 80, 224 or 366.

14. The engineered DNA polymerase of claim 1, comprising an amino acid 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 residues 12 to 604 of an even-numbered SEQ ID NO: 4-488 or to a reference sequence corresponding to an even-numbered SEQ ID NO: 4-488.

15. The engineered DNA polymerase of claim 1, comprising an amino acid 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 residues 12 to 604 of SEQ ID NO: 10, 80, 224 or 366, or to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 10, 80, 224 or 366, or to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366.

16. The engineered DNA polymerase of claim 1, comprising an amino acid 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 residues 12 to 604 of an even numbered SEQ ID NO: 4-488 or to a reference sequence corresponding to an even numbered SEQ ID NO: 4-488, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 10, 80, 224 or 366 or relative to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366.

17. The engineered DNA polymerase of claim 15 or 16, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at the following amino acid positions: 21, 24, 25, 25, 34, 36, 52, 58, 66, 68, 81, 84, 92, 101, 105, 114, 115, 124, 133, 133, 134, 136, 144, 152, 154, 168, 183, 184, 191, 192, 210, 212, 221, 226, 10, 11, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 294, 295, 300, 304, 322, 325, 372, 373, 374, 393, 427, 432, 452, 454, 456, 462, 470, 483, 486, 495, 505, 509, 541, 541, 545, 547, 551, 552, 573, 575, 578, 584, 585 or 593, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366.

18. according to the engineered DNA polymerase described in any one of claims 15-17, wherein the amino acid sequence of said engineered DNA polymerase comprises at least one substitution or amino acid residue 21M / V, 24M, 25L / M, 34D, 36T, 52G, 58K, 66T, 68G, 81R, 84Q, 92G, 101A, 105S, 114S, 115Y, 124L, 133A, 133R, 134R / T, 136P, 144R, 152R, 154W, 168G, 183P, 184T, 191E / R, 192E, 210A, 212V, 221M, 226K, 230G / Q, 241E / Q , 252T, 253R, 276G, 287R, 290G, 294R, 295K, 300A / G / R, 304L, 322C, 325W, 372E / K, 373A, 374E, 393L, 427A, 432K / L, 452R / S, 454G, 456T, 462R, 470S, 483K, 486R, 495F, 505H, 509R, 541G / R, 541T, 545K, 547A / H / V, 551K, 552L, 573R / V, 575V, 578F, 584V / I, 585R or 593S, or a combination thereof, wherein the amino acid positions are relative to the amino acid positions corresponding to SEQ Reference sequence of ID NO: 10, 80, 224 or 366.

19. The engineered DNA polymerase of claim 15 or 16, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at amino acid position 36, 241, 372 or 470 or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366.

20. The engineered DNA polymerase according to any one of claims 15, 16 and 19, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or amino acid residues 36T, 241Q, 372K / E or 470S or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366.

21. The engineered DNA polymerase of claim 15 or 16, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one substitution at the following amino acid positions: 36, 52, 101, 124, 134, 136, 154, 212, 241, 253, 294, 300, 372, 393, 452, 454, 456, 470, 483, 505, 509, 547, 573 or 584 or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366.

22. The engineered DNA polymerase of any one of claims 15, 16 and 21, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or amino acid residue 36T, 52G, 101A, 124L, 134R, 136P, 154W, 212V, 241Q, 253R, 294R, 300R, 372K / E, 393L, 452R, 454G, 456T, 470S, 483K, 505H, 509R, 547H, 573V or 584I / V or 584I / V or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to SEQ ID NO: 10, 80, 224 or 366.

23. The engineered DNA polymerase of claim 15, comprising an amino acid 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 residues 12 to 604 of SEQ ID NO: 10 or to a reference sequence corresponding to SEQ ID NO: 10, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 10 or to a reference sequence corresponding to SEQ ID NO:

10.

24. The engineered DNA polymerase of claim 16, comprising an amino acid 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 residues 12 to 604 of an even numbered SEQ ID NO: 10-218 or a reference sequence corresponding to an even numbered SEQ ID NO: 10-218, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 10 or relative to the reference sequence corresponding to SEQ ID NO:

10.

25. The engineered DNA polymerase of claim 23 or 24, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one set of substitutions at the following amino acid positions: 52 / 101 / 124 / 212 / 294 / 372 / 393 / 452 / 483 / 509, 52 / 124 / 300 / 393 / 452, 154 / 212 / 294 / 300 / 372 / 393 / 452 / 483 / 509, 52 / 101 / 154 / 294 / 300 / 452 / 509 / 593, 52 / 154 / 212 / 294 / 300 / 393 / 452, 212 / 300 / 393 / 452 / 509, 2 / 294 / 300 / 393 / 452 / 483 / 509 / 593、52 / 124 / 294 / 300 / 452 / 509、124 / 300 / 393 / 452 / 483 / 509 / 593、101 / 124 / 212 / 452 / 483 / 509、52 / 393 / 452 / 509 / 593 、212 / 300 / 452 / 509 / 593、52 / 452 / 509 / 593、154 / 212 / 300 / 372 / 452 / 509 / 593、52 / 124 / 212 / 294 / 393 / 452 / 593、124 / 452 / 509、152 / 253 / 287 / 505 / 541 / 5 73 / 584、154 / 212 / 300 / 452、152 / 253 / 287 / 304 / 454 / 545 / 573 / 584、52 / 124 / 154 / 294 / 300 / 372 / 452 / 509、294 / 300 / 372 / 452 / 509 / 593、52 / 212 / 452、12 4 / 294 / 393 / 452、52 / 300 / 509 / 593、152 / 253 / 287 / 454 / 541 / 584、52 / 101 / 452 / 509、52 / 154 / 212 / 300 / 372 / 393 / 509、21 / 253 / 287 / 454 / 584、52 / 393 / 452 、52 / 124 / 154 / 294 / 509、101 / 124 / 212 / 300 / 393、124 / 294 / 300 / 372 / 452 / 509 / 593、52 / 124 / 154 / 294 / 483 / 593、52 / 372 / 393 / 452 / 509 / 593、154 / 300 / 3 93 / 452 / 593、52 / 101 / 294 / 372 / 393 / 452 / 509、21 / 152 / 253 / 287 / 304 / 454 / 505、52 / 300 / 393、101 / 124 / 154 / 294 / 452 / 509、154 / 212 / 372 / 393 / 509 / 593、21 / 253 / 287 / 454 / 505 / 541、287 / 454 / 505 / 584、52 / 124 / 294 / 300 / 372 / 452、287 / 505 / 541 / 573、253 / 505 / 584、84 / 253 / 287 / 456 / 573、52 / 372 / 509 / 593、509 / 593 、154 / 212 / 300 / 372 / 509、52 / 154 / 294 / 509 / 593、152 / 295 / 505 / 545 / 573、253 / 541 / 584、253 / 295 / 304 / 454 / 462 / 573 / 584、253 / 454 / 505 / 573 / 584、154 / 212 / 300 、287 / 454 / 545 / 573、21 / 152 / 287 / 304 / 454、152 / 253 / 295 / 304 / 541 / 573、152 / 253 / 287 / 304 / 454、152 / 454 / 573、124 / 294 / 372 / 509 / 593、152 / 253 / 295 / 454、154 / 300 / 452, 287 / 304 / 505, 152 / 253 / 454 / 505, 253 / 454 / 505, 21 / 253 / 454 / 505 / 545, 152 / 253 / 287 / 505, 505 / 541 / 584 or 152 / 253 / 454 / 584, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

10.

26. The engineered DNA polymerase of any one of claims 23-25, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one substitution set 52G / 101A / 124L / 212V / 294R / 372K / 393L / 452R / 483K / 509R, 52G / 124L / 300A / 393L / 452R, 154W / 212V / 294R / 300G / 372K / 393L / 452R / 483K / 509R, 52G / 101A / 154W / 294R / 300R / 452S / 509R / 593S, 52G / 154W / 212V / 294R / 300G / 393L / 452 R、212V / 300R / 393L / 452R / 509R、52G / 101A / 212V / 294R / 300R / 393L / 452R / 4 83K / 509R / 593S, 52G / 124L / 294R / 300R / 452S / 509R, 124L / 300R / 393L / 452R / 483K / 509R / 593S, 101A / 124L / 212V / 452R / 483K / 509R, 52G / 393L / 452R / 50 9R / 593S, 212V / 300R / 452R / 509R / 593S, 52G / 452R / 509R / 593S, 154W / 212V / 300R / 372K / 452R / 509R / 593S, 52G / 124L / 212V / 294R / 393L / 452R / 593S, 124 L / 452R / 509R, 152R / 253R / 287R / 505H / 541R / 573V / 584V, 154W / 212V / 300R / 452R, 152R / 253R / 287R / 304L / 454G / 545K / 573V / 584V, 52G / 124L / 154W / 294 R / 300A / 372K / 452R / 509R, 294R / 300R / 372K / 452S / 509R / 593S, 52G / 212V / 4 52R, 124L / 294R / 393L / 452R, 52G / 300R / 509R / 593S, 152R / 253R / 287R / 454G / 541R / 584V, 52G / 101A / 452S / 509R, 52G / 154W / 212V / 300R / 372K / 393L / 509 R、21M / 253R / 287R / 454G / 584V、52G / 393L / 452R、52G / 124L / 154W / 294R / 509 R, 101A / 124L / 212V / 300R / 393L, 124L / 294R / 300G / 372K / 452S / 509R / 593S,52G / 124L / 154W / 294R / 483K / 593S, 52G / 372K / 393L / 452S / 509R / 593S, 154W / 300R / 393L / 452S / 593S, 52G / 101A / 294R / 372K / 393L / 45 2S / 509R, 21M / 152R / 253R / 287R / 304L / 454G / 505H, 52G / 300R / 393L, 101A / 124L / 154W / 294R / 452S / 509R, 154W / 212V / 372K / 393L / 509R / 593S、21M / 253R / 287R / 454G / 505H / 541G、287R / 454G / 505H / 584V、52G / 124L / 294R / 300R / 372K / 452S、287R / 505H / 541R / 573V、253R / 505H / 584V, 84Q / 253R / 287R / 456T / 573V, 52G / 372K / 509R / 593S, 509R / 593S, 154W / 212V / 300R / 372K / 509R, 52G / 154W / 294R / 509R / 593 S, 152R / 295K / 505H / 545K / 573V, 253R / 541R / 584V, 253R / 295K / 304L / 454G / 462R / 573V / 584V, 253R / 454G / 505H / 573V / 584V, 154W / 21 2V / 300R, 287R / 454G / 545K / 573V, 21M / 152R / 287R / 304L / 454G, 152R / 253R / 295K / 304L / 541R / 573V, 152R / 253R / 287R / 304L / 454G, 152 R / 454G / 573V, 124L / 294R / 372K / 509R / 593S, 152R / 253R / 295K / 454G, 154W / 300R / 452S, 287R / 304L / 505H, 152R / 253R / 454G / 505H, 253R / 454G / 505H, 21M / 253R / 454G / 505H / 545K, 152R / 253R / 287R / 505H, 505H / 541R / 584V or 152R / 253R / 454G / 584V, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

10.

27. The recombinant reverse transcriptase of claim 15, wherein the engineered DNA polymerase comprises an amino acid 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 residues 12 to 604 of SEQ ID NO: 80 or to a reference sequence corresponding to SEQ ID NO: 80, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 80 or to a reference sequence corresponding to SEQ ID NO:

80.

28. The engineered DNA polymerase of claim 16, comprising an amino acid 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 residues 12 to 604 of an even numbered SEQ ID NO: 220-258 or a reference sequence corresponding to an even numbered SEQ ID NO: 220-258, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 80 or relative to the reference sequence corresponding to SEQ ID NO:

80.

29. The engineered DNA polymerase of claim 27 or 28, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions at one or more of the following amino acid positions: 300 / 454 / 456 / 541 / 584, 21 / 300 / 454 / 545 / 584, 253 / 300 / 454 / 456 / 584, 154 / 253 / 300 / 454 / 456 / 505 / 573, 21 / 154 / 300 / 454 / 456 / 573, 152 / 300 / 454 / 456 / 505 / 584 / 593, 154 / 253 / 300 / 456 / 505 / 545 / 573 / 584, 253 / 300 / 454 / 456 / 584 0 / 454 / 456 / 505 / 573、154 / 253 / 300 / 454 / 456 / 541 / 573 / 584、154 / 300 / 454 / 505、21 / 253 / 300 / 454 / 456 / 545 / 573 / 593、168 / 300 / 454 / 456 / 545 / 573、253 / 300 / 454 / 505 / 573、154 / 300 / 505 / 545 / 584、154 / 253 / 300 / 456 / 541 / 573 / 584、21 / 154 / 300 / 454 / 456 / 505 / 545、154 / 253 / 300 / 454 / 505、21 / 154 / 253 / 300 / 454 / 573、300 / 454 / 505 / 541 / 584、300 / 454 / 505 / 541 / 573、154 / 253 / 300 / 454、154 / 300 / 454 / 573、300 / 454 / 456 / 545 / 573、253 / 300 / 454 / 456、154 / 253 / 300 / 454 / 545 / 573、21 / 152 / 253 / 300 / 454 / 456 / 541 / 573、154 / 253 / 300 / 454 / 456 / 573、152 / 300 / 454、154 / 253 / 300 / 454 / 505 / 541 / 593、21 / 154 / 300 / 454 / 573 / 584、154 / 300 / 456 / 573 / 593、152 / 253 / 300 / 454 / 545 / 573、21 / 300 / 456 / 495 / 505 / 541 / 584、154 / 300 / 454 / 541 / 584、253 / 300 / 505 / 541 / 573 / 584、2 53 / 300 / 584、253 / 300 / 454 / 456 / 545、21 / 253 / 300 / 573、300 / 454 / 545、454 / 456 / 541 / 545 / 573 / 584、152 / 253 / 454 / 505 / 545 / 573、21 / 300 / 454 / 456 / 495、300 / 573、154 / 300 / 454 / 545、154 / 253 / 300 / 545 / 584、21 / 300 / 505、21 / 152 / 253 / 300 / 454 / 456 / 545、154 / 253 / 300 / 456 / 541 / 573、154 / 300 / 454、253 / 300 / 454 / 541、253 / 454 / 456 / 541 / 584、21 / 300 / 573、300 / 454 / 456、300 / 454、154 / 253 / 30 0 / 454 / 456, 154 / 456 / 505 / 593, 454 / 456 / 505, 253 / 454 / 456 / 584, 154 / 253 / 300, 253 / 456 / 505 / 584, 454 / 456, 154 / 454, 584, 432 / 505 / 573, 456 / 584, 21 / 505 / 545 / 584, 21 / 154 / 545 / 573, 454 / 584, 154 / 253 / 505 or 505, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

80.

30. The engineered DNA polymerase of any one of claims 27-29, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions 300R / 454G / 456T / 541R / 584V, 21M / 300R / 454G / 545K / 584V, 253R / 300R / 454G / 456T / 584V, 154W / 253R / 300R / 454G / 456T / 505H / 573V, 21M / 154W / 300R / 454G / 456T / 573V, 152R / 300R / 454G / 456T / 505H / 584V / 593S, 154W / 253 R / 300R / 456T / 505H / 545K / 573V / 584V, 253R / 300R / 454G / 456T / 505H / 573V , 154W / 253R / 300R / 454G / 456T / 541G / 573V / 584V, 154W / 300R / 454G / 505H, 21M / 253R / 300R / 454G / 456T / 545K / 573V / 593S, 168G / 300R / 454G / 456T / 54 5K / 573V, 253R / 300R / 454G / 505H / 573V, 154W / 300R / 505H / 545K / 584V, 154 W / 253R / 300R / 456T / 541R / 573V / 584V, 21M / 154W / 300R / 454G / 456T / 505H / 545K, 154W / 253R / 300R / 454G / 505H, 21M / 154W / 253R / 300R / 454G / 573V, 30 0R / 454G / 505H / 541R / 584V, 300R / 454G / 505H / 541R / 573V, 154W / 253R / 300 R / 454G, 154W / 300R / 454G / 573V, 300R / 454G / 456T / 545K / 573V, 253R / 300R / 454G / 456T、154W / 253R / 300R / 454G / 545K / 573V、21M / 152R / 253R / 300R / 4 54G / 456T / 541R / 573V, 154W / 253R / 300R / 454G / 456T / 573V, 152R / 300R / 45 4G, 154W / 253R / 300R / 454G / 505H / 541R / 593S, 21M / 154W / 300R / 454G / 573V / 584V、154W / 300R / 456T / 573V / 593S、152R / 253R / 300R / 454G / 545K / 573V、21M / 300R / 456T / 495F / 505H / 541R / 584V, 154W / 300R / 454G / 541G / 584V, 253R / 300R / 505H / 541R / 573V / 584V, 25 3R / 300R / 584V, 253R / 300R / 454G / 456T / 545K, 21M / 253R / 300R / 573V, 300R / 454G / 545K, 454G / 456T / 541G / 545K / 573V / 584V, 152R / 253R / 454G / 505H / 545K / 573V, 21M / 300R / 454G / 456T / 495F, 300R / 573V, 154W / 300R / 454G / 5 45K、154W / 253R / 300R / 545K / 584V、21M / 300R / 505H、21M / 152R / 253R / 300R / 454G / 456T / 545K、154W / 253R / 300R / 456T / 541R / 573V, 154W / 300R / 454G, 253R / 300R / 454G / 541R, 253R / 454G / 456T / 541G / 584V, 21M / 300R / 573V, 30 0R / 454G / 456T, 300R / 454G, 154W / 253R / 300R / 454G / 456T, 154W / 456T / 505H / 593S, 454G / 456T / 505H, 253R / 454G / 456T / 584V, 154W / 253R / 300R, 253R / 456T / 505H / 584V, 454G / 456T, 154W / 454G, 584V, 432K / 505H / 573V, 456T / 584V, 21M / 505H / 545K / 584V, 21V / 154W / 545K / 573V, 454G / 584V, 154W / 253R / 505H or 505H, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

80.

31. The engineered DNA polymerase of claim 15, comprising an amino acid 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 residues 12 to 604 of SEQ ID NO: 224 or to a reference sequence corresponding to SEQ ID NO: 224, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 224 or to the reference sequence corresponding to SEQ ID NO:

224.

32. The engineered DNA polymerase of claim 16, comprising an amino acid 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 residues 12 to 604 of an even numbered SEQ ID NO: 360-400 or a reference sequence corresponding to an even numbered SEQ ID NO: 360-400, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 224 or relative to the reference sequence corresponding to SEQ ID NO:

224.

33. The engineered DNA polymerase of claim 31 or 32, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one set of substitutions at the following amino acid positions: 144 / 154 / 505 / 547 / 573 / 584, 154 / 191 / 325 / 505 / 573 / 584, 144 / 154 / 373 / 374 / 505 / 573 / 584, 134 / 136 / 154 / 505 / 547 / 573 / 584 3 / 584、81 / 144 / 154 / 505 / 547 / 573 / 584、154 / 505 / 573 / 584、144 / 154 / 191 / 230 / 322 / 505 / 573 / 584、68 / 144 / 154 / 505 / 573 / 584、144 / 154 / 226 / 230 / 505 / 573 / 584、144 / 154 / 374 / 486 / 505 / 573 / 584、81 / 114 / 144 / 154 / 505 / 573 / 584、144 / 154 / 276 / 505 / 573 / 584、134 / 144 / 154 / 505 / 573 / 584、144 / 154 / 505 / 573 / 584、81 / 144 / 154 / 183 / 505 / 547 / 573 / 584、68 / 81 / 133 / 134 / 144 / 154 / 505 / 547 / 573 / 584、14 or 81 / 154 / 505 / 547 / 573 / 584, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

224.

34. The engineered DNA polymerase of any one of claims 31-33, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one substitution set 144R / 154W / 505H / 547H / 573V / 584I, 154W / 191E / 325W / 505H / 573V / 584I, 144R / 154W / 373A / 374E / 505H / 573V / 584I, 134R / 136P / 154W / 505H / 547H / 573V / 584I, 81R / 144R / 154W / 505H / 547H / 573V / 584I, 154W / 505H / 573V / 584I, 144R / 154W / 191R / 230G / 322C / 505H / 573V / 584I, 68G / 144R / 154W / 505H / 573V / 584I、144R / 154W / 226K / 230Q / 505H / 573V / 584I、144R / 154W / 374E / 486R / 505H / 573V / 584I、81R / 114S / 144R / 154W / 505H / 573V / 584I、144R / 154W / 276G / 505H / 573V / 584I、134T / 144R / 154W / 505H / 573V / 584I、144R / 154W / 505H / 57 3V / 584I, 81R / 144R / 154W / 183P / 505H / 547A / 573V / 584I, 68G / 81R / 133A / 134R / 144R / 154W / 505H / 547H / 573V / 584I, 144R / 154W / 374E / 505H / 573V / 584I, 92G / 133A / 144R / 154W / 505H / 573V / 584I, 144R / 154W / 373A / 505H / 573V / 584I, 114S / 144R / 154W / 505H / 573V / 584I, 154W / 505H / 551K / 573R / 584I / 585R or 81R / 154W / 505H / 547V / 573V / 584I, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

224.

35. The engineered DNA polymerase of claim 15, comprising an amino acid 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 residues 12 to 604 of SEQ ID NO: 366 or to a reference sequence corresponding to SEQ ID NO: 366, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 366 or to a reference sequence corresponding to SEQ ID NO:

366.

36. The engineered DNA polymerase of claim 16, comprising an amino acid 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 residues 12 to 604 of an even numbered SEQ ID NO: 402-488 or a reference sequence corresponding to an even numbered SEQ ID NO: 402-488, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 604 of SEQ ID NO: 366 or relative to the reference sequence corresponding to SEQ ID NO:

366.

37. The engineered DNA polymerase of claim 35 or 36, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions at one or more of the following amino acid positions: 24 / 58 / 432, 58 / 432 / 575, 58 / 115 / 432 / 575, 24 / 115 / 432 / 575, 24 / 25 / 221 / 432, 25 / 58 / 432 / 575, 24 / 25 / 58 / 432 / 575, 221 / 432 / 575, 432 / 575, 24 / 221 / 432, 24 / 115 / 221 / 432, 24 / 115 / 221 / 432 / 575, 432, 24 / 34 / 432, 24 / 432 / 575, 184 / 221 / 432 / 575, 24 / 432, 24 / 25 / 58 / 115 / 432 / 575, 25 / 221 / 432, 25 / 58 / 115 / 432, 221 / 575, 221 / 432, 24 / 58 / 115 / 221 / 575, 24 / 25 / 221 / 432 / 575, 24 / 25 / 58 / 221 / 432, 34 / 58 / 105 / 432, 24 / 25 / 115 / 432, 24 / 221 / 575, 115 / 432, 58 / 2 21, 58 / 575, 25 / 432 / 575, 25 / 432, 24 / 221 / 432 / 575, 24 / 115 / 432, 24 / 25 / 105 / 432, 25 / 34 / 432, 25 / 105 / 221 / 432, 24 / 25 / 432, 24 / 58 / 575 or 24 / 25 / 105 / 432 / 575, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

366.

38. The engineered DNA polymerase of any one of claims 35-37, wherein the amino acid sequence of the engineered DNA polymerase comprises at least one substitution or set of substitutions 24M / 58K / 432L, 58K / 432L / 575V, 58K / 115Y / 432L / 575V, 24M / 115Y / 432L / 575V, 24M / 25L / 221M / 432L, 25L / 58K / 432L / 575V, 24M / 25M / 58K / 432L / 575V, 221M / 432 L / 575V, 432L / 575V, 24M / 221M / 432L, 24M / 115Y / 221M / 432L, 24M / 25M / 221M / 432L, 24M / 115Y / 221M / 432L / 575V, 432L , 24M / 34D / 432L, 24M / 432L / 575V, 184T / 221M / 432L / 575V, 24M / 432L, 24M / 25L / 58K / 115Y / 432L / 575V, 25L / 221M / 432L , 25M / 58K / 115Y / 432L, 221M / 575V, 221M / 432L, 24M / 58K / 115Y / 221M / 575V, 24M / 25M / 221M / 432L / 575V, 24M / 25M / 58K / 221M / 432L, 34D / 58K / 105S / 432L, 24M / 25M / 115Y / 432L, 24M / 221M / 575V, 115Y / 432L, 58K / 221M, 58K / 575V, 25L / 432L / 575V, 25L / 432L, 24M / 221M / 432L / 575V, 24M / 115Y / 432L, 24M / 25M / 105S / 432L, 25M / 34D / 432L, 24M / 25L / 105S / 432L, 25M / 432L, 25M / 105S / 221M / 432L, 24M / 25L / 432L, 24M / 58K / 575V or 24M / 25M / 105S / 432L / 575V, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

366.

39. The engineered DNA polymerase of claim 1, wherein the DNA polymerase comprises an amino acid 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 amino acid sequence comprising a substitution or set of substitutions of an engineered DNA polymerase variant listed in Tables 5.1, 6.1, 7.1, 8.1 and 9.

1.

40. The engineered DNA polymerase of claim 1, wherein the amino acid sequence of the engineered DNA polymerase comprises residues 12 to 604 of an even-numbered SEQ ID NO. in SEQ ID NO: 4-488 or comprises an even-numbered SEQ ID NO. in SEQ ID NO: 4-488, optionally wherein the engineered DNA polymerase has 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions in the amino acid sequence.

41. The engineered DNA polymerase of claim 1, wherein the amino acid sequence of the engineered DNA polymerase comprises residues 12 to 604 of SEQ ID NO: 10, 80, 224, or 366, or wherein the amino acid sequence comprises SEQ ID NO: 10, 80, 224, or 366, optionally wherein the engineered DNA polymerase has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions in the amino acid sequence.

42. The engineered DNA polymerase of claim 40 or 41, wherein the amino acid sequence of the engineered DNA polymerase has 1, 2, 3, 4, up to 5 substitutions in the amino acid sequence.

43. The engineered DNA polymerase of any one of claims 40-42, wherein the substitutions comprise conservative substitutions.

44. The engineered DNA polymerase of any one of claims 1-43, having at least one improved property compared to a reference DNA polymerase.

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 sensitivity to input target RNA or DNA, vii) increased product yield in isothermal amplification reactions, viii) increased salt tolerance, and ix) increased resistance to inhibitors, or any combination of i), ii), iii), iv), v), vi), vii) and ix).

46. ​​The engineered DNA polymerase of claim 44 or 45, wherein the improved property is the use of DNA as a template.

47. The engineered DNA polymerase of claim 44 or 45, wherein the improved property is the use of RNA as a template.

48. The engineered DNA polymerase of any one of claims 44-47, wherein the reference DNA polymerase has an amino acid sequence corresponding to residues 12 to 604 of SEQ ID NO: 2, 10, 80, 224, or 366 or an amino acid sequence corresponding to SEQ ID NO: 2, 10, 80, 224, or 366.

49. The engineered DNA polymerase of claim 48, wherein the reference DNA polymerase has an amino acid sequence corresponding to residues 12 to 604 of SEQ ID NO: 2 or an amino acid sequence corresponding to SEQ ID NO:

2.

50. The engineered DNA polymerase of any one of claims 1-49, wherein the engineered DNA polymerase comprises a fusion polypeptide.

51. The engineered DNA polymerase of any one of claims 1-50, wherein the engineered DNA polymerase is purified.

52. A recombinant polynucleotide comprising a polynucleotide sequence encoding the engineered DNA polymerase of any one of claims 1-50.

53. The recombinant polynucleotide of claim 52, comprising 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 polynucleotide sequence corresponding to nucleotide residues 34 to 1812 of SEQ ID NO: 1, 9, 79, 223 or 365, wherein the recombinant polynucleotide encodes an engineered DNA polymerase.

54. The recombinant polynucleotide of claim 52, comprising 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 polynucleotide sequence corresponding to nucleotide residues 34 to 1812 of an odd-numbered SEQ ID NO: 3-487 or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO: 3-487, wherein the recombinant polynucleotide encodes an engineered DNA polymerase.

55. The recombinant polynucleotide of any one of claims 52-54, wherein the polynucleotide sequence is codon-optimized for expression of the encoded engineered DNA polymerase.

56. The recombinant polynucleotide of claim 52, wherein the polynucleotide sequence comprises nucleotide residues 34 to 1812 of SEQ ID NO: 9, 79, 223, or 365 or comprises SEQ ID NO: 9, 79, 223, or 365.

57. The recombinant polynucleotide of claim 52, wherein the polynucleotide sequence comprises nucleotide residues 34 to 1812 of an odd-numbered SEQ ID NO. in SEQ ID NOs: 3-487 or comprises an odd-numbered SEQ ID NO. in SEQ ID NOs: 3-487.

58. An expression vector comprising at least one recombinant polynucleotide according to any one of claims 52-57.

59. The expression vector of claim 58, wherein the polynucleotide is operably linked to a control sequence.

60. The expression vector of claim 59, wherein the control sequence comprises at least one promoter.

61. A host cell comprising the expression vector according to any one of claims 58-60.

62. The host cell of claim 61, comprising a prokaryotic cell or a eukaryotic cell.

63. The host cell of claim 62, comprising a bacterial cell, a fungal cell, an insect cell or a mammalian cell.

64. A method for producing an engineered DNA polymerase polypeptide in a host cell, the method comprising culturing the host cell of any one of claims 61-63 under suitable culture conditions such that the DNA polymerase is produced.

65. The method of claim 64, further comprising recovering the DNA polymerase from the culture and / or host cell.

66. The method of claim 64 or 65, further comprising purifying the engineered DNA polymerase.

67. A composition comprising the DNA polymerase of any one of claims 1-51.

68. The composition of claim 67, comprising at least one DNA or RNA template.

69. A composition according to claim 67 or 68, further comprising one or more nucleotide substrates.

70. The composition of any one of claims 67-69, further comprising one or more oligonucleotide primer substrates.

71. The composition of any one of claims 67-70, comprising at least one buffer.

72. A method for preparing complementary DNA of a target nucleic acid, the method comprising contacting the target nucleic acid with a DNA polymerase as described in any one of claims 1-51 in the presence of a substrate and a target nucleic acid under conditions suitable for producing a complementary DNA that is complementary to all or part of the target nucleic acid.

73. The method of claim 72, wherein the target nucleic acid is DNA or RNA.

74. A method for detecting the presence of a target nucleic acid, the method comprising contacting a sample suspected of containing the target nucleic acid with a DNA polymerase as described in any one of claims 1 to 51 in the presence of a substrate, under conditions suitable for DNA polymerase-mediated production of DNA complementary to all or part of the target nucleic acid, and detecting the presence of the complementary DNA.

75. The method of claim 74, wherein the target nucleic acid is DNA or RNA.

76. The method of claim 74 or 75, wherein the complementary DNA is detected by amplifying the complementary DNA.

77. The method of claim 76, wherein the amplification is performed by isothermal amplification.

78. The method of claim 77, wherein the isothermal amplification is performed by loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), or recombinase polymerase amplification (RPA).

79. A method for sequencing a target DNA or RNA, the method comprising contacting the target DNA or RNA with a DNA polymerase described in any one of claims 1 to 51 in the presence of a substrate suitable for sequencing, under conditions suitable for DNA polymerase-mediated extension of a complementary DNA of the target DNA or RNA, and determining the sequence of the target DNA or RNA.

80. A kit comprising at least one engineered DNA polymerase according to any one of claims 1-51.

81. The kit of claim 80, comprising one or more of a buffer, a nucleotide substrate, an oligonucleotide primer substrate, and a nucleic acid template.

82. The kit of claim 80 or 81 , further comprising a second DNA polymerase.

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