Engineered DNA polymerase variants

Engineered DNA polymerases with targeted amino acid modifications address the need for high-fidelity thermostable enzymes, enhancing their suitability for PCR and diagnostic uses.

JP7780186B2Active Publication Date: 2025-12-04CODEXIS INC
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Patent Information

Application Number
JP2021523258
Authority / Receiving Office
JP · JP
Patent Type
Patents
Current Assignee / Owner
Priority Date
2018-10-29
Filing Date
2019-10-28
Publication Date
2025-12-04
Estimated Expiration
2039-10-28

AI Technical Summary

Technical Problem

There is a need for thermostable DNA polymerases with high fidelity for applications such as PCR, as existing enzymes do not meet the requirements for accuracy and reliability in these processes.

Method used

Engineered DNA polymerases with specific amino acid substitutions or sets of substitutions at defined positions, based on reference sequences, to enhance fidelity and performance.

Benefits of technology

The engineered DNA polymerases demonstrate improved fidelity and performance, making them suitable for diagnostic and other applications requiring high accuracy and stability.

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Abstract

The present invention provides engineered DNA polymerase polypeptides and compositions thereof, as well as polynucleotides encoding the engineered DNA polymerase polypeptides. The present invention also provides methods for using compositions comprising engineered DNA polymerase polypeptides for diagnostic and other purposes. The present invention also provides polynucleotide sequences encoding the engineered DNA polymerases provided herein. In some embodiments, the polynucleotide sequences encode at least one engineered DNA polymerase provided herein.
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Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 752,215, filed October 29, 2018, which is incorporated by reference in its entirety for all purposes. Reference to a sequence listing, table, or computer program

[0002] The Sequence Listing, which was submitted contemporaneously herewith under 37 C.F.R. § 1.821 via EFS-Web in computer readable form (CRF) under filename CX9-181WO2_ST25.txt, is incorporated herein by reference. The electronic copy of the Sequence Listing was created on October 28, 2019, and is 5,361 kilobytes in size.

[0003] The present invention provides engineered DNA polymerase polypeptides and compositions thereof, as well as polynucleotides encoding the engineered DNA polymerase polypeptides. The present invention also provides methods for the use of compositions comprising the engineered DNA polymerase polypeptides for diagnostic and other purposes. [Background technology]

[0004] DNA polymerases are enzymes that synthesize DNA from deoxyribonucleotides. These enzymes are essential for DNA replication. There are various types of DNA polymerases, generally divided into seven families: A, B, C, D, X, Y, and RT. These families have different properties and are found in different types of organisms. For example, group A polymerases are replication and repair polymerases found in both eukaryotes and prokaryotes (examples include T7 DNA polymerase and E. coli pol I). Group B polymerases are also replication and repair enzymes found in both eukaryotes and prokaryotes (e.g., pol II, pol B, etc.), while group C and group D include replicative polymerases found in prokaryotes and Euryarchaeota, respectively (group C polymerases include pol III, while group D polymerases are less well characterized). Group X, group Y, and group RT polymerases are replication and repair enzymes found in eukaryotes (group X), eukaryotes and prokaryotes (group Y), and viruses, retroviruses, and eukaryotes (group RT). Examples of group X polymerases include pol β, while group Y polymerases include pol IV and pol V, and RT group polymerases include the polymerase of hepatitis B virus. Some of these polymerases, particularly those obtained from thermophilic organisms, have been widely used in various in vitro methods, including, but not limited to, polymerase chain reaction (PCR). The availability of thermophilic polymerases has made automation of the PCR process possible. Thus, they are critical enzymes in applications where PCR is useful. While there are many commercially available enzymes (e.g., Taq and many others), there remains a need in the art for thermostable enzymes with high levels of fidelity. Summary of the Invention [Means for solving the problem]

[0005] The present invention provides engineered DNA polymerase polypeptides and compositions thereof, as well as polynucleotides encoding the engineered DNA polymerase polypeptides. The present invention also provides methods for the use of compositions comprising the engineered DNA polymerase polypeptides for diagnostic and other purposes.

[0006] The present invention provides engineered DNA polymerases comprising a polypeptide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence of SEQ ID NO: 2, 6, 22, 24, 26, 28, and / or 824, or a functional fragment thereof, wherein the polypeptide sequence comprises at least one substitution or set of substitutions, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2, 6, 22, 24, 26, 28, and / or 824.

[0007] The present invention also relates to 21, 21 / 66 / 247 / 282, 247 / 282 / 575, 282 / 575, 283 / 647 / 702 / 743, 339 / 647 / 661 / 664 / 668 / 702 / 712, 372 / 391 / 702, 391, 391 / 647 / 659 / 661 / 668 / 671 / 712 / 716, 391 / 647 / 659 / 661 / 668 / 671 / 716, 391 / 647 / 659 / 664 / 668 / 702 / 728 / 732, 391 / 647 / 659 / 664 / 671 / 702, 391 / 647 / 661 / 664 / 671 / 702 / 716, 391 / 647 / 671 / 728, 391 / 659 / 702 / 716 / 732 / 737, 391 / 661 / 664 / 668 / 671 / 716 / 737, 391 / 671, 391 / 702 / 712 / 716 / 732 / 743, 647 / 659 / 661 / 664 / 668 / 702, 647 / 659 / 664 / 668 / 702 / 712 / 737, 647 / 659 / 668 / 671 / 716 / 728, 647 / 668, 647 / 668 / 671 / 712, 659 / 702 / 743, 661 / 664 / Also provided is an engineered DNA polymerase comprising at least one substitution or set of substitutions at position(s) selected from 668 / 671 / 716, 668 / 702, 671 / 702, 671 / 702 / 716, 702, and 743, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:6.In some embodiments, at least one substitution or set of substitutions is 21E, 21E / 66T / 247G / 282R, 247G / 282K / 575L, 282K / 575L, 283M / 647H / 702A / 743A, 339L / 647H / 661T / 664L / 668E / 702A / 712V, 372S / 391E / 702A, 391E, 391E / 647H / 659E / 661T / 668E / 671P / 712V / 716I, 391E / 647H / 659E / 661T / 668E / 671P / 716I, 391E / 647H / 659E / 664L / 668E / 702A / 728A / 732 E, 391E / 647H / 659E / 664L / 671P / 702A, 391E / 647H / 661T / 664L / 671P / 702A / 716I, 391E / 647H / 671P / 728A, 39 1E / 659E / 702A / 716I / 732E / 737R, 391E / 661T / 664L / 668E / 671P / 716I / 737R, 391E / 671P, 391E / 702A / 712V / 716I / 732E / 743A, 647H / 659E / 661T / 664L / 668E / 702A, 647H / 659E / 664L / 668E / 702A / 712V / 737R, 647H / 659E / 668E / 671P / 716I / 728A, 647H / 668E, 647H / 668E / 671P / 712V, 659E / 702A / 743A, 661T / 664L / 668E / 671P / 716I, 668E / 702A, 671P / 702A, 671P / 702A / 716I, 702A, and 743A, wherein the amino acid positions are numbered with reference to SEQ ID NO:6.

[0008] The present invention also includes 18 / 387, 24 / 719, 43 / 528, 48 / 760, 101 / 646, 108 / 679, 223, 257, 282, 359, 360, 361, 362, 376 / 619, 390, 391, 394, 394 / 399, 420, 421, 478, 502, 506, 514, 515, 521, 528, 583 / 730, 603, 619, 631 , 646, 655, 662, 666, 668, 685, 691, 702, 721, 738, 754, 760, and 761, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:6. In some embodiments, at least one substitution or set of substitutions is 18H / 387C, 24M / 719A, 43L / 528S, 48H / 760H, 101S / 646R, 108C / 679S, 223N, 257R, 257W, 282R, 359C, 360R, 360T, 360V, 361G, 361M, 361W, 362R, 376V / 619F, 390A, 390G, 390Q, 391A, 391G, 394G, 394M / 399R, 394N, 394T, 420A, 420G, 420I, 420K, 420V, 421M , 421Q, 478L, 502A, 506R, 514R, 515F, 515G, 515R, 521P, 521T, 528A, 528S, 583N / 730A, 603R, 619C, 619V, 631G, 646R, 655W, 662C, 666T, 668C, 668L, 685D, 691S, 702A, 721R, 721T, 738V, 754C, 760F, 760G, 761R, and 761W, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:6.In some embodiments, at least one substitution or set of substitutions is Y18H / E387C, K24M / K719A, P43L / T528S, Y48H / E760H, P101S / K646R, R108C / Q679S, D223N, M257R, M257W, N282R, R359C, S360R ... 60T, S360V, S361G, S361M, S361W, T362R, A376V / T619F, Y390A, Y390G, Y390Q, K391 A, K391G, L394G, L394M / L399R, L394N, L394T, R420A, R420G, R420I, R420K, R420V, S and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 6.

[0009] The present invention also relates to 174 / 361 / 394 / 666 / 668 / 721, 360 / 391, 361 / 391 / 659, 361 / 394 / 420 / 528 / 646 / 666 / 721 / 743, 361 / 394 / 420 / 528 / 666, 361 / 394 / 420 / 646 / 666 / 702 / 721 / 743, 361 / 528 / 646 / 666, 361 / 528 / 646 / 702 / 721, 361 / 528 / 666, 361 / 646 , 394 / 420, 502 / 507 / 695, 528 / 646 / 659 / 668 / 743, 528 / 666, 528 / 668, 528 / 743, 619, 666, and 685 / 691 / 743, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:22. In some embodiments, at least one substitution or set of substitutions is 174V / 361G / 394T / 666T / 668L / 721T, 360T / 391G, 361G / 394T / 420A / 528A / 666T, 361G / 394T / 420A / 528S / 646R / 666T / 721T / 743P, 361G / 528A / 646R / 666T, 361G / 528A / 666T, 361G / 528S / 646R / 702T / 721T, 361G / 646R, 361M / 391 A / 659D, 361W / 394T / 420A / 646R / 666T / 702T / 721T / 743P, 394G / 420K, 502I / 507F / 695A, 528S / 646R / 659D / 668L / 743P, 528S / 666T, 528S / 668L, 528S / 743P, 619C, 666T, and 685D / 691S / 743P, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:22.In some embodiments, at least one substitution or set of substitutions is A174V / S361G / L394T / K666T / R668L / S721T, S360T / K391G, S361G / L394T / R420A / T528A / K666T, S361G / L394T / R420A / T528S / K646R / K666T / S721T / A743P, S361G / T528A / K646R / K666T, S361G / T528A / K666T, S361G / T528S / K646R / A702T / S721T, S361G / K646R, S361M / and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:22.

[0010] The present invention also provides engineered DNA polymerases comprising at least one substitution or set of substitutions at positions selected from 100, 277, 280, 281, 283, 339, 401, 468, 479, 480, 482, 489, 490, 491, 496, 497, and 498, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:22. In some embodiments, at least one substitution or set of substitutions is selected from 100Y, 277A, 280Y, 281C, 283V, 339M, 401S, 468N, 479P, 479Q, 480D, 480M, 482Q, 482V, 489V, 490L, 491L, 496A, 497D, and 498C, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:22. In some embodiments, at least one substitution or set of substitutions is selected from H100Y, V277A, T280Y, I281C, L283V, F339M, G401S, G468N, K479P, K479Q, K480D, K480M, K482Q, K482V, E489V, K490L, K491L, R496A, Q497D, and R498C and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 22.

[0011] The present invention also relates to 15 / 134 / 482 / 490 / 497 / 671 / 685, 234 / 497 / 647, 257 / 390 / 420, 257 / 390 / 420 / 647, 257 / 401 / 420, 257 / 401 / 420 / 482 / 647 / 671 / 685, 257 / 482 / 497 / 647, 257 / 647, 257 / 671 / 685 / 702, 281, 281 / 391 / 478, 281 / 391 / 478 / 685, 281 / 391 / 488 / 492, 281 / 391 / 495 / 561 / 659 / 668, 281 / 391 / 659 / 668, 281 / 391 / 668, 281 / 478 / 659 / 685 / 702, 281 / 478 / 668, 281 / 488, 281 / 488 / 492 / 495 / 659 / 668, 281 / 488 / 492 / 668 / 702, 281 / 488 / 495, 281 / 488 / 495 / 668, 281 / 492 / 495 / 668, 281 / 492 / 495 / 668 / 702, 281 / 668, 390 / 401 / 716, 390 / 420, 390 / 491 / 671, 390 / 497, 390 / 671 / 685, 391, 391 / 478, 391 / 478 / 479 / 668, 391 / 478 / 4 92 / 668, 391 / 479 / 659 / 668, 391 / 488 / 492 / 659 / 685, 391 / 488 / 492 / 668, 391 / 488 / 495 / 668 / 685 / 702, 391 / 492 / 495, 391 / 492 / 495 / 659, 391 / 492 / 515 / 6 59 / 685, 391 / 495 / 659, 401, 401 / 482 / 659 / 671 / 702, 401 / 490, 401 / 490 / 659 / 671, 401 / 671, 420, 420 / 482 / 659 / 702, 420 / 490, 420 / 490 / 659 / 661 / 671, 42 0 / 659 / 702, 420 / 661 / 671, 420 / 685, 478, 478 / 479, 478 / 479 / 668, 478 / 479 / 702, 478 / 488 / 659, 478 / 488 / 668 / 685 / 702, 478 / 515, 479 / 492, 479 / 659 / 678 , 482 / 497 / 647 / 716, 482 / 497 / 671 / 685, 482 / 671 / 702 / 716, 488, 488 / 492, 488 / 492 / 495, 488 / 495, 488 / 495 / 685, 490 / 497 / 661 / 671 / 685 / 702 / 716, 492,Also provided is an engineered DNA polymerase comprising at least one substitution or set of substitutions at a position or positions selected from 492 / 495 / 659 / 668, 492 / 659 / 685, 492 / 668 / 685 / 712, 492 / 668 / 712, 495, 495 / 659, 495 / 659 / 685, 497 / 647, 497 / 647 / 659 / 671, 497 / 659 / 691 / 716, 497 / 661, 497 / 661 / 671, 497 / 671 / 702, 497 / 671 / 716, 497 / 685, 497 / 702, 515, 659, 659 / 691, and 671, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:24. In some embodiments, at least one permutation or set of permutations is selected from the group consisting of 15N / 134N / 482Q / 490L / 497D / 671P / 685K, 234V / 497D / 647H, 257W / 390H / 420Q, 257W / 390Q / 420Q / 647H, 257W / 401S / 420Q, 257W / 401S / 420Q / 482Q / 647H / 671P / 685K, 257W / 482Q / 497D / 647H, 257W / 647H, 257W / 671P / 685K / 702T, 281C, 281C / 391E / 478L / 685K, 281C / 391E / 488R / 492V, 281C / 391G / 478L, 281C / 391G / 495N / 561A / 659D / 668E, 281C / 391G / 659D / 668E, 281C / 391G / 668E, 281C / 478L / 659D / 685K / 702T, 281C / 478L / 668E, 281C / 488R, 281C / 488R / 492V / 495N / 659D / 668E, 281C / 488R / 492V / 668E / 702T, 281C / 488R / 495N, 281C / 488R / 495N / 668E, 281C / 492V / 495N / 668E, 281C / 492V / 495N / 668E / 702T, 281C / 668E, 390Q / 401S / 716I, 390Q / 420Q, 390Q / 491D / 671P, 390Q / 497D, 390Q / 671P / 685K, 391E, 391E / 478L, 391E / 478L / 479P / 668E, 391E / 488R / 492V / 659D / 685K, 391E / 488R / 492V / 668E,391E / 492V / 495N / 659D, 391G / 478L / 492V / 668E, 391G / 479P / 659D / 668E, 391G / 488R / 495N / 668E / 685K / 702T, 391G / 492V / 495N, 391G / 492 V / 515L / 659D / 685K, 391G / 495N / 659D, 401S, 401S / 482Q / 659D / 671P / 702T, 401S / 490L, 401S / 490L / 659D / 671P, 401S / 671P, 420G, 420Q, 4 20Q / 482Q / 659D / 702T, 420Q / 490L, 420Q / 490L / 659D / 661T / 671P, 420Q / 659D / 702T, 420Q / 661T / 671P, 420Q / 685K, 478L, 478L / 479P, 478L / 479P / 668E, 478L / 479P / 702T, 478L / 488R / 659D, 478L / 488R / 668E / 685K / 702T, 478L / 515L, 479P / 492V, 479P / 659D / 678G, 482Q / 497D / 647 H / 716I, 482Q / 497D / 671P / 685K, 482Q / 671P / 702T / 716I, 488R, 488R / 492V, 488R / 492V / 495N, 488R / 495N, 488R / 495N / 685K, 490L / 497D / 6 61T / 671P / 685K / 702T / 716I, 492V, 492V / 495N / 659D / 668E, 492V / 659D / 685K, 492V / 668E / 685K / 712V, 492V / 668E / 712V, 495N, 495N / 659D , 495N / 659D / 685K, 497D / 647H, 497D / 647H / 659D / 671P, 497D / 659D / 691G / 716I, 497D / 661T, 497D / 661T / 671P, 497D / 671P / 702T, 497D / 671P / 716I, 497D / 685K, 497D / 702T, 515L, 659D, 659D / 691G, and 671P, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:24. In some embodiments, at least one substitution or set of substitutions is D15N / D134N / K482Q / K490L / Q497D / L671P / D685K, A234V / Q497D / D647H,M257W / Y390H / R420Q、M257W / Y390Q / R420Q / D647H、M257W / G401S / R420Q、M257W / G401S / R420Q / K482Q / D647H / L671P / D685K、M257W / K482Q / Q497D / D647H、M257W / D647H、M257W / L671P / D685K / A702T、I281C、I281C / K391E / K478L / D685K、I281C / K391E / I488R / M492V、I281C / K391G / K478L、I281C / K391G / Y495N / T561A / E659D / R668E、I281C / K391G / E659D / R668E、I281C / K391G / R668E、I281C / K478L / E659D / D685K / A702T、I281C / K478L / R668E、I281C / I488R、I281C / I488R / M492V / Y495N / E659D / R668E、I281C / I488R / M492V / R668E / A702T、I281C / I488R / Y495N、I281C / I488R / Y495N / R668E、I281C / M492V / Y495N / R668E、I281C / M492V / Y495N / R668E / A702T、I281C / R668E、Y390Q / G401S / L716I、Y390Q / R420Q、Y390Q / K491D / L671P、Y390Q / Q497D、Y390Q / L671P / D685K、K391E、K391E / K478L、K391E / K478L / K479P / R668E、K391E / I488R / M492V / E659D / D685K、K391E / I488R / M492V / R668E、K391E / M492V / Y495N / E659D、K391G / K478L / M492V / R668E、K391G / K479P / E659D / R668E、K391G / I488R / Y495N / R668E / D685K / A702T、K391G / M492V / Y495N、K391G / M492V / K515L / E659D / D685K、K391G / Y495N / E659D、G401S、G401S / K482Q / E659D / L671P / A702T、G401S / K490L、G401S / K490L / E659D / L671P、G401S / L671P、R420G、R420Q、R420Q / K482Q / E659D / A702T, R420Q / K490L, R420Q / K490L / E659D / V661T / L671P, R420Q / E659D / A702T, R420Q / V661T / L6 71P, R420Q / D685K, K478L, K478L / K479P, K478L / K479P / R668E, K478L / K479P / A702T, K478L / I488R / E659D, K478L / I488 R / R668E / D685K / A702T, K478L / K515L, K479P / M492V, K479P / E659D / E678G, K482Q / Q497D / D647H / L716I, K482Q / Q497D / L671P / D685K, K482Q / L671P / A702T / L716I, I488R, I488R / M492V, I488R / M492V / Y495N, I488R / Y495N, I488R / Y495N / D68 5K, K490L / Q497D / V661T / L671P / D685K / A702T / L716I, M492V, M492V / Y495N / E659D / R668E, M492V / E659D / D685K, M492V / R668E / D685K / I712V, M492V / R668E / I712V, Y495N, Y495N / E659D, Y495N / E659D / D685K, Q497D / D647H, Q497D / D647H / E and L671P, Q497D / E659D / S691G / L716I, Q497D / V661T, Q497D / V661T / L671P, Q497D / L671P / A702T, Q497D / L671P / L716I, Q497D / D685K, Q497D / A702T, K515L, E659D, E659D / S691G, and L671P, wherein the amino acid positions are numbered with reference to SEQ ID NO: 24.

[0012] The present invention also includes 55 / 579, 108, 108 / 521, 156 / 451, 236 / 755, 240, 247, 248, 256, 298, 299, 299 / 319, 302, 309, 316, 319, 350, 356, 357, 358, 370, 384, 385, 386, 389, 406, 407, 411, 415, 440, 443, 447, 450, 451, 520, 536, 539, 540, 544, 550 / 575, 566, 568, 575, 579, Also provided are engineered DNA polymerases comprising at least one substitution or set of substitutions at position(s) selected from 579 / 767, 600, 601, 601 / 638, 609 / 648, 624, 634, 648, 656, 672, 758, 765, 767, 772, 777, 778, 779, 780, 782, 784, and 785, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:24.In some embodiments, at least one substitution or set of substitutions is 55E / 579V, 55G / 579A, 108A, 108C, 108F, 108G, 108S, 108V / 521R, 108Y, 156L / 451C, 236R / 755T, 240A, 240Y, 247I, 247S, 248P, 256A, 298E, 299A, 299A / 319G, 299E, 299Q, 299R, 319G ... 02F, 309V, 316G, 319E, 319H, 319S, 350V, 356N, 356P, 356V, 357S, 358I, 370D, 370S, 370T, 384R, 385L, 386 G, 386P, 386V, 389Q, 389R, 406V, 407A, 407L, 407R, 407S, 407Y, 411H, 415V, 440H, 443V, 447A, 447L, 450L, 4 50Y, 451G, 520C, 536N, 536Q, 536T, 539G, 539H, 539Q, 539S, 539V, 540G, 544G, 550S / 575Q, 566G, 566Q, 568 G, 568L, 575F, 575T, 579A, 579M, 579Q, 579Q / 767Q, 579R, 579S, 600A, 601I, 601L / 638L, 601M, 601V, 609C / 6 and 785G, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:24.In some embodiments, at least one substitution or set of substitutions is selected from the group consisting of D55E / N579V, D55G / N579A, R108A, R108C, R108F, R108G, R108S, R108V / K521R, R108Y, F156L / V451C, K236R / V755T, R240A, R240Y, K247I, K247S, E248P, R256A, K298E, T299A, T299A / K319G, T299E, T299Q, T299R, K30 2F, A309V, E316G, K319E, K319H, K319S, I350V, D356N, D356P, D356V, V357S, S358I, L370D, L370S, L370T, K384R, P385L, D38 6G, D386P, D386V, E389Q, E389R, P406V, E407A, E407L, E407R, E407S, E407Y, W411H, I415V, E440H, E443V, I447A, I447L, I450 L, I450Y, V451G, S520C, E536N, E536Q, E536T, I539G, I539H, I539Q, I539S, I539V, K540G, E544G, V550S / R575Q, K566G, K566 Q, E568G, E568L, R575F, R575T, N579A, N579M, N579Q, N579Q / E767Q, N579R, N579S, G600A, F601I, F601L / A638L, F601M, F601V , A609C / G648Q, V624C, V624S, K634R, G648Q, G648R, I656A, I656Y, E672G, I758V, R765D, E767G, E767T, Q772G, T777D, G778Q, L779D, D780A, D780W, W782S, W782V, K784-, and R785G, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:24.

[0013] The present invention also provides engineered DNA polymerases comprising at least one substitution or set of substitutions at position(s) selected from 248, 281, 281 / 302, 281 / 492, 302 / 401, 339 / 491 / 492 / 579 / 712, 390 / 466 / 539 / 712, and 661, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:26. In some embodiments, at least one substitution or set of substitutions is selected from 248P, 281I, 281I / 302F, 281I / 492S, 302F / 401S, 339A / 491D / 492V / 579A / 712V, 390Q / 466A / 539S / 712V, and 661T, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:26. In some embodiments, at least one substitution or set of substitutions is selected from E248P, C281I, C281I / K302F, C281I / M492S, K302F / G401S, F339A / K491D / M492V / N579A / I712V, Y390Q / I466A / I539S / I712V, and V661T, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:26.

[0014] The present invention also provides engineered DNA polymerases comprising at least one substitution or set of substitutions at position(s) selected from 240 / 579, 240 / 579 / 702, 248 / 391 / 539 / 579 / 659 / 702, 248 / 391 / 659, 302 / 391 / 579, 339 / 390 / 420 / 425 / 466 / 490 / 491 / 515 / 702, 391, 391 / 482, 391 / 659, 420 / 515, 579, 579 / 659 / 702, 579 / 702, and 659 / 702, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:28. In some embodiments, at least one substitution or set of substitutions is selected from 240A / 579A, 240A / 579A / 702A, 248P / 391G / 539S / 579A / 659D / 702A, 248P / 391G / 659D, 302F / 391G / 579A, 339A / 390Q / 420G / 425R / 466A / 490L / 491P / 515L / 702A, 391G, 391G / 482Q, 391G / 659D, 420G / 515F, 579A, 579A / 659D / 702A, 579A / 702A, and 659D / 702A, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:28. In some embodiments, at least one substitution or set of substitutions is selected from the group consisting of R240A / N579A, R240A / N579A / T702A, E248P / K391G / I539S / N579A / E659D / T702A, E248P / K391G / E659D, K302F / K391G / N579A, F339A / Y390Q / R420G / S425R / I466A / K490L / and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:28.

[0015] The present invention also provides engineered DNA polymerases comprising at least one substitution or set of substitutions at position(s) selected from 257, 420, 515, and 521, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 6. In some embodiments, the at least one substitution or set of substitutions is selected from 257W, 420Q, 515L, and 521S, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 6. In some embodiments, the at least one substitution or set of substitutions is selected from M257W, R420Q, K515L, and K521S, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 6.

[0016] The present invention also provides engineered DNA polymerases comprising at least one substitution or set of substitutions at position(s) selected from 71 / 361 / 702 / 721 / 738, 277, 281, 339, 391 / 491, 401, 479, 480, 482, 488, 490, 491, 492, 495, 497, 528 / 646 / 659 / 668 / 743, 702 / 743, and 743, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:22. In some embodiments, at least one substitution or set of substitutions is selected from 71D / 361M / 702T / 721R / 738V, 277A, 281C, 339M, 391N / 491Q, 401S, 479P, 480M, 482Q, 482V, 488R, 490L, 490Y, 491D, 492V, 495N, 497D, 528S / 646R / 659D / 668L / 743P, 702T / 743P, and 743P, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:22. In some embodiments, at least one substitution or set of substitutions is selected from G71D / S361M / A702T / S721R / K738V, V277A, I281C, F339M, K391N / K491Q, G401S, K479P, K480M, K482Q, K482V, I488R, K490L, K490Y, K491D, M492V, Y495N, Q497D, T528S / K646R / E659D / R668L / A743P, A702T / A743P, and A743P, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:22.

[0017] The present invention also provides engineered DNA polymerases comprising at least one substitution or set of substitutions at position(s) selected from 240, 370, 385, 539, 540, 550 / 575, 634, and 777, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 24. In some embodiments, the at least one substitution or set of substitutions is selected from 240A, 370T, 385L, 539V, 540G, 540Q, 550S / 575Q, 634R, and 777D, and 743P, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 24. In some embodiments, at least one substitution or set of substitutions is selected from R240A, L370T, P385L, I539V, K540G, K540Q, V550S / R575Q, K634R, and T777D, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:24.

[0018] The present invention also provides engineered DNA polymerases comprising at least one substitution or set of substitutions at position(s) selected from 390 / 391, 482, and 515, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 28. In some embodiments, the at least one substitution or set of substitutions is selected from 390Q / 391G, 482Q, 515F, and 515L, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 28. In some embodiments, the at least one substitution or set of substitutions is selected from Y390Q / K391G, K482Q, K515F, and K515L, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 28.

[0019] The present invention also provides engineered DNA polymerases comprising at least one substitution or set of substitutions at position(s) selected from 281, 281 / 579, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 28. In some embodiments, the at least one substitution or set of substitutions is selected from 281I and 281I / 579A, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 28. In some embodiments, the at least one substitution or set of substitutions is selected from C281I and C281I / N579A, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 28.

[0020] The present invention also relates to the use of the compounds of formula (I) in the compositions of formula (I) above. , 563, 564, 564 / 572, 567, 569, 575, 580, 601, 603, 619, 620, 648, 667, 673, 690, 705, 719, 731, 758, 761, 772, 774, 775, 778, 783, and 784, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:824.In some embodiments, at least one substitution or set of substitutions is 13T, 15G, 15W, 19S, 26S, 52M, 55K, 55P, 61A, 61R, 80G, 81T, 82Q, 95R, 111A, 111V, 118V, 141R, 141S, 148P, 152T, 156R, 162Q, 163A, 163G, 163K, 163P, 163Q, 1 63W, 179G, 181R, 187L, 189G, 191A, 191N, 196A, 196R, 208C, 221G, 229S, 231H, 242L, 258L, 258R , 258S, 274I, 274L, 274V, 297F, 313F, 314V, 317P, 317R, 317T, 325Q, 326K, 333R, 349I, 377W, 387 A, 387S, 394G, 394R, 395H, 411T, 447V, 450V, 451Y, 453R, 469H, 469L, 482V, 496S, 502W, 520C, 5 21V, 537G, 537K, 563L, 564D / 572G, 564Q, 567G, 569G, 569L, 569T, 575H, 575W, 580A, 580I, 601I, 603R, 619L, 619V, 620K, 648F, 667N, 667T, 673M, 690L, 705L, 719A, 731G, 758V, 761P, 772S, 774R, 775F, 775G, 778P, 778R, 783Q, 783R, and 784E, wherein the amino acid positions are numbered with reference to SEQ ID NO: 824.In some additional embodiments, at least one substitution or set of substitutions is I13T, D15G, D15W, I19S, I26S, L52M, D55K, D55P, E61A, E61R, V80G, K81T, V82Q, K95R, I111A, I111V, I118V, E141R, E141S, L148P, D152T, F156R, E162Q, F163A, F163G, F163K, F163P, F163Q , F163W, A179G, V181R, I187L, L189G, Y191A, Y191N, S196A, S196R, V208C, N221G, Y229S, I231H, V242L, G258L, G2 58R, G258S, F274I, F274L, F274V, G297F, E313F, T314V, S317P, S317R, S317T, S325Q, M326K, Y333R, L349I, R377W, E387A, E387S, L394G, L394R, R395H, W411T, I447V, I450V, V451Y, Y453R, D469H, D469L, K482V, R496S, L502W, S52 0C, K521V, M537G, M537K, P563L, G564D / K572G, G564Q, P567G, I569G, I569L, I569T, R575H, R575W, Y580A, Y580I, and selected from F601I, V603R, T619L, T619V, R620K, G648F, Y667N, Y667T, K673M, I690L, I705L, K719A, L731G, I758V, A761P, Q772S, S774R, K775F, K775G, G778P, G778R, L783Q, L783R, and K784E, wherein the amino acid positions are numbered with reference to SEQ ID NO: 824.

[0021] The present invention also provides engineered DNA polymerases comprising at least one substitution or set of substitutions at position(s) selected from 15 / 447 / 569 / 775 / 783 / 784, 82 / 242 / 569, 82 / 450 / 567 / 569, 313, 314 / 447 / 569 / 783 / 784, 537 / 667, 567 / 569 / 667, and 569, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:824. In some embodiments, at least one substitution or set of substitutions is selected from 15W / 447V / 569T / 775F / 783Q / 784E, 82Q / 242L / 569L, 82Q / 450V / 567G / 569G, 313F, 314V / 447V / 569T / 783Q / 784E, 537K / 667N, 567G / 569G / 667N, and 569T, where the amino acid positions are numbered with reference to SEQ ID NO: 824. In some additional embodiments, the at least one substitution or set of substitutions is selected from D15W / I447V / I569T / K775F / L783Q / K784E, V82Q / V242L / I569L, V82Q / I450V / P567G / I569G, E313F, T314V / I447V / I569T / L783Q / K784E, M537K / Y667N, P567G / I569G / Y667N, and I569T, wherein the amino acid positions are numbered with reference to SEQ ID NO: 824.

[0022] The present invention also provides engineered DNA polymerases comprising a polypeptide sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the sequence of at least one engineered DNA polymerase variant listed in Tables 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.1, 4.2, 4.3, 4.4, 4.5, 6.2, and / or 6.3. In some embodiments, the engineered DNA polymerase has DNA polymerase activity. In some embodiments, the engineered DNA polymerase has at least one improved property compared to a wild-type DNA polymerase. In some embodiments, the wild-type DNA polymerase is selected from Pfu DNA polymerase from Pyrococcus furiosus, Group B DNA polymerase from Thermococcus sp. strain 2319x1, and Taq DNA polymerase from Thermus aquaticus. In some embodiments, the engineered DNA polymerase has at least one improved property compared to the wild-type DNA polymerase, the improved property being selected from increased product production in polymerase chain reactions, higher fidelity, and higher thermostability. In some embodiments, the engineered DNA polymerase results in higher product yields in polymerase chain reactions than the wild-type DNA polymerase. In some embodiments, the wild-type DNA polymerase is selected from Pfu DNA polymerase from Pyrococcus furiosus, Group B DNA polymerase from Thermococcus sp. strain 2319x1, and Taq DNA polymerase from Thermus aquaticus. In some additional embodiments, the engineered DNA polymerase exhibits greater fidelity than the wild-type DNA polymerase.In some embodiments, the wild-type DNA polymerase is selected from Pfu DNA polymerase from Pyrococcus furiosus, Group B DNA polymerase from Thermococcus sp. strain 2319x1, and Taq DNA polymerase from Thermus aquaticus. In still some additional embodiments, the engineered DNA polymerase exhibits greater thermostability than the wild-type DNA polymerase. In some further embodiments, the wild-type DNA polymerase is selected from Pfu DNA polymerase from Pyrococcus furiosus, Group B DNA polymerase from Thermococcus sp. strain 2319x1, and Taq DNA polymerase from Thermus aquaticus. In still some further embodiments, the engineered DNA polymerase is purified.

[0023] The present invention also provides polynucleotide sequences encoding the engineered DNA polymerases provided herein. In some embodiments, the polynucleotide sequence encodes at least one engineered DNA polymerase provided herein. In some additional embodiments, the polynucleotide sequence comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence of SEQ ID NO: 1, 5, 21, 23, 25, 27, and / or 823, or comprises a functional fragment thereof, wherein the engineered polypeptide comprises at least one substitution at one or more amino acid positions. In some additional embodiments, the polynucleotide sequence encodes at least one engineered DNA polymerase comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence of SEQ ID NO: 2, 6, 22, 24, 26, 28, and / or 824. In some further embodiments, the polynucleotide sequence comprises SEQ ID NO: 1, 5, 21, 23, 25, 27, and / or 823. In some additional embodiments, the polynucleotide sequence is operably linked to a regulatory sequence. In yet some further embodiments, the polynucleotide sequence is codon-optimized.

[0024] The present invention also provides expression vectors comprising at least one polynucleotide sequence provided herein. The present invention also provides host cells transformed with at least one expression vector provided herein.

[0025] The present invention also provides methods for producing an engineered DNA polymerase polypeptide in a host cell, the method comprising culturing a host cell provided herein under suitable culture conditions such that at least one engineered DNA polymerase is produced. In some embodiments, the method further comprises recovering the at least one engineered DNA polymerase from the culture and / or host cell. In some additional embodiments, the method further comprises purifying the at least one engineered DNA polymerase. The present invention also provides compositions comprising at least one engineered DNA polymerase provided herein.

[0026] The present invention also provides a high-throughput assay system for determining the fidelity of a DNA polymerase. The present invention also provides a method for determining the fidelity of a DNA polymerase, the method comprising: (i) providing a reporter plasmid containing at least one DNA polymerase, a gene encoding a first reporter protein, a second reporter protein, and a selectable marker; an amplification system containing a thermocycler and reagents for performing a polymerase chain reaction; a purification system; a transformation system containing competent host cells; and a flow cytometer; (ii) exposing the DNA polymerase and the reporter plasmid to the amplification system under conditions such that the reporter construct is amplified by the DNA polymerase to produce a PCR product; (iii) circularizing the PCR amplicon to obtain a circularized PCR amplicon; (vi) transforming the PCR amplicon using the transformation system to produce a transformed cell; (vii) analyzing the transformed cell using a flow cytometer; and (viii) determining the fidelity of the DNA polymerase. In some embodiments, the method comprises at least one DNA polymerase provided herein (e.g., provided in any of the Examples and Tables). In some embodiments, the method further comprises inducing the transformed cells. In some additional embodiments, the first reporter protein comprises green fluorescent protein. In yet some further embodiments, the second reporter protein comprises dsRed. In still further embodiments, the selectable marker comprises chloramphenicol acetyltransferase. In some further embodiments, circularization of the PCR amplicon is performed using at least one ligase. In some embodiments, the PCR amplicon is purified. In some additional embodiments, the method further comprises determining a fold improvement in polymerase fidelity compared to a reference DNA polymerase. In some embodiments, the reference DNA polymerase is a wild-type polymerase.In some further embodiments, the wild-type polymerase is selected from Pfu DNA polymerase from Pyrococcus furiosus, Group B DNA polymerase from Thermococcus sp. strain 2319x1, and Taq DNA polymerase from Thermus aquaticus. In some embodiments, the relative error rate of each variant is calculated by dividing the frequency of the first fluorescent protein (e.g., green only) of that variant by the frequency of the parental control. In some additional embodiments, the fold improvement in polymerase fidelity is reported to determine the relative error rate. In certain embodiments, for example, the following items are provided: (Item 1) 1. An engineered DNA polymerase comprising a polypeptide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence of SEQ ID NO: 2, 6, 22, 24, 26, 28, and / or 824, or a functional fragment thereof, wherein the polypeptide sequence comprises at least one mutation, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2, 6, 22, 24, 26, 28, or 824. (Item 2) At least one substitution or substitution set is 21, 21 / 66 / 247 / 282, 247 / 282 / 575, 282 / 575, 283 / 647 / 702 / 743, 339 / 647 / 661 / 664 / 668 / 702 / 712, 372 / 391 / 702, 391, 391 / 647 / 659 / 661 / 668 / 671 / 712 / 716, 391 / 647 / 659 / 661 / 668 / 671 / 716, 391 / 647 / 659 / 664 / 668 / 702 / 728 / 732, 391 / 647 / 659 / 664 / 671 / 702, 391 / 647 / 661 / 664 / 671 / 702 / 716, 391 / 647 / 671 / 728, 391 / 659 / 702 / 716 / 732 / 737, 391 / 661 / 664 / 668 / 671 / 716 / 737, 391 / 671, 391 / 702 / 712 / 716 / 732 / 743, 647 / 659 / 661 / 664 / 668 / 702, 647 / 659 / 664 / 668 / 702 / 712 / 737, 647 / 659 / 668 / 671 / 716 / 728, 647 / 668, 647 / 668 / 671 2. The engineered DNA polymerase of claim 1, wherein the amino acid positions are selected from the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ (Item 3) At least one substitution or set of substitutions is: 18 / 387, 24 / 719, 43 / 528, 48 / 760, 101 / 646, 108 / 679, 223, 257, 282, 359, 360, 361, 362, 376 / 619, 390, 391, 394, 394 / 399, 420, 421, 478, 502, 506, 514, 515, 521, 528, 583 / 73 2. The engineered DNA polymerase of item 1, comprising a substitution at an amino acid position selected from 0, 603, 619, 631, 646, 655, 662, 666, 668, 685, 691, 702, 721, 738, 754, 760, and 761, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:6. (Item 4) At least one substitution or set of substitutions is 174 / 361 / 394 / 666 / 668 / 721, 360 / 391, 361 / 391 / 659, 361 / 394 / 420 / 528 / 646 / 666 / 721 / 743, 361 / 394 / 420 / 528 / 666, 361 / 394 / 420 / 646 / 666 / 702 / 721 / 743, 361 / 528 / 646 / 666, 361 / 528 / 646 / 702 / 721, 361 / 528 / 2. The engineered DNA polymerase of item 1, comprising a substitution at an amino acid position selected from 666, 361 / 646, 394 / 420, 502 / 507 / 695, 528 / 646 / 659 / 668 / 743, 528 / 666, 528 / 668, 528 / 743, 619, 666, and 685 / 691 / 743, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 22. (Item 5) 2. The engineered DNA polymerase of item 1, wherein at least one substitution or set of substitutions comprises a substitution at an amino acid position selected from 100, 277, 280, 281, 283, 339, 401, 468, 479, 480, 482, 489, 490, 491, 496, 497, and 498, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:22. (Item 6) At least one substitution or set of substitutions is: 15 / 134 / 482 / 490 / 497 / 671 / 685, 234 / 497 / 647, 257 / 390 / 420, 257 / 390 / 420 / 647, 257 / 401 / 420, 257 / 401 / 420 / 482 / 647 / 671 / 685, 257 / 482 / 497 / 647, 257 / 647, 257 / 671 / 685 / 702, 281, 281 / 391 / 478, 281 / 391 / 478 / 685, 281 / 391 / 488 / 492, 281 / 391 / 495 / 561 / 659 / 668 ... 91 / 659 / 668, 281 / 391 / 668, 281 / 478 / 659 / 685 / 702, 281 / 478 / 668, 281 / 488, 281 / 488 / 492 / 495 / 659 / 668, 281 / 488 / 492 / 668 / 702, 281 / 488 / 495, 281 / 488 / 495 / 668, 281 / 492 / 495 / 668, 281 / 492 / 495 / 668 / 702, 281 / 668, 390 / 401 / 716, 390 / 420, 390 / 491 / 671, 390 / 497, 390 / 671 / 685, 391, 391 / 478, 39 1 / 478 / 479 / 668, 391 / 478 / 492 / 668, 391 / 479 / 659 / 668, 391 / 488 / 492 / 659 / 685, 391 / 488 / 492 / 668, 391 / 488 / 495 / 668 / 685 / 702, 391 / 492 / 495, 391 / 492 / 495 / 659, 391 / 492 / 515 / 659 / 685, 391 / 495 / 659, 401, 401 / 482 / 659 / 671 / 702, 401 / 490, 401 / 490 / 659 / 671, 401 / 671, 420, 420 / 482 / 659 / 702, 420 / 490, 420 / 490 / 659 / 661 / 671, 420 / 659 / 702, 420 / 661 / 671, 420 / 685, 478, 478 / 479, 478 / 479 / 668, 478 / 479 / 702, 478 / 488 / 659, 478 / 488 / 668 / 685 / 702, 478 / 515, 479 / 492, 479 / 659 / 678, 482 / 497 / 647 / 716, 482 / 497 / 671 / 685, 482 / 671 / 702 / 716, 488, 488 / 492, 488 / 492 / 495, 488 / 495, 488 / 495 / 685,490 / 497 / 661 / 671 / 685 / 702 / 716, 492, 492 / 495 / 659 / 668, 492 / 659 / 685, 492 / 668 / 685 / 712, 492 / 668 / 712, 495, 495 / 659, 495 / 659 / 685, 497 / 647, 497 / 647 / 659 / 671, 497 / 659 / 691 / 716, 497 / 661, 497 / 2. The engineered DNA polymerase of item 1, comprising a substitution at an amino acid position selected from 661 / 671, 497 / 671 / 702, 497 / 671 / 716, 497 / 685, 497 / 702, 515, 659, 659 / 691, and 671, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 24. (Item 7) At least one substitution or set of substitutions is present in 55 / 579, 108, 108 / 521, 156 / 451, 236 / 755, 240, 247, 248, 256, 298, 299, 299 / 319, 302, 309, 316, 319, 350, 356, 357, 358, 370, 384, 385, 386, 389, 406, 407, 411, 415, 440, 443, 447, 450, 451, 520, 536, 539, 540, 544, 550 / 575, 566 779, 780, 782, 784, and 785, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:24. (Item 8) 2. The engineered DNA polymerase of item 1, wherein at least one substitution or set of substitutions comprises a substitution at an amino acid position selected from 248, 281, 281 / 302, 281 / 492, 302 / 401, 339 / 491 / 492 / 579 / 712, 390 / 466 / 539 / 712, and 661, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:26. (Item 9) 2. The engineered DNA polymerase of item 1, wherein at least one substitution or set of substitutions comprises a substitution at an amino acid position selected from 240 / 579, 240 / 579 / 702, 248 / 391 / 539 / 579 / 659 / 702, 248 / 391 / 659, 302 / 391 / 579, 339 / 390 / 420 / 425 / 466 / 490 / 491 / 515 / 702, 391, 391 / 482, 391 / 659, 420 / 515, 579, 579 / 659 / 702, 579 / 702, and 659 / 702, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:28. (Item 10) 2. The engineered DNA polymerase of item 1, wherein at least one substitution or set of substitutions comprises a substitution at an amino acid position selected from 257, 420, 515, and 521, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:6. (Item 11) 2. The engineered DNA polymerase of item 1, wherein at least one substitution or set of substitutions comprises a substitution at an amino acid position selected from 71 / 361 / 702 / 721 / 738, 277, 281, 339, 391 / 491, 401, 479, 480, 482, 488, 490, 491, 492, 495, 497, 528 / 646 / 659 / 668 / 743, 702 / 743, and 743, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 22. (Item 12) 2. The engineered DNA polymerase of item 1, wherein at least one substitution or set of substitutions comprises a substitution at an amino acid position selected from 240, 370, 385, 539, 540, 550 / 575, 634, and 777, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:24. (Item 13) 2. The engineered DNA polymerase of item 1, wherein at least one substitution or set of substitutions comprises a substitution at an amino acid position selected from 390 / 391, 482, and 515, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:28. (Item 14) 2. The engineered DNA polymerase of item 1, wherein at least one substitution or set of substitutions comprises a substitution at an amino acid position selected from 281, 281 / 579, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:28. (Item 15) At least one substitution or substitution set is: 13, 15, 19, 26, 52, 55, 61, 80, 81, 82, 95, 111, 118, 141, 148, 152, 156, 162, 163, 179, 181, 187, 189, 191, 196, 208, 221, 229, 231, 242, 258, 274, 297, 313, 314, 317, 325, 326, 333, 349, 377, 387, 394, 395, 411, 447, 450, 451, 453, 469, 482, 496, 50 2, 520, 521, 537, 563, 564, 564 / 572, 567, 569, 575, 580, 601, 603, 619, 620, 648, 667, 673, 690, 705, 719, 731, 758, 761, 772, 774, 775, 778, 783, and 784, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 824. (Item 16) 2. The engineered DNA polymerase of item 1, wherein at least one substitution or set of substitutions comprises a substitution at an amino acid position selected from 15 / 447 / 569 / 775 / 783 / 784, 82 / 242 / 569, 82 / 450 / 567 / 569, 313, 314 / 447 / 569 / 783 / 784, 537 / 667, 567 / 569 / 667, and 569, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 824. (Item 17) 10. The engineered DNA polymerase of item 1, comprising a polypeptide sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered DNA polymerase variant described in Tables 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.1, 4.2, 4.3, 4.4, 4.5, 6.2, and / or 6.3. (Item 18) 18. The engineered DNA polymerase according to any of items 1 to 17, having DNA polymerase activity. (Item 19) 19. The engineered DNA polymerase of any of items 1 to 18, having at least one improved property compared to a wild-type DNA polymerase selected from Pfu DNA polymerase derived from Pyrococcus furiosus, Group B DNA polymerase derived from Thermococcus sp. strain 2319x1, and Taq DNA polymerase derived from Thermus aquaticus. (Item 20) 20. The engineered DNA polymerase of item 19, having at least one improved property compared to a wild-type DNA polymerase, the improved property being selected from: producing increased product in a polymerase chain reaction, higher fidelity, and higher thermostability. (Item 21) 21. The engineered DNA polymerase of any of items 1 to 20, which results in a higher product yield in a polymerase chain reaction than a wild-type DNA polymerase, wherein the wild-type DNA polymerase is selected from Pfu DNA polymerase from Pyrococcus furiosus, Group B DNA polymerase from Thermococcus sp. strain 2319x1, and Taq DNA polymerase from Thermus aquaticus. (Item 22) 22. The engineered DNA polymerase of any of items 1 to 21, which exhibits greater fidelity than a wild-type DNA polymerase, wherein the wild-type DNA polymerase is selected from Pfu DNA polymerase from Pyrococcus furiosus, Group B DNA polymerase from Thermococcus sp. strain 2319x1, and Taq DNA polymerase from Thermus aquaticus. (Item 23) 23. The engineered DNA polymerase of any of items 1 to 22, which exhibits greater thermostability than a wild-type DNA polymerase, wherein the wild-type DNA polymerase is selected from Pfu DNA polymerase from Pyrococcus furiosus, Group B DNA polymerase from Thermococcus sp. strain 2319x1, and Taq DNA polymerase from Thermus aquaticus. (Item 24) 24. The engineered DNA polymerase of any of items 1 to 23, wherein the polymerase is purified. (Item 25) 25. A polynucleotide sequence encoding at least one engineered DNA polymerase according to any of items 1 to 24. (Item 26) A polynucleotide sequence comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence of SEQ ID NO: 1, 5, 21, 23, 25, 27, 823, and / or a functional fragment thereof, wherein the engineered polypeptide comprises at least one substitution at one or more amino acid positions. (Item 27) 27. The polynucleotide sequence of items 25 and / or 26, encoding at least one engineered DNA polymerase comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence of SEQ ID NO: 2, 6, 22, 24, 26, 28, and / or 824. (Item 28) 28. The polynucleotide sequence of item 27, wherein the sequence comprises SEQ ID NO: 1, 5, 21, 23, 25, 27, and / or 823. (Item 29) 29. The polynucleotide sequence of any of items 25 to 28, operably linked to a regulatory sequence. (Item 30) 30. The polynucleotide sequence of any of items 25 to 29, which is codon-optimized. (Item 31) 31. An expression vector comprising at least one polynucleotide sequence according to any of items 25 to 30. (Item 32) A host cell transformed with at least one expression vector of item 31. (Item 33) 33. A method for producing an engineered DNA polymerase polypeptide in a host cell, comprising culturing the host cell of claim 32 under suitable culture conditions such that at least one engineered DNA polymerase is produced. (Item 34) 34. The method of claim 33, further comprising recovering the at least one engineered DNA polymerase from the culture and / or host cell. (Item 35) 35. The method of claim 34, further comprising purifying the at least one engineered DNA polymerase. (Item 36) 25. A composition comprising at least one engineered DNA polymerase according to any of items 1 to 24. (Item 37) A high-throughput assay system for determination of DNA polymerase fidelity. (Item 38) 25. A method for high-throughput fidelity determination of a DNA polymerase, the method comprising: i) providing at least one DNA polymerase according to any one of items 1 to 24, a reporter plasmid comprising a gene encoding a first reporter protein, a second reporter protein, and a selectable marker, an amplification system comprising a thermocycler and reagents for performing a polymerase chain reaction, and a purification system, a transformation system comprising competent host cells, and a flow cytometer; ii) exposing the DNA polymerase and the reporter plasmid to the amplification system under conditions such that the reporter construct is amplified by the DNA polymerase to produce a PCR product; iii) circularizing the PCR product to obtain a circularized PCR amplicon; vi) transforming the PCR amplicon using the transformation system to produce a transformed cell; vii) analyzing the transformed cell using the flow cytometer; and viii) determining the fidelity of the DNA polymerase. (Item 39) 39. The method of item 38, further comprising the step of inducing the transformed cells. (Item 40) 40. The method of claim 38, wherein the first reporter protein comprises green fluorescent protein. (Item 41) 41. The method of any of items 38 to 40, wherein the second reporter protein comprises dsRed. (Item 42) 42. The method of any of items 38 to 41, wherein the selectable marker comprises chloramphenicol acetyltransferase. (Item 43) 43. The method of any of items 38 to 42, wherein the circularization of the PCR amplicon is carried out using at least one ligase. (Item 44) 44. The method of any of items 38 to 43, wherein the PCR amplicon is purified. (Item 45) 45. The method of any of items 38 to 44, further comprising determining the fold improvement in polymerase fidelity compared to a reference DNA polymerase. (Item 46) 46. ​​The method of claim 45, wherein the reference DNA polymerase is a wild-type polymerase. (Item 47) 47. The method of claim 46, wherein the wild-type polymerase is selected from Pfu DNA polymerase from Pyrococcus furiosus, Group B DNA polymerase from Thermococcus sp. strain 2319x1, and Taq DNA polymerase from Thermus aquaticus. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 provides a graph showing the relative error rates of the polymerases tested, as described in Example 5.

[0028] [Figure 2]Figure 2 provides a graph showing coverage uniformity for microbial whole-genome resequencing of an organism with low GC content (Staphylococcus epidermidis, 32% GC). Normalized coverage is plotted as a function of the GC content of the respective genome. The theoretical optimum for normalized coverage is plotted as a dashed line (1.0).

[0029] [Figure 3] Figure 3 provides a graph showing coverage uniformity of microbial whole-genome resequencing of an organism with high GC content (Rhodobacter sphaeroides, 69% GC). Normalized coverage is plotted as a function of the GC content of the respective genome. The theoretical optimum of normalized coverage is plotted as a dashed line (1.0). DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention provides engineered DNA polymerase polypeptides and compositions thereof, as well as polynucleotides encoding the engineered DNA polymerase polypeptides. The present invention also provides methods for using compositions comprising the engineered DNA polymerase polypeptides for diagnostic and other purposes. In some embodiments, the engineered DNA polymerase polypeptides are optimized to provide enhanced polymerization activity with high replication fidelity, particularly under conditions involving low DNA input concentrations, high-throughput analysis, and / or sequencing reactions. In some embodiments, the present invention provides methods and compositions comprising the engineered DNA polymerases for diagnostic and research purposes. The present invention also provides engineered DNA polymerase polypeptides, their mutants, biologically active fragments, and analogs, and compositions comprising the same.

[0031] In some embodiments, the engineered DNA polymerases of the present invention are used in diagnostic and research applications using small amounts of DNA from patient samples, including cell-free DNA, circulating tumor DNA, DNA isolated from circulating tumor cells, circulating fetal DNA, DNA isolated from virus-infected cells, fine needle aspirates, or single cells isolated by FACS (fluorescence-activated cell sorting), laser capture microscopy, or microfluidic devices. However, it is not intended that the samples used in the present invention be limited to any particular sample type, as any suitable sample, including those with low DNA concentrations, can be used in the present invention.

[0032] In some embodiments, the engineered DNA polymerases of the invention are used in the construction of DNA sequencing libraries for medium to high concentration DNA samples.

[0033] In some embodiments, the engineered DNA polymerases of the invention find use in molecular cloning applications, particularly where DNA concentrations are low compared to the Km of the naturally occurring enzyme. In some embodiments, this also applies to high-throughput cloning applications where samples are prepared in small amounts, or to any low-concentration DNA sample, such as environmental samples, patient samples, or ancient DNA.

[0034] In some embodiments, the engineered DNA polymerases of the present invention are used in simplified molecular biology workflows, including automated workflows, thereby eliminating cleanup steps between operations. Because the engineered DNA polymerases are active against low concentrations of substrate, small (or diluted) amounts of substrate samples containing inhibitors can be added to ligation reactions. Relevant inhibitor-containing DNA samples can include DNA in PCR buffer, DNA in electrophoresis buffer, or DNA in crude extracts. The engineered DNA polymerases of the present invention can ligate diluted samples more efficiently than native DNA polymerases. Alternatively, in other embodiments, the engineered DNA polymerases of the present invention are used in undiluted samples containing inhibitors.

[0035] In some embodiments, the engineered DNA polymerases of the invention are used in single-pot multi-enzyme reactions performed in microfluidic droplets or well plates. The high specific activity of the DNA polymerase allows for the selection of buffer formulations with respect to the performance of other enzymes in the reaction, thereby achieving ligation performance that does not limit the overall workflow.

[0036] In some embodiments, the engineered DNA polymerases of the present invention are used in the construction of DNA libraries. These libraries can be used for DNA sequencing, high-throughput screening, genetic selection, phage display, yeast display, ribosome display, cell-based assays, biochemical assays, or imaging-based high-content screening. In some embodiments, the engineered DNA polymerases of the present invention are particularly useful when the size, diversity, or fidelity of a library is limited by the ligation substrate concentration when using wild-type DNA polymerases. Abbreviations and definitions:

[0037] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures of cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry, and nucleic acid chemistry described below are well known and widely used in the art. Such techniques are well known and described in numerous documents and references known to those skilled in the art. Standard techniques, or modifications thereof, are used for chemical synthesis and chemical analysis.

[0038] All patents, patent applications, articles and publications mentioned herein, both supra and infra, are expressly incorporated herein by reference.

[0039] Although any suitable methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, some methods and materials are described herein.It should be understood that the present invention is not limited to specific methods, protocols, and reagents, as these may vary depending on the circumstances under which those skilled in the art use them.Therefore, the terms defined immediately below are more fully explained by referring to this application as a whole.All patents, patent applications, papers, and publications described herein, both above and below, are expressly incorporated herein by reference.

[0040] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0041] Numerical ranges are inclusive of the numbers defining the range. Accordingly, every numerical range disclosed herein is intended to include every narrower numerical range that falls within such broad numerical range, as if such narrower numerical ranges were all expressly written herein. Every maximum (or minimum) numerical limit disclosed herein is also intended to include every lower (or higher) numerical limit, as if such lower (or higher) numerical limit were all expressly written herein.

[0042] The term "about" refers to an acceptable error for a particular value. In some instances, "about" refers to a range of within 0.05%, 0.5%, 1.0%, or 2.0% of a given value. In some instances, "about" refers to within 1, 2, 3, or 4 standard deviations of a given value.

[0043] Furthermore, the headings provided herein are not limitations on the various aspects and embodiments of the invention, which can be had by reference to this application as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to this application as a whole. Regardless, to facilitate understanding of the invention, certain terms are defined below.

[0044] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0045] As used herein, the term "comprising" and its cognates are used in their inclusive sense (i.e., equivalent to the term "including" and its corresponding cognates).

[0046] As used herein, "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 numbering system for enzymes based on the chemical reaction they catalyze.

[0047] As used herein, "ATCC" refers to the American Type Culture Collection, whose biorepository collection includes genes and strains.

[0048] As used herein, "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

[0049] As used herein, the term "DNA" refers to deoxyribonucleic acid.

[0050] As used herein, the term "RNA" refers to ribonucleic acid.

[0051] As used herein, the terms "fusion protein" and "chimeric protein" and "chimera" refer to a hybrid protein created by the joining of two or more genes that originally encoded separate proteins. In some embodiments, fusion proteins are created by recombinant techniques (e.g., molecular biology techniques known in the art).

[0052] As used herein, the term "polymerase" refers to a class of enzymes that polymerize nucleoside triphosphates. Polymerases use a template nucleic acid strand to synthesize a complementary nucleic acid strand. The template strand and the synthesized nucleic acid strand can independently be either DNA or RNA. Polymerases known in the art include, but are not limited to, DNA polymerases (e.g., E. coli DNA pol I, T. aquaticus DNA polymerase [Taq], DNA-dependent RNA polymerase, and reverse transcriptase). As used herein, a polymerase is a polypeptide or protein that contains sufficient amino acids to perform the desired enzymatic function of the polymerase. In some embodiments, a polymerase does not contain all of the amino acids found in a naturally occurring enzyme, but only enough to enable the polymerase to perform the desired catalytic activity, including, but not limited to, 5'-3' polymerization, 5'-3' exonuclease, and 3'-5' exonuclease activity.

[0053] As used herein, the terms "DNA polymerase activity," "synthetic activity," and "polymerase activity" are used interchangeably herein and refer to the ability of an enzyme to synthesize new DNA strands by the incorporation of deoxynucleoside triphosphates.

[0054] As used herein, the terms "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 and C pairs with G), arranged in an antiparallel manner in the 5' to 3' direction, and held together by hydrogen bonds between the nucleobases (i.e., adenine [A], guanine [G], cytosine [C], and thymine [T]).

[0055] As used herein, the term "blunt" refers to the ends of self-complementary DNA duplexes or single-stranded ("ss") DNA that have no 5' or 3' overhangs. Blunt ends may have a 5' phosphate on one or both strands, making them compatible with ligation via a ligase, e.g., T4 DNA ligase.

[0056] As used herein, the term "end repair" refers to a method for repairing DNA (e.g., fragmented or damaged DNA or DNA molecules that are incompatible with other DNA molecules). In some embodiments, the process involves two functions: 1) converting double-stranded DNA with overhangs to double-stranded DNA without overhangs by an enzyme, such as T4 DNA polymerase and / or Klenow fragment, and 2) adding a phosphate group to the 5' end of the DNA (single-stranded or double-stranded) by an enzyme, such as polynucleotide kinase.

[0057] "Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer composed of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

[0058] "Amino acids" are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. Abbreviations used for genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glutamine (Gln or Q), 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 a three-letter abbreviation is used, an amino acid is referred to as an amino acid having an α-carbon (C α ) may be in either the L- or D-configuration with respect to the α-carbon. For example, "Ala" represents alanine without specifying the configuration with respect to the α-carbon, while "D-Ala" and "L-Ala" represent D-alanine and L-alanine, respectively. When single-letter abbreviations are used, uppercase letters represent amino acids in the L-configuration with respect to the α-carbon, and lowercase letters represent amino acids in the D-configuration with respect to the α-carbon. For example, "A" represents L-alanine and "a" represents D-alanine. When a polypeptide sequence is represented by a string of one-letter or three-letter abbreviations (or combinations thereof), the sequence is presented, according to convention, in the amino (N) to carboxy (C) direction.

[0059] Abbreviations used for genetically encoded nucleosides are conventional and are as follows: adenosine (A), guanosine (G), cytidine (C), thymidine (T), and uridine (U). Unless otherwise specified, nucleosides designated by abbreviations can be either ribonucleosides or 2'-deoxyribonucleosides. Nucleosides may be designated individually or collectively as either ribonucleosides or 2'-deoxyribonucleosides. When nucleic acid sequences are presented as a string of single-letter abbreviations, the sequences are presented in the 5' to 3' direction, according to convention, and the phosphate is not indicated.

[0060] The terms "engineered," "recombinant," "non-naturally occurring," and "variant," when used with respect to a cell, polynucleotide, or polypeptide, refer to material that has been modified in a way that would not otherwise occur in nature, or material that corresponds to a native or original form of that material that is identical but produced or derived from synthetic material and / or by manipulation using recombinant techniques.

[0061] As used herein, "wild-type" and "naturally occurring" refer to forms found in nature. For example, a wild-type polypeptide or polynucleotide sequence is one that is present in an organism that can be isolated from a source in nature and that has not been intentionally modified by human manipulation.

[0062] As used herein, "coding sequence" refers to that portion of a nucleic acid (eg, a gene) that codes for the amino acid sequence of a protein.

[0063] As used herein, the term "percent sequence identity (%)" refers to the comparison between polynucleotides and polypeptides, and is determined by comparing two optimally aligned sequences across a comparison window, where the portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence due to the optimal alignment of the two sequences.Percentages can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue exists in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions within the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.Alternatively, percentages can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue exists in both sequences, or where the nucleic acid base or amino acid residue is aligned with a gap, to obtain the number of matching positions, dividing the number of matching positions by the total number of positions within the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Those skilled in the art will recognize that there are many established algorithms available for aligning two sequences.The alignment of the sequences that are best suited for comparison can be carried out as known in the art, for example, by Smith and Waterman's local homology algorithm (Smith and Waterman, Adv. Appl. Math., 2:482

[1981] ), by Needleman and Wunsch's homology alignment algorithm (Needleman and Wunsch, J. Mol. Biol., 48:443

[1970] ), by Pearson and Lipman's similarity search method (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444

[1988] ), by computer implementations of these algorithms (for example, GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package), or by visual inspection.Examples of suitable algorithms for determining percent sequence identity and sequence similarity include, but are not limited to, the BLAST and BLAST 2.0 algorithms (see, e.g., Altschul et al., J. Mol. Biol., 215:403-410

[1990] and Altschul et al., Nucleic Acids Res., 3389-3402

[1977] ). Software for performing BLAST analyses is publicly available through the website of the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short lengths of words "W" in the query sequence that, when aligned with words of the same length in a database sequence, either match or meet a certain positive threshold score "T." T is referred to as the neighborhood word score threshold (see Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters "M" (reward score for a pair of matching residues, always greater than 0) and "N" (penalty score for mismatching residues, always less than 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score falls by an amount "X" from its maximum achieved value, when the cumulative score becomes zero or lower due to the accumulation of one or more negative-scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915

[1989] ). For exemplary sequence alignment and determination of percent sequence identity, the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison WI) can be used with the default parameters provided.

[0064] As used herein, a "reference sequence" refers to a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of a larger sequence, e.g., a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotides or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length, or the full length of a nucleic acid or polypeptide. Because two polynucleotides or polypeptides may each contain (1) sequences that are similar between the two sequences (i.e., a portion of the complete sequence) and (2) sequences that are diverse between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. In some embodiments, a "reference sequence" may be based on a primary amino acid sequence, in which case the reference sequence may have one or more alterations within the primary sequence. For example, the phrase "a reference sequence based on SEQ ID NO: 6 having a valine at the residue corresponding to X712" (or "a reference sequence based on SEQ ID NO: 6 having a valine at the residue corresponding to position 712") refers to a reference sequence in which the residue corresponding to position X712 in SEQ ID NO: 6 (e.g., an isoleucine) has been changed to a valine.

[0065] As used herein, a "comparison window" refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues, where a sequence can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and the portion of the sequence within the comparison window may contain 20 percent or less additions or deletions (i.e., gaps) compared to the reference sequence (which contains no additions or deletions) for optimal alignment of the two sequences. The comparison window may be greater than 20 contiguous residues, and may include a window of 30, 40, 50, 100, or longer, as desired.

[0066] As used herein, "corresponding to," "with reference to," and "compared to," when used in the context of the numbering of a given amino acid or polynucleotide sequence, refer to the numbering of residues in the reference sequence when comparing the given amino acid or polynucleotide sequence to the reference sequence. In other words, the residue number or residue position of a given polymer is specified relative to the reference sequence, not by the actual numerical position of the residue in 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 to a reference sequence by introducing gaps to optimize residue matching between the two sequences. In these cases, although gaps exist, the numbering of residues in the given amino acid or polynucleotide sequence is performed relative to the reference sequence to which it is aligned. In some embodiments, the sequence is tagged (e.g., with a histidine tag).

[0067] As used herein, a "mutation" refers to an alteration in a nucleic acid sequence. In some embodiments, the mutation results in a change in the encoded polypeptide sequence (i.e., compared to the original sequence not having the mutation). In some embodiments, the mutation comprises a substitution, resulting in a different amino acid being produced (e.g., replacement of an aspartic acid with a tryptophan). In some alternative embodiments, the mutation comprises an addition, resulting in an amino acid being added to the original polypeptide sequence. In some further embodiments, the mutation comprises a deletion, resulting in an amino acid being deleted from the original polypeptide sequence. Any number of mutations may be present in a given sequence.

[0068] As used herein, "amino acid difference" and "residue difference" refer to a difference in an amino acid residue at a position in a polypeptide sequence compared to the amino acid residue at the corresponding position in a reference sequence. The position of an amino acid difference is generally designated herein as "Xn," where n refers to the corresponding position in the reference sequence to which the residue difference is based. For example, "a residue difference at position X15 compared to SEQ ID NO:824" (or "a residue difference at position 15 compared to SEQ ID NO:824") refers to an amino acid residue difference at the polypeptide position corresponding to position 15 of SEQ ID NO:824. Thus, if the reference polypeptide of SEQ ID NO:824 has an aspartic acid at position 15, then "a residue difference at position X15 compared to SEQ ID NO:824" refers to an amino acid substitution of any residue that is not aspartic acid at the polypeptide position corresponding to position 15 of SEQ ID NO:824. In most cases herein, a specific amino acid residue difference at a position is designated as "XnY," where "Xn" indicates the corresponding residue and position in the reference polypeptide (as described above), and "Y" is the single-letter identifier of the amino acid found in the engineered polypeptide (i.e., the residue that differs from that in the reference polypeptide). In some cases (e.g., in the tables in the Examples), the present disclosure also provides specific amino acid differences designated by the conventional notation "AnB," where A is the single-letter identifier of the residue in the reference sequence, "n" is the number of the residue position in the reference sequence, and B is the single-letter identifier of the residue substitution in the sequence of the engineered polypeptide. In some cases, the polypeptides of the present disclosure may include one or more amino acid residue differences compared to the reference sequence, as indicated by a listing of the designated positions at which the residue difference occurs compared to the reference sequence. In some embodiments, when more than one amino acid can be used at a particular residue position in a polypeptide, the various amino acid residues that can be used are separated by a " / " (e.g., X775F / X775G, X775F / G, or K775F / G).The present disclosure includes engineered polypeptide sequences that contain one or more amino acid differences, including either or both conservative and non-conservative amino acid substitutions, as well as amino acid insertions and deletions in the sequence (e.g., a deletion at position 784).

[0069] As used herein, the terms "amino acid substitution set" and "substitution set" refer to a group of amino acid substitutions within a polypeptide sequence. In some embodiments, a substitution set includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. In some embodiments, a substitution set refers to a set of amino acid substitutions present in any of the variant DNA polymerase polypeptides listed in any of the tables in the Examples. In these substitution sets, individual substitutions are separated by a semicolon (";", e.g., P567G; I569G; Y667N) or a slash (" / ", e.g., P567G / I569G / Y667N). In some embodiments, a "substitution" includes an amino acid deletion.

[0070] As used herein, a "conservative amino acid substitution" refers to the replacement of one residue with a different residue having a similar side chain, and thus typically includes the substitution of an amino acid in a polypeptide with an amino acid within the same or a similar defined class of amino acids. By way of example, and not limitation, an amino acid having an aliphatic side chain can be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine), an amino acid having a hydroxyl side chain can be substituted with another amino acid having a hydroxyl side chain (e.g., serine and threonine), an amino acid having an aromatic side chain can be substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine), an amino acid having a basic side chain can be substituted with another amino acid having a basic side chain (e.g., lysine and arginine), an amino acid having an acidic side chain can be substituted with another amino acid having an acidic side chain (e.g., aspartic acid or glutamic acid), and a hydrophobic or hydrophilic amino acid can be substituted with another hydrophobic or hydrophilic amino acid, respectively.

[0071] As used herein, a "non-conservative substitution" refers to the replacement of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions may use amino acids between, rather than within, defined groups and may affect (a) the structure of the peptide backbone in the area of ​​the substitution (e.g., proline for glycine), (b) the charge or hydrophobicity, and / or (c) the bulk of the side chain. By way of example and not limitation, exemplary non-conservative substitutions include an acidic amino acid substituted with a basic or aliphatic amino acid, an aromatic amino acid substituted with a small amino acid, and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0072] As used herein, "deletion" refers to a modification to a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions can include removing one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids constituting the reference enzyme, while retaining the enzymatic activity and / or improved properties of the engineered polymerase enzyme. Deletions can be directed to internal and / or terminal portions of the polypeptide. In various embodiments, deletions can include contiguous segments or can be discontinuous. Deletions are indicated by "-" and can be present in a substitution set.

[0073] As used herein, "insertion" refers to a modification to a polypeptide by adding one or more amino acids from a reference polypeptide. The insertion may be in the internal part of the polypeptide or at the carboxy or amino terminus. As used herein, an insertion includes fusion proteins, which are known in the art. The insertion may be a continuous segment of amino acids, or may be separated by one or more amino acids in a naturally occurring polypeptide.

[0074] As used herein, "functional fragment" and "biologically active fragment" are used interchangeably herein to refer to a polypeptide that has amino-terminal and / or carboxy-terminal deletion(s), and / or internal deletion(s), but where the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is being compared (e.g., a full-length engineered DNA polymerase of the invention), and which retains substantially all of the activity of the full-length polypeptide.

[0075] As used herein, an "isolated polypeptide" refers to a polypeptide that has been substantially separated from other contaminants (e.g., proteins, lipids, and polynucleotides) that naturally accompany it. The term encompasses polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., a host cell or in vitro synthesis). Recombinant DNA polymerase polypeptides may be present intracellularly, in cell culture medium, or prepared in various forms, for example, as a lysate or isolated preparation. Thus, in some embodiments, the recombinant DNA polymerase polypeptides provided herein are isolated polypeptides.

[0076] As used herein, "substantially pure polypeptide" refers to a composition in which the polypeptide species is the predominant species present (i.e., it is more abundant than any other individual macromolecular species in the composition, on a molar or weight basis). A composition is generally substantially purified when the desired species constitutes at least about 50 percent of the macromolecular species present, on a molar or weight percent basis. Generally, a substantially pure DNA polymerase composition constitutes about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species present in the composition, on a molar or weight percent basis. In some embodiments, the desired species is purified to essential homogeneity (i.e., contaminating species cannot be detected in the composition by conventional detection methods), and the composition essentially consists of a single macromolecular species. Solvent species, small molecules (less than 500 daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant DNA polymerase polypeptide is a substantially pure polypeptide composition.

[0077] As used herein, "improved enzymatic properties" refers to an engineered DNA polymerase polypeptide that exhibits an improvement in any enzymatic property compared to a reference DNA polymerase polypeptide, e.g., a wild-type DNA polymerase polypeptide (e.g., the wild-type DNA polymerase of SEQ ID NO: 2) or another engineered DNA polymerase polypeptide. Improved properties include, but are not limited to, increased protein expression, increased thermal activity, increased thermostability, increased stability, increased enzymatic activity, increased substrate specificity and / or affinity, increased specific activity, increased resistance to substrate and / or end-product inhibition, increased chemical stability, improved chemoselectivity, improved solvent stability, increased tolerance to acidic pH, increased tolerance to proteolytic activity (i.e., reduced susceptibility to proteolysis), increased solubility, and altered temperature profile.

[0078] As used herein, "increased enzymatic activity" and "enhanced catalytic activity" refer to improved properties of an engineered DNA polymerase polypeptide and can be expressed in terms of an increase in specific activity (e.g., product produced / time / weight of protein) and / or an increase in percent substrate-to-product conversion (e.g., percent conversion of starting substrate mass to product over a specified period of time using a specified amount of DNA polymerase) compared to a reference DNA polymerase enzyme (e.g., a wild-type DNA polymerase and / or another engineered DNA polymerase). Exemplary methods for determining enzymatic activity are provided in the Examples. K, a change that can result in increased enzymatic activity, can be expressed in terms of an increase in specific activity (e.g., product produced / time / weight of protein) and / or percent substrate-to-product conversion (e.g., percent conversion of starting substrate mass to product over a specified period of time using a specified amount of DNA polymerase). Exemplary methods for determining enzymatic activity are provided in the Examples. m , V max , or k cat Any property associated with enzymatic activity can be affected, including the classical enzymatic property of being able to produce a DNA polymerase that is 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, or more, that is greater than the enzymatic activity of a naturally occurring DNA polymerase or another engineered DNA polymerase from which the DNA polymerase polypeptide is derived.

[0079] The terms "proteolytic activity" and "proteolysis," used interchangeably herein, refer to the breakdown of proteins into smaller polypeptides or amino acids. Protein breakdown is generally the result of hydrolysis of peptide bonds by protease (proteinase) enzymes. Protease enzymes include, but are not limited to, pepsin, trypsin, chymotrypsin, elastase, carboxypeptidase A and B, and peptidases (e.g., aminopeptidase, dipeptidase, and enteropeptidase).

[0080] The phrases "reduced susceptibility to proteolysis" and "reduced proteolytic susceptibility" are used interchangeably herein and mean that an engineered DNA polymerase polypeptide according to the invention has increased enzymatic activity in a standard assay (e.g., one disclosed in the Examples) after treatment with one or more proteases compared to a reference DNA polymerase.

[0081] As used herein, "conversion" refers to the enzymatic conversion (or bioconversion) of a substrate to a corresponding product. "Percent conversion" refers to the percent of a substrate that is converted to a product within a period of time under specified conditions. Thus, the "enzymatic activity" or "activity" of a DNA polymerase polypeptide can be expressed as the "percent conversion" of substrate to product over a specified period of time.

[0082] As used herein, "hybridization stringency" refers to the hybridization conditions, such as washing conditions, in nucleic acid hybridization. Generally, hybridization reaction is carried out under low stringency conditions, followed by washing under variable but high stringency conditions. The term "moderately stringent hybridization" refers to the conditions that allow target DNA to bind to the complementary nucleic acid with about 60% identity to target DNA, preferably about 75% identity, about 85% identity, with more than about 90% identity to target polynucleotide. Exemplary moderately stringent conditions are those that are equivalent to hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.2x SSPE, 0.2% SDS at 42°C. "High stringency hybridization" generally refers to hybridization that is performed at a thermal melting temperature, T, determined under solution conditions for a given polynucleotide sequence. m To about 10°C or less. In some embodiments, high stringency conditions refer to conditions that allow hybridization of only nucleic acid sequences that form stable hybrids at 65°C and 0.018M NaCl (i.e., if a hybrid is not stable at 65°C and 0.018M NaCl, it is not stable under the high stringency conditions contemplated herein). High stringency conditions are provided, for example, by hybridization under conditions equivalent to 50% formamide, 5x Denhardt's solution, 5x SSPE, and 0.2% SDS at 42°C, followed by washing in 0.1x SSPE and 0.1% SDS at 65°C. Another high stringency condition includes hybridization under conditions equivalent to hybridization in 5x SSC containing 0.1% (w:v) SDS at 65°C, followed by washing in 0.1x SSC containing 0.1% SDS at 65°C. Other high stringency hybridization conditions, as well as moderately stringent conditions, are described in the references cited above.

[0083] As used herein, "codon-optimized" refers to changing the codons of a polynucleotide encoding a protein to those preferentially used in a particular organism, so that the encoded protein is more efficiently expressed in that organism. Although the genetic code is degenerate in that most amino acids are represented by multiple codons, referred to as "synonymous" or "synonymous" codons, it is well known that the codon usage frequency of a particular organism is not random but is biased toward certain codon triplets. This codon usage bias may be higher for a given gene, for common functional or ancestral genes, for highly expressed proteins compared to low-copy proteins, and for aggregated protein-coding regions in the genome of an organism. In some embodiments, a polynucleotide encoding a DNA polymerase enzyme is codon-optimized for optimal production from the host organism selected for expression.

[0084] As used herein, the term "control sequences" refers to all components necessary or beneficial for the expression of the polynucleotides and / or polypeptides of the present disclosure. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter sequence, signal peptide sequence, initiation sequence, and transcription terminator. At a minimum, control sequences include a promoter, and transcriptional and translational stop signals. In some embodiments, control sequences are provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding the polypeptide.

[0085] "Operably linked" is defined herein as a configuration in which a control sequence is suitably positioned (i.e., in a functional relationship) with respect to a polynucleotide encoding a polypeptide of interest so that the control sequence induces or regulates expression of the polynucleotide encoding the polypeptide of interest.

[0086] As used herein, "promoter sequence" refers to a nucleic acid sequence 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 expression of the polynucleotide of interest. The promoter may be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell, including mutant promoters, truncated promoters, and hybrid promoters, and may be derived from a gene encoding an extracellular or intracellular polypeptide, either homologous or heterologous to the host cell.

[0087] As used herein, "suitable reaction conditions" refers to the conditions of an enzyme conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffers, co-solvents, etc.) under which a DNA polymerase polypeptide of the disclosure can convert a substrate into a desired product compound; exemplary "suitable reaction conditions" are provided herein (see Examples).

[0088] As used herein, "loading," as in "compound loading" or "enzyme loading," refers to the concentration or amount of a component in the reaction mixture at the start of the reaction. "Substrate," in the context of an enzymatic conversion reaction process, refers to a compound or molecule that is acted upon by a DNA polymerase polypeptide.

[0089] As used herein, "product" in the context of an enzymatic conversion process refers to a compound or molecule that results from the action of a DNA polymerase polypeptide on a substrate.

[0090] As used herein, "culturing" refers to growing a population of microbial cells under suitable conditions using any suitable medium (e.g., liquid, gel, or solid).

[0091] Recombinant polypeptides (e.g., DNA polymerase enzyme variants) can be produced using any suitable method known in the art. For example, there are a variety of different mutagenesis techniques well known to those of skill in the art. In addition, mutagenesis kits are also available from many commercial molecular biology suppliers. Methods are available for making specific substitutions at predetermined amino acids (site-directed), specific or random mutations in localized regions of a gene (region-directed), or random mutagenesis of the entire gene (e.g., saturation mutagenesis). Many suitable methods for generating enzyme variants are known to those of skill in the art, including, but not limited to, site-directed mutagenesis of single- or double-stranded DNA using PCR, cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, and chemical saturation mutagenesis, or any other suitable method known in the art. Non-limiting examples of methods used for DNA and protein engineering are provided in the following patents: U.S. Patent No. 6,117,679, U.S. Patent No. 6,420,175, U.S. Patent No. 6,376,246, U.S. Patent No. 6,586,182, U.S. Patent No. 7,747,391, U.S. Patent No. 7,747,393, U.S. Patent No. 7,783,428, and U.S. Patent No. 8,383,346. After variants are produced, they can be screened for any desired properties (such as high or increased activity, or low or reduced activity, increased thermal activity, increased thermostability, and / or acidic pH stability). In some embodiments, "recombinant DNA polymerase polypeptides" (also referred to herein as "engineered DNA polymerase polypeptides," "engineered DNA polymerases," "variant DNA polymerase enzymes," and "DNA polymerase variants") are used.

[0092] As used herein, a "vector" is a DNA construct for introducing a DNA sequence into a cell. In some embodiments, the vector is an expression vector operably linked to a suitable control sequence capable of effecting expression in a suitable host of a polypeptide encoded in the DNA sequence. In some embodiments, an "expression vector" has a promoter sequence operably linked to a DNA sequence (e.g., a transgene) to drive expression in a host cell, and in some embodiments, also includes a transcription termination sequence.

[0093] As used herein, the term "expression" includes any step involved in producing a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from the cell.

[0094] As used herein, the term "produce" refers to the production of a protein and / or other compound by a cell. The term is intended to encompass any step involved in producing a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses the secretion of a polypeptide from the cell.

[0095] As used herein, an amino acid or nucleotide sequence (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) is "heterologous" to another sequence to which it is operably linked if the two sequences are not essentially related.

[0096] As used herein, the terms "host cell" and "host strain" refer to a suitable host for an expression vector containing DNA (e.g., a polynucleotide sequence encoding at least one DNA polymerase variant) provided herein. In some embodiments, a host cell is a prokaryotic or eukaryotic cell that has been transformed or transfected with a vector constructed using recombinant DNA techniques known in the art.

[0097] As used herein, the term "analog" refers to a polypeptide having greater than 70% but less than 100% sequence identity (e.g., greater than 75%, greater than 78%, greater than 80%, greater than 83%, greater than 85%, greater than 88%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99% sequence identity) with a reference polypeptide. In some embodiments, analogs include non-naturally occurring amino acid residues, including, but not limited to, homoarginine, ornithine, and norvaline, as well as naturally occurring amino acids. In some embodiments, analogs also include one or more D-amino acid residues and a non-peptide linkage between two or more amino acid residues.

[0098] As used herein, the term "effective amount" means an amount sufficient to bring about a desired result. One of ordinary skill in the art can determine an effective amount using routine experimentation.

[0099] The terms "isolated" and "purified" are used to refer to a molecule (e.g., an isolated nucleic acid, polypeptide, etc.) or other component that is removed from at least one other component with which it is naturally associated. The term "purified" does not require absolute purity; rather, it is intended to be a relative definition.

[0100] As used herein, "composition" and "formulation" encompass products that contain at least one.

[0101] As used herein, "cell-free DNA" refers to DNA that circulates freely in the bloodstream and is not contained in or associated with cells. In some embodiments, cell-free DNA includes DNA originally derived from and released from normal somatic or germline cells, cancer cells, fetal cells, microbial cells, or viruses.

[0102] As used herein, "amplification" refers to nucleic acid replication. In some embodiments, the term refers to the duplication of a particular template nucleic acid.

[0103] As used herein, "polymerase chain reaction" and "PCR" refer to the methods described in U.S. Pat. Nos. 4,683,195 and 4,6884,202, which are incorporated herein by reference. These methods are used to increase the concentration of a segment of a target sequence or the entire target sequence in a mixture of purified DNA without the need for cloning or purification. A series of denaturation, annealing, and extension constitutes a "cycle." The steps of denaturation, primer annealing, and polymerase extension can be repeated multiple times (i.e., multiple cycles are used) to obtain a high concentration of amplified DNA. This process is well known in the art, and many variations have been developed over the years since the method was first described. PCR allows single copies of a specific target sequence to be isolated by hybridization with a labeled probe, incorporation of a biotinylated primer, followed by avidin enzyme-conjugated detection, and subsequent amplification of the amplified segment. 32 It can be amplified to detectable levels by several different techniques, including, but not limited to, incorporation of P-labeled deoxyribonucleotide triphosphates (e.g., dCTP or dATP). In addition to genomic DNA, any oligonucleotide sequence suitable for amplification can be copied using PCR with an appropriate primer set. The PCR product can also serve as a template for amplification.

[0104] As used herein, "target" when used in reference to PCR refers to the region of nucleic acid to which the primers used in the PCR method bind. A "target" is separated from other nucleic acids present in the sample used in the PCR method. A "segment" is a region of nucleic acid within the target sequence.

[0105] As used herein, "sample template" refers to nucleic acid derived from a sample that is analyzed for the presence of a target nucleic acid. In contrast, "background template" refers to nucleic acid other than the sample template that may or may not be present in the sample. Background template may be included in the sample incidentally, may arise from carryover contamination, or may result from the presence of nucleic acid contaminants from which the target nucleic acid is purified. For example, in some embodiments, nucleic acid derived from organisms other than those to be detected may be present as background in a test sample. However, it is not intended that the present invention be limited to any particular nucleic acid sample or template.

[0106] As used herein, "amplifiable nucleic acid" is used in reference to a nucleic acid that can be amplified by any amplification method, including but not limited to PCR. In most embodiments, the amplifiable nucleic acid comprises a sample template.

[0107] As used herein, "PCR product," "PCR fragment," and "amplification product" refer to the resulting compound typically obtained after two or more PCR amplification cycles (or other amplification methods, if indicated by context), which include denaturation, annealing, and extension steps. These terms encompass situations in which there is amplification of one or more segments of one or more target sequences.

[0108] As used herein, "amplification reagents" and "PCR reagents" refer to the reagents required for amplification (e.g., deoxyribonucleotide triphosphates, buffers, etc.) excluding primers, nucleic acid template, and amplification enzymes. Typically, amplification reagents, along with other reaction components, are placed and contained in a reaction vessel (e.g., test tube, microwell, etc.). It is not intended that the present invention be limited to any particular amplification reagents, as any suitable reagents may be used in the present invention.

[0109] As used herein, "restriction endonucleases" and "restriction enzymes" refer to enzymes that cut double-stranded nucleic acids at or near specific nucleotide sequences (i.e., "restriction sites"). In some embodiments, the restriction enzymes are bacterial enzymes, and in some additional embodiments, the nucleic acid is DNA.

[0110] As used herein, a "primer" refers to an oligonucleotide (i.e., a series of nucleotides), whether naturally occurring or produced synthetically, recombinantly, or by amplification, that can act as a point of initiation for nucleic acid synthesis when placed under conditions that induce synthesis of a primer extension product that is complementary to a nucleic acid strand (i.e., in the presence of nucleotides and an inducing agent, such as a DNA polymerase, and at a suitable temperature and pH). In most embodiments, a primer is single-stranded, but in some embodiments, it is double-stranded. In some embodiments, a primer is sufficiently long to prime the synthesis of an extension product in the presence of a DNA polymerase. The exact length of a primer will depend on many factors, as known to those of skill in the art.

[0111] As used herein, "probe" refers to an oligonucleotide (i.e., a series of nucleotides) that can hybridize to another oligonucleotide of interest, whether naturally occurring or produced synthetically, recombinantly, or by amplification. Probes are used to detect, identify, and / or isolate specific gene sequences of interest. In some embodiments, probes are labeled with a "reporter molecule" (also referred to as a "label") that aids in detection of the probe in a suitable detection system (e.g., fluorescent, radioactive, luminescent, enzymatic, and other systems). The present invention is not intended to be limited to any particular detection system or label. Primers, deoxyribonucleotides, and deoxyribonucleosides can include labels. Indeed, the labeling compositions of the present invention are not intended to be limited to any particular components. Exemplary labels include: 32 P, 35S, and fluorescent molecules (e.g., fluorescent dyes, including but not limited to green fluorescent protein).

[0112] As used herein, "fidelity" in reference to a polymerase is intended to refer to the accuracy of incorporation of a complementary base into a template-guided synthesized DNA strand compared to the template strand. Typically, fidelity is measured based on the frequency of incorrect base incorporation in the newly synthesized nucleic acid strand. Incorporation of an incorrect base can result in a point mutation, insertion, or deletion. Fidelity can be calculated according to any method known in the art (see, for example, Tindall and Kunkel, Biochem., 27:6008-6013

[1988] and Barnes, Gene 112:29-35

[1992] ). A polymerase or polymerase variant can exhibit either high fidelity or low fidelity. As used herein, "high fidelity" refers to a polymerase having a frequency of correct base incorporation above a predetermined value. As used herein, "low fidelity" refers to a polymerase having a frequency of correct base incorporation below a predetermined value. In some embodiments, the predetermined value is a desired frequency of correct base incorporation or the fidelity of a known polymerase (i.e., a reference polymerase).

[0113] As used herein, "altered fidelity" refers to a fidelity of a polymerase variant that differs from the fidelity of the parent polymerase from which the polymerase variant is derived. In some embodiments, the altered fidelity is higher than the fidelity of the parent polymerase, while in some other embodiments, the altered fidelity is lower than the fidelity of the parent polymerase. The altered fidelity can be determined by assaying the parent and variant polymerases and comparing their activities using any suitable assay known in the art.

[0114] As used herein, the term "ligase" generally refers to a class of enzymes used to join multiple polynucleotides together or to join the ends of a single polynucleotide. Ligases include ATP-dependent double-stranded polynucleotide ligase, NADPH-dependent double-stranded polynucleotide ligase, and NADPH-dependent double-stranded polynucleotide ligase. + These include ligases that are dependent on double-stranded DNA or RNA and single-stranded polynucleotide ligases. In some embodiments, the present invention provides bacteriophage ligases (e.g., T3 DNA ligase, T4 DNA ligase, and T7 DNA ligase), as well as variants thereof. In some further embodiments, the present invention provides fusion ligases or chimeric ligases. DNA ligases are often used in conjunction with restriction enzymes for the insertion of DNA fragments (e.g., genes) into plasmids. For ligation of sticky-ended fragments, controlling the optimal temperature is important for efficient recombination. T4 DNA ligase is most active at 37°C, but for optimal ligation efficiency with sticky-ended fragments, the optimal temperature of the enzyme must be balanced with the melting temperature of the ends to be ligated; the shorter the overhang, the lower the melting temperature of the fragment. Ligation reactions tend to be most efficient when the sticky ends are already stably annealed. For the ligation of blunt-ended DNA fragments, the melting temperature is not a factor to consider if the reaction occurs within the normal temperature range used for ligation. In these reactions, the limiting factor is not the ligase activity but the number of possible alignments between the ends of the DNA fragments. Therefore, the most efficient temperature for ligating blunt-ended DNA fragments is the temperature at which the number of possible alignments in the reaction is maximized.

[0115] As used herein, the term "adapter" refers to a single- or double-stranded oligonucleotide with ligation-compatible DNA ends. The adapter ends may be single- or double-stranded and may contain overhangs compatible with complementary overhangs in the processed library insert DNA. The adapter may have both single- and double-stranded regions. In some embodiments, the term "adapter" refers to a full-length adapter used in NGS (i.e., next-generation sequencing) reactions, which may include primer binding sites, barcodes, and other features, as well as a simplified model adapter used in HTP screening and ligation assays, which has the same ligation-compatible ends as a full-length adapter but lacks these additional features. NGS adapters designed for use in the Illumina® sequencing platform have deoxythymidine 3' overhangs compatible with ligation to the deoxyadenosine 3' overhangs present in A-tailed insert fragments. T-tailed adapters are not efficiently ligated to each other due to the preference of wild-type T4 DNA ligase for non-complementary DNA ends. Extreme ligation conditions, including long incubation periods, high adapter concentrations, or high crowding agent concentrations, result in adapter dimerization. Importantly, nuclease contaminants in the ligation reaction can remove overhanging adapter ends, resulting in blunt-ended substrates that are compatible with self-ligation.

[0116] As used herein, the term "compatible ends" refers to the ends of two double-stranded DNA fragments with 5' or 3' overhangs that hybridize in a 5' to 3' antiparallel orientation, such that all bases on the overhangs are complementary. In the context of ligation, at least one DNA fragment must have a 5' phosphate on a nucleotide that, upon hybridization of the 3' or 5' overhang, is positioned adjacent to the 3' hydroxyl of a nucleotide from another molecule. Ligation results in covalent linkage of two substrate molecules at compatible ends. In some embodiments, including library preparation for DNA sequencing, two DNA molecules, such as adapter and insert fragments, must have compatible ends, and both strands of the adapter / insert hybrid must be ligated to enable productive library amplification by PCR or sequencing by polymerase extension of primers hybridized to the adapter.

[0117] As used herein, the term "overhang" refers to one or more unpaired polynucleotide regions that occur at the ends of a double-stranded DNA fragment. Either the 5' or 3' DNA end can be present in the unpaired region. A double-stranded DNA fragment can be a duplex of two complementary single-stranded polynucleotides, or it can be a single polynucleotide with self-complementarity that forms a double-stranded DNA region.

[0118] The term "subject" includes mammals, e.g., humans, non-human primates, livestock, companion animals, and laboratory animals (e.g., rodents and lagamorphs). The term is intended to include females as well as males.

[0119] As used herein, the term "patient" means any subject being evaluated for, undergoing treatment for, or experiencing a disease. Engineered DNA polymerase polypeptides:

[0120] It should be understood that when referring to a particular DNA polymerase variant (i.e., an engineered DNA polymerase polypeptide) by reference to a modification of a particular amino acid residue in the sequence of a wild-type or reference DNA polymerase, other DNA polymerase variants that are modified at the equivalent position (as determined by amino acid sequence alignment, if necessary, between the respective amino acid sequences) are encompassed herein.

[0121] The engineered DNA polymerase polypeptide variants of the present invention perform polymerase reactions, including those useful in the polymerase chain reaction (PCR) and other reactions that utilize a polymerase to produce DNA.

[0122] The engineered DNA polymerase variants of the present invention are used in the efficient generation of DNA libraries suitable for NGS and other diagnostic methods. These DNA polymerase variants are used in solution as well as immobilized embodiments.

[0123] In some additional embodiments, engineered DNA polymerase polypeptides of the invention include polypeptides comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 2, 6, 22, 24, 26, 28, and / or 824.

[0124] In some embodiments, the engineered DNA polymerase polypeptide is produced by culturing a microorganism comprising at least one polynucleotide sequence encoding at least one engineered DNA polymerase polypeptide under conditions conducive to production of the engineered DNA polymerase polypeptide, hi some embodiments, the engineered DNA polymerase polypeptide is subsequently recovered from the resulting culture medium and / or cells.

[0125] The present invention provides exemplary engineered DNA polymerase polypeptides with DNA polymerase activity. The examples provide tables showing sequence-structure information correlating specific amino acid sequence characteristics with the functional activity of the engineered DNA polymerase polypeptides. This structure-function correlation information is provided in the form of specific amino acid residue differences compared to the reference engineered polypeptides of SEQ ID NOs: 2, 6, 22, 24, 26, 28, and / or 824, as well as experimentally determined activity data for the related exemplary engineered DNA polymerase polypeptides.

[0126] In some embodiments, an engineered DNA polymerase polypeptide of the invention having DNA polymerase activity comprises an amino acid sequence having at least 85% sequence identity to a reference sequence SEQ ID NO: 2, 6, 22, 24, 26, 28, and / or 824, and exhibits at least one improved property compared to the reference sequence (e.g., a wild-type DNA polymerase). In some embodiments, the improved property is an increase in product produced during PCR, while in some additional embodiments, the improved property is increased fidelity, and in yet some additional embodiments, the improved property is increased thermostability.

[0127] In some embodiments, an engineered DNA polymerase polypeptide exhibiting at least one improved property has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more amino acid sequence identity with SEQ ID NO:2, 6, 22, 24, 26, 28, and / or 824 and an amino acid residue difference at one or more amino acid positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 20, or more amino acid positions) compared to SEQ ID NO:2, 6, 22, 24, 26, 28, and / or 824. In some embodiments, the engineered DNA polymerase polypeptide is a polypeptide listed in a table provided in the examples (e.g., Tables 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 4.1, 4.2, 4.3, 4.4, 4.5, 6.2, and / or 6.3).

[0128] In some embodiments, the present invention provides functional fragments of engineered DNA polymerase polypeptides. In some embodiments, a functional fragment comprises at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the activity of the engineered DNA polymerase polypeptide (i.e., the parent engineered DNA polymerase) from which it is derived. In some embodiments, a functional fragment comprises at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the parent sequence of the engineered DNA polymerase. In some embodiments, a functional fragment is a derivative that is truncated by fewer than 5, fewer than 10, fewer than 15, fewer than 10, fewer than 25, fewer than 30, fewer than 35, fewer than 40, fewer than 45, and fewer than 50 amino acids.

[0129] In some embodiments, the present invention provides functional fragments of engineered DNA polymerase polypeptides. In some embodiments, the functional fragment comprises at least about 95%, 96%, 97%, 98%, or 99% of the activity of the engineered DNA polymerase polypeptide from which it is derived (i.e., the parent engineered DNA polymerase). In some embodiments, the functional fragment comprises at least 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 by fewer than 5, fewer than 10, fewer than 15, fewer than 10, fewer than 25, fewer than 30, fewer than 35, fewer than 40, fewer than 45, fewer than 50, fewer than 55, fewer than 60, fewer than 65, or fewer than 70 amino acids.

[0130] In some embodiments, an engineered DNA polymerase polypeptide exhibiting at least one improved property has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more amino acid sequence identity with SEQ ID NO:2, 6, 22, 24, 26, 28, and / or 824 and an amino acid residue difference at one or more amino acid positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, or more amino acid positions) compared to SEQ ID NO:2, 6, 22, 24, 26, 28, and / or 824. In some embodiments, the engineered DNA polymerase comprises at least 90% sequence identity to SEQ ID NOs: 2, 6, 22, 24, 26, 28, and / or 824 and comprises amino acid differences at at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid positions. In some embodiments, the engineered DNA polymerase polypeptide consists of the sequence of SEQ ID NOs: 6, 22, 24, 26, 28, and / or 824. Polynucleotides encoding engineered polypeptides, expression vectors, and host cells:

[0131] The present invention provides polynucleotides encoding the engineered DNA polymerase polypeptides described herein. In some embodiments, the polynucleotides are operably linked to one or more heterologous regulatory sequences that control gene expression to create recombinant polynucleotides capable of expressing the polypeptides. In some embodiments, an expression construct comprising at least one heterologous polynucleotide encoding an engineered DNA polymerase polypeptide is introduced into a suitable host cell to express the corresponding DNA polymerase polypeptide(s).

[0132] As will be apparent to those skilled in the art, the availability of protein sequences and knowledge of the codons corresponding to various amino acids provides a description of all polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, in which the same amino acid is coded for by alternative or synonymous codons, makes it possible to create a vast number of nucleic acids, all of which encode engineered DNA polymerase polypeptides. Thus, the present invention provides methods and compositions for producing any and all possible variations of DNA polymerase polynucleotides encoding the DNA polymerase polypeptides described herein by selecting combinations based on potential codon choices, and all such variations are considered to be specifically disclosed with respect to any polypeptide described herein, including the amino acid sequences presented in the Examples (e.g., Tables 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 4.1, 4.2, 4.3, 4.4, and / or 4.5).

[0133] In some embodiments, codons are preferably optimized for utilization by the host cell selected for protein production. For example, preferred codons used in bacteria are typically used for expression in bacteria. As a result, a polynucleotide encoding an engineered DNA polymerase polypeptide that has been codon-optimized contains preferred codons at about 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% of the codon positions in the full-length coding region.

[0134] In some embodiments, the DNA polymerase polynucleotide encodes an engineered polypeptide having DNA polymerase activity with the properties disclosed herein, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequence selected from SEQ ID NOs: 2, 6, 22, 24, 26, 28, and / or 824, or the amino acid sequence of any variant (e.g., those provided in the Examples), and comprises one or more residue differences (e.g., at one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid residue positions) compared to the amino acid sequence of the reference polynucleotide of SEQ ID NOs: 2, 6, 22, 24, 26, 28, and / or 824, or any variant disclosed in the Examples. In some embodiments, the reference sequence is selected from SEQ ID NOs: 2, 6, 22, 24, 26, 28, and / or 824. In some embodiments, the engineered DNA polymerase variant comprises a polypeptide sequence set forth in SEQ ID NOs: 6, 22, 24, 26, 28, and / or 824. In some embodiments, the engineered DNA polymerase variant comprises a substitution or set of substitutions of a variant DNA polymerase provided in the Examples.

[0135] The present invention provides polynucleotides encoding the engineered DNA polymerase variants provided herein. In some embodiments, the polynucleotides comprise a nucleotide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequence selected from SEQ ID NOs: 1, 5, 21, 23, 25, 27, and / or 823, or the nucleic acid sequence of any variant (e.g., those provided in the Examples), and contain one or more residue differences (e.g., at one, two, three, four, five, six, seven, eight, nine, ten, or more positions) compared to the reference polynucleotide of SEQ ID NOs: 1, 5, 21, 23, 25, 27, and / or 823, or the nucleic acid sequence of any variant disclosed in the Examples. In some embodiments, the reference sequence is selected from SEQ ID NOs: 1, 5, 21, 23, 25, 27, and / or 823. In some embodiments, a polynucleotide is capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from SEQ ID NOs: 1, 5, 21, 23, 25, 27, and / or 823, or its complement, or to a polynucleotide sequence encoding any of the variant DNA polymerase polypeptides provided herein. In some embodiments, a polynucleotide capable of hybridizing under highly stringent conditions encodes a DNA polymerase polypeptide comprising an amino acid sequence having one or more residue differences compared to SEQ ID NOs: 2, 22, 24, 26, 28, and / or 824. In some embodiments, the engineered DNA polymerase variant is encoded by a polynucleotide sequence set forth in SEQ ID NOs: 1, 5, 21, 23, 25, 27, and / or 823.

[0136] In some embodiments, an isolated polynucleotide encoding any of the engineered DNA polymerase polypeptides herein is engineered by various means to promote expression of the DNA polymerase polypeptide. In some embodiments, the polynucleotide encoding the DNA polymerase polypeptide comprises an expression vector in which one or more regulatory sequences are present to regulate expression of the DNA polymerase polynucleotide and / or polypeptide. Manipulation of the isolated polynucleotide prior to insertion into a vector may be desirable or necessary depending on the expression vector used. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. In some embodiments, regulatory sequences include, among others, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators. In some embodiments, a suitable promoter is selected based on the choice of host cell.For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the disclosure include promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase genes (e.g., Villa-Kamaroff et al., Proc. Natl. Acad. Sci. USA 75: 3727-3731).

[1978] ), and the tac promoter (see, for example, DeBoer et al., Proc. Natl. Acad. Sci. USA 80: 21-25

[1983] ).Exemplary promoters for filamentous fungal host cells include promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., International Publication No. WO 96 / 00787), and the NA2-tpi promoter (Aspergillus niger neutral alpha-amylase and Aspergillus Examples of useful promoters for yeast host cells include, but are not limited to, promoters derived from the 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 genes. Other useful promoters for yeast host cells are known in the art (see, for example, Romanos et al., Yeast 8:423-488

[1992] ).

[0137] In some embodiments, the control sequence is also a suitable transcription terminator sequence (i.e., a sequence recognized by a host cell to terminate transcription). In some embodiments, the terminator sequence is operably linked to the 3' end of the nucleic acid sequence encoding the DNA polymerase polypeptide. Any suitable terminator that is functional in the host cell of choice finds use in the present invention. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (see, e.g., Romanos et al., supra).

[0138] In some embodiments, the control sequence is also a suitable leader sequence (i.e., a nontranscribed region of an mRNA that is important for translation by the host cell). In some embodiments, the leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the DNA polymerase polypeptide. Any suitable leader sequence that is functional in the host cell of choice finds use in the present invention. Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0139] In some embodiments, the control sequence is also a polyadenylation sequence (i.e., a sequence operably linked to the 3' end of a nucleic acid sequence that, upon transcription, is recognized by a host cell as a signal for the addition of polyadenosine residues to the transcribed mRNA). Any suitable polyadenylation sequence that is functional in the host cell of choice finds use in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are known (see, e.g., Guo and Sherman, Mol. Cell. Biol., 15:5983-5990

[1995] ).

[0140] In some embodiments, the control sequence is also a signal peptide (i.e., a coding region that encodes an amino acid sequence linked to the amino terminus of a polypeptide that directs the encoded polypeptide into the secretory pathway of a cell). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region that is naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence. Any suitable signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a selected host cell is used in expressing the engineered polypeptide(s). Effective signal peptide coding regions for bacterial host cells include, but are not limited to, those obtained from the genes for Bacillus NC1B11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are known in the art (see, e.g., Simonen and Palva, Microbiol. Rev., 57:109-137

[1993] ). In some embodiments, effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to, signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase.Useful signal peptides for yeast host cells include, but are not limited to, those derived from the genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase.

[0141] In some embodiments, the control sequence is also a propeptide-coding region that encodes an amino acid sequence located at the amino terminus of a polypeptide. The resulting polypeptide is referred to as a "proenzyme," "propolypeptide," or "zymogen." A propolypeptide can be converted to a mature, active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide-coding region can be obtained from any suitable source, including, but not limited to, the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase (see, e.g., International Publication No. WO 95 / 33836). When both a signal peptide and a propeptide are present at the amino terminus of a polypeptide, the propeptide region is located adjacent to the amino terminus of the polypeptide, and the signal peptide region is located adjacent to the amino terminus of the propeptide region.

[0142] In some embodiments, regulatory sequences may also be utilized. These sequences facilitate regulation of polypeptide expression relative to host cell growth. Examples of regulatory systems are those that result in gene expression being switched on or off in response to chemical or physical stimuli, including the presence of a regulatory compound. For prokaryotic host cells, suitable regulatory sequences include, but are not limited to, the lac, tac, and trp operator systems. For yeast host cells, suitable regulatory systems include, but are not limited to, the ADH2 system or the GAL1 system. For filamentous fungi, suitable regulatory sequences include, but are not limited to, the TAKA alpha-amylase promoter, the Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter.

[0143] In another aspect, the present invention is directed to recombinant expression vectors comprising a polynucleotide encoding an engineered DNA polymerase polypeptide and, depending on the type of host into which they are to be introduced, one or more expression control regions, such as a promoter and terminator, an origin of replication, etc. In some embodiments, the various nucleic acids and control sequences described herein are joined together to produce a recombinant expression vector containing one or more convenient restriction sites, thereby allowing for the insertion or substitution of a nucleic acid sequence encoding a DNA polymerase polypeptide at such sites. Alternatively, in some embodiments, the nucleic acid sequences of the present invention are expressed by inserting the nucleic acid sequence or a nucleic acid construct containing the sequence into an appropriate vector for expression. In some embodiments involving the creation of an expression vector, the coding sequence is positioned within the vector such that the coding sequence is operably linked to appropriate control sequences for expression.

[0144] The recombinant expression vector may be any suitable vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and that can result in expression of the DNA polymerase polynucleotide sequence. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

[0145] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, extrachromosomal element, minichromosome, or artificial chromosome). The vector may contain any means for ensuring self-replication. In some alternative embodiments, the vector, upon introduction into a host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, in some embodiments, a single vector or plasmid, or two or more vectors or plasmids, and / or transposons, which together contain the total DNA to be introduced into the genome of the host cell, are utilized.

[0146] In some embodiments, the expression vector contains one or more selectable markers that allow for easy selection of transformed cells. A "selectable marker" is a gene the product of which provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc. Examples of bacterial selectable markers include, but are not limited to, the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance, such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase, e.g., from A. nidulans or A. orzyae), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase, e.g., from S. hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase, e.g., from A. nidulans or A. orzyae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), and equivalents thereof. In another aspect, the invention provides a host cell comprising at least one polynucleotide encoding at least one engineered DNA polymerase polypeptide of the invention, wherein the polynucleotide is operably linked to one or more control sequences for expression of the engineered DNA polymerase enzyme in the host cell.Suitable host cells for use in expressing polypeptides encoded by the expression vectors of the invention are well known in the art and include, but are not limited to, bacterial cells such as E. coli, Vibrio fluvialis, Streptomyces, and Salmonella typhimurium cells, fungal cells such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)), insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells, and plant cells. Exemplary host cells also include various Escherichia coli strains (e.g., W3110(ΔfhuA) and BL21).

[0147] Thus, in another aspect, the invention provides methods for producing an engineered DNA polymerase polypeptide, the method comprising culturing a host cell capable of expressing a polynucleotide encoding the engineered DNA polymerase polypeptide under conditions suitable for expression of the polypeptide. In some embodiments, the method further comprises isolating and / or purifying the DNA polymerase polypeptide described herein.

[0148] Suitable culture medium and growth conditions of host cells are well known in the art.Any suitable method for introducing polynucleotide into cell for expressing DNA polymerase polypeptide is contemplated to 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.

[0149] An engineered DNA polymerase polypeptide having the properties disclosed herein can be obtained by subjecting a polynucleotide encoding a naturally occurring or engineered DNA polymerase polypeptide to any suitable mutagenesis and / or directed evolution method known in the art and / or described herein. Exemplary directed evolution techniques are mutagenesis and / or DNA shuffling (see, e.g., Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751

[1994] , International Publication Nos. WO95 / 22625, WO97 / 0078, WO97 / 35966, WO98 / 27230, WO00 / 42651, WO01 / 75767, and U.S. Patent No. 6,537,746). Other directed evolution procedures that can be used include, among others, the staggered extension process (StEP), in vitro recombination (see, e.g., Zhao et al., Nat. Biotechnol., 16:258-261

[1998] ), mutagenic PCR (see, e.g., Caldwell et al., PCR Methods Appl., 3:S136-S140

[1994] ), and cassette mutagenesis (see, e.g., Black et al., Proc. Natl. Acad. Sci. USA 93:3525-3529

[1996] ).

[0150] Mutagenesis and directed evolution methods can be readily applied to polynucleotides encoding DNA polymerases to generate libraries of variants that can be expressed, screened, and assayed. Any suitable mutagenesis and directed evolution method can be used in the present invention and is well known in the art (e.g., U.S. Patents 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, 6,406,910, 6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675, 6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,483,011, 6,484,105, 6,489,146, 6,500,617, 6,500,639, 6,506,602, 6,506,603, 6,518, 065, 6,519,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098, 6,576,467, 6,579,678, 6,586,182, 6,602,986, 6,605,430, 6,613,514, 6,653,072, 6,686,515, 6,703,240, 6,716,631, 6,825,001, 6,902,922, 6,917,882, 6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515、7,105,297、7,148,054、7,220,566、7,288,375、7,384,387、7,421,347、7,430,477、7,462,469、7,534,564、7,620,500、7,620,502、7,629,170、7,702,464、7,747,391、7,747,393、7,751,986、7,776,598、7,783,428、7,795,030、7,853,410、7,868,138、7,783,428、7,873,477、7,873,499、7,904,249、7,957,912、7,981,614、8,014,961、8,029,988、8,048,674、8,058,001、8,076,138、8,108,150、8,170,806、8,224,580、8,377,681、8,383,346、8,457,903、8,504,498、8,589,085、8,762,066、8,768,871、 9,593,326、9,665,694、9,684,771,and all relevant PCT and non-US counterparts, Ling et al., Anal. Biochem., 254(2):157-78

[1997] ; Dale et al., Meth. Mol. Biol., 57:369-74

[1996] ; Smith, Ann. Rev. Genet., 19:423-462

[1985] ; Botstein et al., Science, 229:1193-1201

[1985] ; Carter, Biochem. J., 237:1-7

[1986] ; Kramer et al., Cell, 38:879-887

[1984] ; Wells et al., Gene, 34:315-323

[1985] ; Minshull et al., Curr. Op. Chem. Biol., 3:284-290

[1999] , Christians et al., Nat. Biotechnol., 17:259-264

[1999] , Crameri et al., Nature, 391:288-291

[1998] , Crameri, et al., Nat. Biotechnol., 15:436-438

[1997] , Zhang et al., Proc. Nat. Acad. Sci. USA, 94:4504-4509

[1997] , Crameri et al., Nat. Biotechnol., 14:315-319

[1996] , Stemmer, Nature, 370:389-391

[1994] , Stemmer, Proc. Nat. Acad. Sci. USA, 91:10747-10751 See, for example,

[1994] , European Patent No. EP3049973, International Publication Nos. WO95 / 22625, WO97 / 0078, WO97 / 35966, WO98 / 27230, WO00 / 42651, WO01 / 75767, WO2009 / 152336, and WO2015 / 048573, all of which are incorporated herein by reference.

[0151] In some embodiments, enzyme clones obtained after mutagenesis treatment are screened by subjecting the enzyme preparation to a predetermined temperature (or other assay conditions) and measuring the amount of enzyme activity remaining after the heat treatment or other suitable assay conditions. Clones containing polynucleotides encoding DNA polymerase polypeptides are then isolated from the gene, sequenced to identify nucleotide sequence changes (if any), and used to express the enzyme in host cells. Measurement of enzyme activity from expression libraries can be performed using any suitable method known in the art (e.g., standard biochemical techniques, e.g., HPLC analysis).

[0152] For engineered polypeptides of known sequence, polynucleotides encoding the enzymes can be prepared by standard solid-phase methods according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be synthesized separately and then joined (e.g., by enzymatic or chemical ligation methods, or polymerase-mediated methods) to form any desired contiguous sequence. For example, the polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using the classical phosphoramidite method (see, e.g., Beaucage et al., Tet. Lett., 22:1859-69

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

[1984] ), as typically performed in automated synthesis methods. By the phosphoramidite method, oligonucleotides are synthesized (e.g., purified, annealed, ligated, and cloned in an appropriate vector in an automated DNA synthesizer).

[0153] Thus, in some embodiments, a method for preparing an engineered DNA polymerase polypeptide may include (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the amino acid sequences of any variant described herein, and (b) expressing the 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 more (e.g., up to 3, 4, 5, or up to 10) deletions, insertions, and / or substitutions of amino acid residues. In some embodiments, the amino acid sequence has deletions, insertions, and / or substitutions of 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 15, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, or 1 to 50 amino acid residues, as appropriate. In some embodiments, the amino acid sequence optionally has deletions, insertions, and / or substitutions of 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 residues. In some embodiments, the amino acid sequence optionally has deletions, insertions, and / or substitutions of 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 residues. In some embodiments, the substitutions are conservative or non-conservative substitutions.

[0154] The expressed engineered DNA polymerase polypeptide can be evaluated for any desired improved property or combination of properties (e.g., activity, selectivity, fidelity, stability, thermostability, tolerance to various pH levels, protease susceptibility, etc.) using any suitable assay known in the art, including but not limited to the assays and conditions described herein.

[0155] In some embodiments, any of the engineered DNA polymerase polypeptides expressed in the host cells are recovered from the cells and / or culture medium using any one or more of well-known techniques for protein purification, including lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography, among others.

[0156] Chromatographic techniques for isolating 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 particular enzyme depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those skilled in the art. In some embodiments, affinity techniques can be used to isolate improved DNA polymerase enzymes. For affinity chromatography purification, any antibody that specifically binds to the DNA polymerase polypeptide of interest can be used. For antibody production, various host animals, including but not limited to rabbits, mice, rats, etc., are immunized by injection with the DNA polymerase polypeptide or a fragment thereof. In some embodiments, the DNA polymerase polypeptide or fragment is bound to a suitable carrier, e.g., BSA, by a side chain functional group or a linker attached to the side chain functional group.

[0157] In some embodiments, the engineered DNA polymerase polypeptide is produced in a host cell by a method comprising culturing a host cell (e.g., an E. coli strain) comprising a polynucleotide sequence encoding an engineered DNA polymerase polypeptide described herein under conditions conducive to production of the engineered DNA polymerase polypeptide, and recovering the engineered DNA polymerase polypeptide from the cell and / or culture medium. In some embodiments, the host cell produces more than one engineered DNA polymerase polypeptide.

[0158] In some embodiments, the invention provides methods for producing an engineered DNA polymerase polypeptide, the method comprising culturing recombinant bacterial cells comprising a polynucleotide sequence encoding an engineered DNA polymerase polypeptide having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a reference sequence SEQ ID NO: 2, 6, 26, 24, 26, 28, and / or 824, and having one or more amino acid residue differences, under suitable culture conditions that allow for the production of the engineered DNA polymerase polypeptide, and optionally recovering the engineered DNA polymerase polypeptide from the culture and / or the cultured bacterial cells. In some embodiments, the host cells produce more than one engineered DNA polymerase polypeptide.

[0159] In some embodiments, once the engineered DNA polymerase polypeptides are recovered from the recombinant host cells and / or culture medium, they are further purified by any suitable method(s) known in the art. In some additional embodiments, the purified engineered DNA polymerase polypeptides are combined with other components and compounds to provide compositions and formulations comprising the engineered DNA polymerase polypeptides that are suitable for various applications and uses (e.g., diagnostic methods and compositions). [Example]

[0160] The following examples, including the experiments and results obtained, are provided for illustrative purposes only and are not to be construed as limiting the invention.

[0161] In the experimental disclosure that follows, the following abbreviations apply: ppm (parts per million), M (molar), mM (millimolar), uM and μM (micromolar), nM (nanomolar), mol (mole), gm and g (grams), mg (milligram), ug and μg (micrograms), L and l (liters), ml and mL (milliliters), cm (centimeters), mm (millimeters), um and μm (micrometers), sec. (seconds), min (minutes), h and hr (hours), Ω (ohms), μf (microfarads), U (units), 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 E. coli strain, available from the Coli Genetic Stock Center [CGSC], New Haven, CT), HTP (high-throughput), HPLC (high-performance liquid chromatography), MCYP (microcyp), ddH2O (double-distilled water), PBS (phosphate-buffered saline), BSA (bovine serum albumin), DTT (dithiothreitol), CAM (chloramphenicol), CAT (chloramphenicol acetyltransferase), IPTG (isopropyl β-D-1-thiogalactopyranoside), GFP (green fluorescent protein), eGFP (enhanced GFP), DsRed (red fluorescent protein isolated from Discosoma sp.), FIOPC (fold improvement over positive control), LB (Luria-Bertani), SPRI (solid-phase reversible immobilization), Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO), Perkin Elmer (Perkin Elmer, Inc., Waltham, MA), Harvard Apparatus (Harvard Apparatus, Holliston, MA), Millipore (Millipore, Corp., Billerica MA), Covaris (Covaris, Inc.)., Woburn, MA), MagBio (MagBio Genomics, Inc., Gaithersburg, MD), Qiagen (Qiagen Inc., Germantown, MD), Illumina (Illumina, Inc., San Diego, CA), BD Biosciences (BD Biosciences, San Jose, CA), Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, MI), Kuhner (Adolf Kuhner, AG, Basel, Switzerland), Zymo (Zymo Research, Irvine, CA), Agilent (Agilent Technologies, Inc., Santa Clara, CA), Thermo Scientific (part of Thermo Fisher Scientific, Waltham, MA), GE Healthcare (GE Healthcare Bio-Sciences, Piscataway, NJ), and Bio-Rad (Bio-Rad Laboratories, Hercules, CA). . Example 1 Obtaining DNA polymerase gene and constructing expression vector

[0162] The group B polymerase encoded by the genome of Thermococcus sp. strain 2319x1 (Unprot identifier A0A0U3SCT0; SEQ ID NOS: 1 and 2, polynucleotide and polypeptide sequences, respectively) shares 73% protein sequence identity with Pyrococcus furiosus DNA polymerase (SEQ ID NO: 4). This polymerase (SEQ ID NO: 2) is referred to herein as "Pol3." For clarity, this enzyme is not the same as the DNA polymerase III holoenzyme involved in prokaryotic DNA replication. A synthetic gene (SEQ ID NO: 5) encoding a 6-histidine-tagged version of wild-type (WT) Pol3 polymerase (SEQ ID NO: 6) was constructed and subcloned into the Escherichia coli expression vector pCK100900i (see, e.g., U.S. Patent No. 7,629,157 and U.S. Patent Application Publication No. 2016 / 0244787, both of which are incorporated herein by reference). These plasmid constructs were transformed into E. coli strains derived from W3110. A library of gene variants was generated from these plasmids using directed evolution techniques commonly known to those skilled in the art (see, for example, U.S. Patent No. 8,383,346 and International Publication No. WO2010 / 144103, both of which are incorporated herein by reference). Substitutions in the enzyme variants described herein are indicated with reference to the 6-histidine tagged enzyme (i.e., SEQ ID NO: 6) or variants thereof, as indicated. Example 2 High-throughput (HTP) Pol3 DNA polymerase expression and lysate preparation

[0163] This example describes the methods used for HTP growth and lysate preparation of polymerase variants. High-throughput growth of Pol3 polymerase and variants

[0164] Transformed E. coli cells were selected by plating on LB agar plates containing 1% glucose and 30 μg / ml chloramphenicol. After overnight incubation at 37°C, colonies were placed into wells of a 96-well shallow, flat-bottom NUNC™ microplate (Thermo-Scientific) filled with 180 μl of LB medium per well supplemented with 1% glucose and 30 μg / ml chloramphenicol. Cultures were grown overnight for 18-20 hours on a shaker (200 rpm, 30°C, and 85% relative humidity, Kuhner). Samples of overnight growth (20 μL) were transferred to Costar 96-well deep plates filled with 380 μL of Terrific Broth supplemented with 30 μg / ml chloramphenicol. OD 600 Plates were incubated for 120 minutes on a shaker (250 rpm, 30°C, 85% relative humidity, Kuhner) until the RI reached 0.4-0.8. Cells were then induced with 40 μL of 10 mM IPTG in sterile water and incubated overnight on a shaker (250 rpm, 30°C, and 85% relative humidity, Kuhner). Cells were pelleted (4000 rpm x 20 minutes), the supernatant discarded, and the cells were frozen at -80°C prior to analysis. Dissolution of HTP pellets

[0165] The cell pellet was thawed and resuspended by shaking in 300 μl / well of lysis buffer (20 mM NaCl, 50 mM Tris-HCl, pH 7.5) for 10 minutes at room temperature. 150 μl of the resuspended pellet was then transferred to a HARDSHELL® PCR plate (Bio-Rad). Cell lysis and heat treatment were achieved in a single thermocycler incubation step at 93°C for 60 minutes. Cell debris and heat-insoluble material were pelleted (4000 rpm x 10 minutes), and the clarified lysate supernatant was used for PCR assays, as described in the following examples. Example 3 PCR product yield assay

[0166] Selection of Pol3 variants was achieved by measuring PCR product yield in an end-point PCR assay using a short extension time relative to the template length used. Each variant was screened in a 30 μL reaction mixture consisting of 80 pg / μL MCYP template DNA (SEQ ID NO: 7), 0.2 mM dNTPs, 400 nM each of MCYP forward (SEQ ID NO: 10) and reverse (SEQ ID NO: 11) primers, 20 mM Tris buffer, pH 8.8, 10 mM KCl, 2 mM MgSO4, 10 mM (NH4)2SO4, 0.1% v / v Triton x-100, and 0.1 g / L BSA. The lysate was diluted in 20 mM Tris, pH 8.8, and 5 μL of the diluted lysate was added to a PCR master mix to a final concentration of 0.12–0.58% (v / v) lysate, as indicated in the conditions below each table in the following examples. PCR cycling included an initial denaturation at 95°C for 2 minutes, followed by 25 cycles of 95°C for 25 seconds, annealing at 51-53°C for 30 seconds, and extension at 72°C for 10 seconds to 2.25 minutes. Lysate concentrations, annealing temperatures, and extension times are included in each table in the examples. Upon completion of the reaction, 70 μL of ddH2O was added to each reaction. The 3 kb MCYP PCR product was quantified using the DNA 5k assay on a LABCHIP® GX capillary electrophoresis instrument (Perkin-Elmer). For Table 3.2, product yields were qualitatively ranked after electrophoresis on an E-Gel 96 1% agarose gel (ThermoFisher). [Table 3-1-1] [Table 3-1-2] [Table 3-2-1] [Table 3-2-2] [Table 3-2-3] [Table 3-3] [Table 3-4-1] [Table 3-4-2] [Table 3-5-1] [Table 3-5-2] [Table 3-5-3] [Table 3-6-1] [Table 3-6-2] [Table 3-6-3] [Table 3-6-4] [Table 3-7] [Table 3-8-1] [Table 3-8-2] Example 4 High-throughput polymerase fidelity assay

[0167] Colony-based reporter assays are well-established methods for determining polymerase fidelity. In these assays, a reporter gene, such as lacZ (see Barnes, Gene 112:29-35

[1992] ), lacI (Jozwiakowksi and Connolly, Nucl. Acids Res., 37: e102

[2009] ), and rpsL (Kitabayashi et al., Biosci. Biotechnol. Biochem., 66: 2194-2200

[2002] ), is cloned, and the frequency of gene-inactivating mutations observed in the clones is proportional to the error rate of the DNA polymerase used to clone the reporter gene. Error rates are reported as the fraction of colonies with a blue or white phenotype on X-gal (5-bromo-4-chloro-3-indolyl BD-galactopyranoside) plates for lacI or lacZ, or by the ratio of colonies growing on selective ampicillin or streptomycin agar plates for rpsL. The error rates of proofreading DNA polymerases are exceptionally low (e.g., approximately 3 × 10 -3 ), these techniques require assaying a large number of colonies to reduce the effect of sampling error on the observed error rate. Although simple and affordable compared to direct Sanger sequencing of individual cloned amplicons, these assays have limited throughput.

[0168] A high-throughput assay for DNA polymerase fidelity was developed for use in the present invention using a cell-based flow cytometry assay. A reporter plasmid (SEQ ID NO: 18) was constructed encoding genes for two fluorescent proteins, eGFP (SEQ ID NO: 14) and wild-type dsRed (SEQ ID NO: 16), under the control of an inducible LacI promoter. The plasmid also encodes a chloramphenicol acetyltransferase gene for selection. When this reporter plasmid was transformed into E. coli and induced with IPTG, both fluorescent proteins were expressed in the majority of cells within the population. E. coli populations expressing a single fluorescent protein (e.g., dsRed) exhibit a broad log-normal distribution of fluorescence intensity due to variations in induction and noise in gene expression. Therefore, mutations that inactivate dsRed would be indistinguishable from noise in gene expression. Although there is a wide range of gene expression in cells within a dual-labeled (eGFP / dsRed) population, the two proteins covary in their expression. As a result, cells that strongly express eGFP without expressing dsRed are extremely rare, and cells expressing a reporter plasmid with an inactivating mutation in dsRed (but retaining eGFP expression) are easily distinguished from background.

[0169] PCR reactions were performed using a variant polymerase and adjacent 5'-phosphorylated primers to replicate the entire sequence of the reporter plasmid. During PCR amplification, polymerase-induced errors were introduced into one or both of the fluorescent reporter proteins encoded by the reporter plasmid. The replication products were circularized by ligation and transformed into E. coli, where a mixed population of wild-type and error-containing transformants was induced to express the dual reporter. The induced cell population was then analyzed using flow cytometry to determine the fraction of cells that had lost dsRed expression due to the PCR error but still expressed GFP. Importantly, when isolated clones of the wild-type reporter plasmid were induced for 48-72 hours and analyzed by flow cytometry, the background of cells expressing only eGFP was extremely low.

[0170] The reporter construct was amplified using 5'-phosphorylated forward (SEQ ID NO: 19) and reverse (SEQ ID NO: 20) primers as described for the PCR reaction in Example 2. Typically, a final concentration of 0.25% v / v of HTP lysate was used for each DNA polymerase. 50 μl of reaction was assembled with the fidelity reporter construct (SEQ ID NO: 18) at a final concentration of 120 pg / μl. A 5-minute extension time was used during cycling. To remove background DNA not amplified by the DNA polymerase variants by PCR, the remaining methylated full-length reporter plasmid PCR template (SEQ ID NO: 18) was fragmented by adding DpnI restriction enzyme and incubating at 37°C for 15 minutes.

[0171] Linear ssDNA PCR amplicons were purified by column purification using a ZR-96 DNA Clean and Concentrator (Zymo). Briefly, 200 μl of the provided binding buffer was added to a 50 μl PCR reaction, and the sample was processed according to the manufacturer's protocol. Samples were eluted in 10-50 μl of nuclease-free water.

[0172] The purified linear amplicon was then circularized in a 200 μl ligation reaction with final component concentrations of 66 mM Tris-HCl, pH 8.0, 1 mM ATP, 10 mM MgCl, 1 mM DTT, 50 ng / μl DNA ligase (SEQ ID NO: 38 of U.S. Patent Application No. 15 / 972,919) for 1 hour at 20°C.

[0173] The circularized amplicons were then purified and concentrated using a ZR-96 DNA Clean and Concentrator (Zymo). Briefly, 600 μl of the provided binding buffer was added to 200 μl of the ligation reaction, and the sample was processed according to the manufacturer's protocol. The sample was eluted in 12 μl of nuclease-free water.

[0174] The circularized amplicons were transformed into E. coli using a BTX ECM® 630 / HT-100 96-well electroporation apparatus (BTX, Harvard Apparatus). Electrocompetent W3110 E. coli cells (Agilent) were diluted with an equal volume of ice-cold sterile water. 50 μl of the diluted cell suspension was then added to wells containing 3 μl of circularized amplicon eluate and mixed. The mixture was transferred to an uncoated 96-well disposable electroporation plate (2 mm gap, BTX). The plate was cooled on ice and pulsed using standard settings for E. coli transformation (2500 volts, 200 Ω, 25 μf). Cells were harvested from the wells and added to 500 μl of SOC recovery medium (Invitrogen, see Hanahan, J. Mol. Biol., 166:557-580

[1983] ) and incubated with shaking at 37°C for 1 hour to allow cell recovery and expression of the antibiotic resistance marker (chloramphenicol acetyltransferase) present on the reporter plasmid. After the 1-hour incubation, 500 μl of LB broth containing chloramphenicol (60 μg / ml) was added to the wells for selection of the reporter plasmid during overnight growth at 30°C or 37°C. Also at the 1-hour time point, a portion of the grown cells was diluted 1:100 in LB, and 5 μl of the diluted culture was added to LB broth. + CAM + The transformation efficiency was checked by pipetting onto 1% (v / v) glucose plates. Spots with five or more colonies were counted as at least 10 5 transformants, with some wells containing up to 10 6 Transformants were observed. Blank control wells were inoculated with E. coli expressing the eGFP / dsRed reporter construct (SEQ ID NO: 18) and a positive control expressing eGFP alone.

[0175] The next day, plates were subcultured by adding 20 μl of the overnight culture to 380 μl of LB medium and grown at 30°C with shaking. After 2 hours of incubation, IPTG was added to each plate to a final concentration of 1 mM. Plates were incubated at 30°C with shaking for 40-72 hours to allow induction and complete maturation of wild-type dsRed protein. The induced cultures were pelleted by centrifugation, the supernatant was decanted, and the cells were resuspended in 400 μl of 1x PBS by vortexing. For flow cytometry, cells were further diluted 100-fold in PBS.

[0176] Cells were analyzed using an ACCURI™ C6 flow cytometer (BD Biosciences) with an autosampler unless otherwise indicated in the table below. Both eGFP and dsRed were excited by a 488 nm laser, and fluorescence compensation was used to remove spectral overlap in the eGFP and dsRed emission channels. Gates for single eGFP-expressing cells (green-only) and dual eGFP / dsRed-expressing cells were defined using corresponding control cultures in each plate. Typically, the frequency of background green-only events was 1 x 10 in the eGFP / dsRed-expressing control population. -5 On the other hand, 1×10 -3 ~3×10 -3 Background subtraction was not applied because a frequency of green-only events of 0.001 was observed in PCR-amplified populations using high-fidelity polymerases. To minimize sampling error, wells were split into 500 total green-only events, or a maximum of 10 total events per sample. 6Events were analyzed. At a flow rate of 14 ul / min, this required 15-4 minutes per sample, depending on polymerase fidelity. The green-only frequency of each variant was calculated by dividing the fraction of gated green-only events by the total number of gated fluorescent cell events. The relative error rate of each variant was calculated by dividing the green-only frequency of the variant by the frequency of the parental control. Finally, the fold improvement in polymerase fidelity reported in the table below is the reciprocal of the relative error rate. [Table 4-1] [Table 4-2] [Table 4-3-1] [Table 4-3-2] [Table 4-4] [Table 4-5] Example 5 Relative comparison of polymerase fidelity

[0177] The error rates of the variant DNA polymerases were compared to those of commercially available DNA polymerases used in PCR using a high-throughput flow cytometry assay. The variant polymerases obtained in this study were used to amplify fidelity reporter plasmids and assayed as described in Example 4. The reporter constructs were amplified using commercially available polymerases in the polymerase buffer (without added magnesium), with thermal cycling times and temperatures according to the manufacturer's recommendations for a 4.5 kb plasmid template. The buffers used, dNTP concentrations, annealing temperatures, and extension times used for each polymerase are listed in Table 5.1. The error rate for each sample relative to PLATINUM SUPERFI™ DNA polymerase was then calculated, and the relative error rate compared to Taq DNA polymerase in KCl buffer was then calculated. Figure 1 shows the relative error rates of these polymerases. [Table 5-1] Example 6 Simultaneous screening of multiple polymerase properties

[0178] Robust polymerase performance across a wide range of applications was selected based on the amplification of amplicons of diverse sizes and GC content from plasmid and genomic DNA templates. Subsequent rounds of screening were performed in buffer M6a: 30 mM Tris pH 8.8, 10 mM (NH4)2SO4, 13.2 mM KCl, 0.4% (v / v) Triton x-100, 0.5 mg / ml BSA, 1.5 mM MgSO4, 4.5% v / v DMSO. PCR conditions for the challenge conditions are shown in Table 6.1. Product yield was determined by capillary electrophoresis as described in Example 3, and fidelity was measured as described in Example 4. Different templates were used in these performance challenge experiments. Table 6.1 shows the reaction conditions, primers, and templates for each of the challenges. "ARX" refers to the human arx gene, "MCYP" refers to microcyp, "KCL" refers to challenge with additional KCl (4.5 mM) using the microcyp template, and "BRCA" refers to the human BRCA2 gene. [Table 6-1] [Table 6-2-1] [Table 6-2-2] [Table 6-2-3] [Table 6-2-4] [Table 6-3] Example 7 Coverage uniformity in next-generation sequencing

[0179] Whole-genome sequencing of microbial genomes was used to test the uniformity of coverage of amplified libraries in next-generation sequencing applications. Genomic DNA from two bacteria, Staphylococcus epidermidis (ATCC 12228, 2.5 MB, GC 32.1%) and Rhodobacter sphaeroides (ATCC 17025, 3.22 MB, GC 68.5%), was used in these experiments. DNA from each organism was sheared to an average fragment length of 400 bp using sonication (Covaris). 100 ng of genomic DNA was then used as input for the KAPA Hyper library preparation workflow using KAPA dual-index adapters according to the manufacturer's instructions (Roche, product KR0961). Ligated library fragments were purified using MagBio HighPrep™ SPRI beads, and 10 ng of input DNA was used as a template for PCR amplification using the purified polymerase of SEQ ID NO: 1082. Eight cycles of PCR amplification were performed in M34b buffer (30 mM Tris pH 8.8, 7 mM (NH4)2SO4, 17 mM KCl, 0.05% (v / v) TWEEN®-20 surfactant, 0.5 mg / ml BSA, 2 mM MgSO4, 8% v / v DMSO, 15 μM ZnSO4). Amplified material was cleaned using HighPrep SPRI beads, normalized, and pooled for multiplex sequencing. Library pools were sequenced using the Miseq Reagent Kit v2 (2 × 250 bp) on a MiSeq instrument (Illumina). Reads were demultiplexed, adapter sequences were trimmed, and then aligned to their respective genomes using CLC Genomics (Qiagen) software. Coverage uniformity was determined using the CLC Genomics read mapping QC method. Figures 2 and 3 show the results of these experiments.

[0180] While the invention has been described with reference to specific embodiments, various modifications may be made and equivalents may be substituted to adapt to a particular situation, material, composition of matter, process, process step or steps, thereby achieving benefits of the invention without departing from the scope of the claims.

[0181] Any and all publications and patent documents cited in this disclosure are incorporated herein by reference for all purposes in the United States 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 intended as an indication that any such document is pertinent prior art, nor does it constitute an admission as to the contents or date thereof.

Claims

1. 1. An engineered DNA polymerase comprising a polypeptide sequence having at least 90% or greater sequence identity to a reference sequence of SEQ ID NO:6, wherein the polypeptide sequence comprises at least substitutions 691S, 495N, 515L, 479P / Q, 567G, 488R, 569G or 685D, or a combination thereof, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:6, and wherein the engineered DNA polymerase produces increased product in a polymerase chain reaction and / or has higher fidelity compared to the wild-type DNA polymerase of SEQ ID NO:

6.

2. 2. The engineered DNA polymerase of claim 1, wherein the polypeptide sequence comprises at least the substitution 515L.

3. 2. The engineered DNA polymerase of claim 1, wherein the polypeptide sequence comprises at least the substitution 691S.

4. 2. The engineered DNA polymerase of claim 1, wherein the polypeptide sequence comprises at least the substitution 495N.

5. 2. The engineered DNA polymerase of claim 1, wherein the polypeptide sequence comprises at least the substitution 479P / Q.

6. 2. The engineered DNA polymerase of claim 1, wherein the polypeptide sequence comprises at least the substitution 567G.

7. 2. The engineered DNA polymerase of claim 1, wherein the polypeptide sequence comprises at least the substitution 488R.

8. 2. The engineered DNA polymerase of claim 1, wherein the polypeptide sequence comprises at least the substitution 569G.

9. 2. The engineered DNA polymerase of claim 1, wherein the polypeptide sequence comprises at least the substitution 685D.

10. 2. The engineered DNA polymerase of claim 1, wherein the polypeptide sequence comprises at least the substitutions 515L, 691S, 479P, 488R, 495N, 567G, 569G, or 685D, or any combination thereof.

11. 11. The engineered DNA polymerase of any one of claims 1 to 10, wherein the polypeptide sequence has at least 91%, 92%, 93%, or 94%, or more, sequence identity to the reference sequence of SEQ ID NO:

6.

12. 11. The engineered DNA polymerase of any one of claims 1 to 10, wherein the polypeptide sequence has at least 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NO:

6.

13. The polypeptide sequence may further comprise amino acid positions 21, 21 / 66 / 247 / 282, 247 / 282 / 575, 282 / 575, 283 / 647 / 702 / 743, 339 / 647 / 661 / 664 / 668 / 702 / 712, 372 / 391 / 702, 391, 391 / 647 / 659 / 661 / 668 / 671 / 712 / 716 ... 59 / 661 / 668 / 671 / 716, 391 / 647 / 659 / 664 / 668 / 702 / 728 / 732, 391 / 647 / 659 / 664 / 671 / 702, 391 / 647 / 661 / 664 / 671 / 702 / 716, 391 / 647 / 671 / 728, 391 / 659 / 702 / 716 / 732 / 737, 391 / 661 / 664 / 668 / 671 / 716 / 737, 391 / 671, 391 / 702 / 712 / 716 / 732 / 743, 647 / 659 / 661 / 664 / 668 / 702, 647 / 659 / 664 / 668 / 702 / 712 / 737, 647 / 659 / 668 / 671 / 716 / 728, 647 / 668, 647 / 668 / 671 / 712, 659 / 702 / 743, 661 13. The engineered DNA polymerase of any one of claims 1 to 12, wherein the amino acid positions are numbered with reference to SEQ ID NO:

6.

14. The polypeptide sequence may further comprise at least one substitution or substitution set 21E, 21E / 66T / 247G / 282R, 247G / 282K / 575L, 282K / 575L, 283M / 647H / 702A / 743A, 339L / 647H / 661T / 664L / 668E / 702A / 712V, 372S / 391E / 702A, 391E, 391E / 647H / 659E / 661T / 668E / 671P / 712V / 716I, 391E / 647H / 659E / 661T / 668E / 671P / 716I, 391E / 647H / 659E / 664L / 668E / 702A / 728A / 732E, 391E / 647 H / 659E / 664L / 671P / 702A, 391E / 647H / 661T / 664L / 671P / 702A / 716I, 391E / 647H / 671P / 728A, 391E / 659E / 702A / 7 16I / 732E / 737R, 391E / 661T / 664L / 668E / 671P / 716I / 737R, 391E / 671P, 391E / 702A / 712V / 716I / 732E / 743A, 647 H / 659E / 661T / 664L / 668E / 702A, 647H / 659E / 664L / 668E / 702A / 712V / 737R, 647H / 659E / 668E / 671P / 716I / 728A, 6 14. The engineered DNA polymerase of claim 13, comprising 47H / 668E, 647H / 668E / 671P / 712V, 659E / 702A / 743A, 661T / 664L / 668E / 671P / 716I, 668E / 702A, 671P / 702A, 671P / 702A / 716I, 702A, or 743A, or a combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:

6.

15. The polypeptide sequence may further comprise amino acid positions 18 / 387, 24 / 719, 43 / 528, 48 / 760, 101 / 646, 108 / 679, 223, 257, 282, 359, 360, 361, 362, 376 / 619, 390, 391, 394, 394 / 399, 420, 421, 478, 502, 506, 514, 521, 528, 583 / 730, 6 13. The engineered DNA polymerase of any one of Claims 1-12, wherein the amino acid positions are numbered with reference to SEQ ID NO:

6.

16. The polypeptide sequence may further comprise at least one substitution or set of substitutions: 18H / 387C, 24M / 719A, 43L / 528S, 48H / 760H, 101S / 646R, 108C / 679S, 223N, 257R, 257W, 282R, 359C, 360R, 360T, 360V, 361G, 361M, 361W, 362R, 376V / 619F, 390A, 390G, 390Q, 391A, 391G, 394G, 394M / 399R, 394N, 394T, 420A, 420G, 420I, 420K ...

16. The engineered DNA polymerase of Claim 15, comprising 20V, 421M, 421Q, 478L, 502A, 506R, 514R, 521P, 521T, 528A, 528S, 583N / 730A, 603R, 619C, 619V, 631G, 646R, 655W, 662C, 666T, 668C, 668L, 702A, 721R, 721T, 738V, 754C, 760F, 760G, 761R, or 761W, or a combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:

6.

17. 13. The engineered DNA polymerase of any one of Claims 1 to 12, wherein the polypeptide sequence further comprises at least one substitution at amino acid position 257, 420, or 521, or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:

6.

18. 18. The engineered DNA polymerase of Claim 17, wherein the polypeptide sequence comprises at least one substitution 257W, 420Q, or 521S, or any combination thereof, and the amino acid positions are numbered with reference to SEQ ID NO:

6.

19. The engineered DNA polymerase is selected from the group consisting of SEQ ID NOs: 24, 26, 28, 128, 174, 240, 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, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 6, 408, 410, 412, 414, 416, 418, 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, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 5 94, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 65 6, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718 , 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780,782, 784, 786, 788, 792, 796, 800, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 8 82, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962 , 964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 986, 988, 990, 992, 994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 103 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, or 1082.

20. 2. The engineered DNA polymerase of claim 1, wherein the engineered DNA polymerase comprises a polypeptide sequence comprising SEQ ID NO: 24, 26, 28, 824, or 1082.

21. 21. The engineered DNA polymerase of claim 19 or 20, wherein the engineered DNA polymerase has an 11 or 12 amino acid deletion at the amino terminus of the polypeptide sequence.

22. 22. The engineered DNA polymerase of any one of claims 1 to 21, which has higher thermostability compared to a wild-type DNA polymerase selected from Pfu DNA polymerase from Pyrococcus furiosus, Group B DNA polymerase from Thermococcus sp. strain 2319x1, and Taq DNA polymerase from Thermus aquaticus.

23. 23. The engineered DNA polymerase of any one of claims 1 to 22, wherein the polymerase is purified.

24. 23. A polynucleotide comprising a polynucleotide sequence encoding at least one engineered DNA polymerase of any one of claims 1 to 22.

25. 25. The polynucleotide of Claim 24, wherein the polynucleotide sequence comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence of SEQ ID NO: 5, 21, 23, 25, 27, or 823, and wherein the polynucleotide encodes an engineered DNA polymerase.

26. 26. The polynucleotide of claim 24 or 25, wherein the polynucleotide sequence is codon optimized.

27. the polynucleotide sequence comprises SEQ ID NO:23, 25, 27, or 823, or the polynucleotide sequence is SEQ ID NO:23, 25, 27, 127, 173, 239, 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, 373, 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, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 5 13, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 5 75, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 63 7, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699 ,701,703,705,707,709,711,713,715,717,719,721,723,725,727,729,731,733,735,737,739,741,743,745,747,749,751,753,755,757,759,761,763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 791, 795, 799, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 86 5, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 799, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 9 49, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 102 25. The polynucleotide of claim 24, comprising: 1) a cDNA fragment containing 10, 2, 3, 4, 5, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073, 1075, 1077, 1079, or 1081.

28. 28. The polynucleotide of any one of claims 24 to 27, wherein the polynucleotide sequence is operably linked to a control sequence.

29. 29. An expression vector comprising at least one polynucleotide according to any one of claims 24 to 28.

30. 30. A host cell comprising at least one expression vector according to claim 29.

31. 31. The host cell of claim 30, wherein the host cell is a bacterial cell, a fungal cell, an insect cell, or a mammalian cell.

32. 32. A method for producing an engineered DNA polymerase polypeptide in a host cell, comprising culturing the host cell of claim 30 or 31 under suitable culture conditions such that at least one engineered DNA polymerase is produced.

33. 33. The method of claim 32, further comprising recovering the at least one engineered DNA polymerase from the culture or host cell.

34. 34. The method of claim 32 or 33, further comprising purifying the at least one engineered DNA polymerase.

35. 24. A composition comprising at least one engineered DNA polymerase according to any one of claims 1 to 23.

36. 24. A method for high-throughput fidelity determination of a DNA polymerase, the method comprising: i) providing at least one DNA polymerase according to any one of claims 1 to 23, a reporter plasmid comprising a gene encoding a first reporter protein, a second reporter protein, and a selectable marker, an amplification system comprising a thermocycler and reagents for performing a polymerase chain reaction, and a purification system, a transformation system comprising competent host cells, and a flow cytometer; ii) exposing the DNA polymerase and the reporter plasmid to the amplification system under conditions such that a reporter construct is amplified by the DNA polymerase to produce a PCR product; iii) circularizing the PCR product to obtain a circularized PCR amplicon; vi) transforming the PCR amplicon using the transformation system to produce a transformed cell; vii) analyzing the transformed cell using the flow cytometer; and viii) determining the fidelity of the DNA polymerase.

37. 37. The method of claim 36, further comprising the step of inducing the transformed cells.

38. (a) the first reporter protein comprises green fluorescent protein or dsRed; (b) the selectable marker comprises chloramphenicol acetyltransferase; (c) said circularization of said PCR amplicon is carried out using at least one ligase, and / or 38. The method of claim 36 or 37, wherein (d) the PCR amplicon is purified.

39. 39. The method of any one of claims 36-38, further comprising determining the fold improvement in polymerase fidelity compared to a reference DNA polymerase selected from Pfu DNA polymerase from Pyrococcus furiosus, Group B DNA polymerase from Thermococcus sp. strain 2319x1, and Taq DNA polymerase from Thermus aquaticus.

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