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WO2025235589A1 - Variants d'adn polymérase - Google Patents

Variants d'adn polymérase

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Publication number
WO2025235589A1
WO2025235589A1 PCT/US2025/028112 US2025028112W WO2025235589A1 WO 2025235589 A1 WO2025235589 A1 WO 2025235589A1 US 2025028112 W US2025028112 W US 2025028112W WO 2025235589 A1 WO2025235589 A1 WO 2025235589A1
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WO
WIPO (PCT)
Prior art keywords
seq
amino acid
dna polymerase
sequence corresponding
reference sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/028112
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English (en)
Inventor
Marina Mikhailovna Bakhtina
Ericka Bermudez
Nikki D. Kruse
Marissa Greene MacAvoy
Vesna Mitchell
Jovana Nazor
David Christensen
Christopher C. Marohnic
Anthony S. Muerhoff
Scotty WALKER
Andrew WOOLERY
Qingbei ZHANG
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Codexis Inc
Original Assignee
Codexis Inc
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Filing date
Publication date
Application filed by Codexis Inc filed Critical Codexis Inc
Publication of WO2025235589A1 publication Critical patent/WO2025235589A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

  • the present disclosure provides engineered DNA polymerase polypeptides and compositions thereof, as well as polynucleotides encoding the engineered DNA polymerase polypeptides.
  • the disclosure also provides methods for use of the recombinant DNA polymerase or compositions thereof for diagnostic, molecular biological tools, and other purposes.
  • DNA polymerases are enzymes that synthesize DNA from deoxyribonucleotides. These enzymes are essential for DNA replication. There are various types of DNA polymerases displaying different properties and found in different types of organisms. Polymerases obtained from thermophilic organisms have found wide-ranging uses in various in vitro methods, including but not limited to, the polymerase chain reaction (PCR), nucleic sequencing, and other diagnostic, molecular biological, and forensic applications. While there are numerous commercially available thermostable DNA polymerases, such as Taq and Pfu DNA polymerases, a need remains in the art for thermostable enzymes with improved properties, such as enhanced sensitivity, processivity, and/or fidelity.
  • the present disclosure provides engineered DNA polymerase polypeptides and compositions thereof, as well as polynucleotides encoding the engineered DNA polymerase polypeptides.
  • the present disclosure also provides methods of using the engineered DNA polymerase polypeptides and compositions thereof for diagnostic and other purposes.
  • the present disclosure provides engineered DNA polymerases, or a functional fragment thereof, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 4, 6, or 224, or to a reference sequence corresponding to SEQ ID NO: 2, 4, 6, or 224, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 4, 6, or 224, or relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 6, or 224.
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or to the reference sequence corresponding to SEQ ID NO: 2 or 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6 or 224, or to the reference sequence corresponding to SEQ ID NO: 6 or 224, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO. of SEQ ID NOs: 6-796, or to a reference sequence corresponding to an even numbered SEQ ID NO. of SEQ ID NOs: 6-796, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution at amino acid position 13, 14, 15, 16, 17, 18, 19, 20, 23, 25, 26, 27, 30, 37, 38, 40, 41, 43, 50, 53, 60, 62, 64, 67, 69, 70, 71, 80, 85, 86, 89, 90, 94, 97, 99, 102, 103, 107, 108, 109, 111, 113, 118, 119, 121, 123, 125, 131, 132, 138, 139, 142, 144, 151, 155, 164, 166, 167, 171, 172, 179, 190, 191,
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution at amino acid position 25, 291, 302, 402, 604, 660, or 672, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution set at amino acid positions 291/402/604/660, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution set at amino acid positions 25/302/672, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the amino acid sequence of the engineered DNA polymerase further comprises at least a substitution at amino acid position 2, 3, 21, 39, 51, 66, 68, 72, 73, 78, 79, 87, 124, 147, 178, 181, 185, 199, 216, 224, 233, 239, 241, 251, 257, 262, 271, 275, 288, 291, 303, 316, 317, 327,
  • amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution or substitution set at amino acid position(s) 291/402/604/660, 147/291/402/475/514/604/660/687/760, 191/402, 147/257/402/660/687/760, 196/402/475/515/604/760, 147/402/604/660/687/760, 147/402/475/660/760, 661, 829, 514/604/660/662/687/760, 37, 67, 86, 80, 333, 26, 121, 171/242, 672, 695, 119, 341, 660, 223, 71, 123, 25, 64, 138, 94, 263, 90, 89, 144, 302, 151, 107, 23, 109, 248, 103, 139, 70, 118, 155, 113, 253, 250, 85, 69, 260, 252, 142, 102, 62,
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution provided in Tables 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution or substitution set provided in Tables 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the amino acid sequence of the engineered DNA polymerase comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence having a substitution or substitution set provided in Table 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2, wherein the amino acid position(s) are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6 or 224, or to the reference sequence corresponding to SEQ ID NO: 6 or 224, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6 or 224, or relative to the reference sequence corresponding to SEQ ID NO: 6 or 224.
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO. of SEQ ID NOs: 212-796, or to a reference sequence corresponding to an even numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6 or 224, or relative to the reference sequence corresponding to SEQ ID NO: 6 or 224.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution at amino acid position 13, 14, 15, 16, 17, 18, 19, 20, 23, 25, 26, 27, 30, 37, 38, 40, 41, 43, 50, 53, 60, 62, 64, 67, 69, 70, 71, 80, 85, 86, 89, 90, 94, 97, 99, 102, 103, 107, 108, 109, 111, 113, 118, 119, 121, 123, 125, 131, 132, 138, 139, 142, 144, 151, 155, 164, 166, 167, 171, 172, 179, 190, 191, 193, 196, 203, 207, 213, 219, 223, 238, 240, 242, 244, 246, 248, 250, 252, 253, 260, 263, 269, 273, 280, 284, 292, 301, 302, 305, 306, 307, 310, 320, 323, 325
  • the amino acid sequence of the engineered DNA polymerase further comprises at least a substitution at amino acid position 2, 3, 21, 39, 51, 66, 68, 72, 73, 78, 79, 87, 124, 147, 178, 181, 185, 199, 216, 224, 233, 239, 241, 251, 257, 262, 271, 275, 288, 291, 303, 316, 317, 327,
  • amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6 or 224, or relative to the reference sequence corresponding to SEQ ID NO: 6 or 224.
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6, or to the reference sequence corresponding to SEQ ID NO: 6, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 844 of an even-numbered SEQ ID NO of SEQ ID NOs: 212-314, or to the reference sequence corresponding to an even-numbered SEQ ID NO of SEQ ID NOs: 212-314, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution or substitution set at amino acid position(s) 554, 580, 623, 521, 688, 672, 25/302/672, 520, 526, 25/121/242/672, 597, 586, 589, 284, 551, 675, 25/86/121/333/672/829, 25/121, 25/86/90/121/829, 581, 399, 576, 121/242/672, 99, 302/672/829, 756, 121/333, 528, 555, 121/333/672/829, 556, 280, 519, 99/263/661, 748/749, 496, 25/86/90/263/672, 754, 121, 121/302/672, 660/829, 223/660/661/829, or 684, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6, or relative to the reference sequence corresponding to
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 224, or to the reference sequence corresponding to SEQ ID NO: 224, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 224, or relative to the reference sequence corresponding to SEQ ID NO: 224.
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 844 of an even-numbered SEQ ID NO of SEQ ID NOs: 316-796, or to the reference sequence corresponding to SEQ ID NO: 316-796, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 224, or relative to the reference sequence corresponding to SEQ ID NO: 224.
  • the amino acid sequence of the engineered DNA polymerase comprises a substitution or substitution set at amino acid position(s) 13, 307, 18, 830, 16, 60, 472, 40, 791, 567, 771, 306, 460, 179, 783, 238, 323, 320, 616, 446, 20, 436, 305, 132, 451, 446, 843, 350, 504, 131, 486, 485, 352, 355, 647, 240, 780, 354, 39, 361, 338, 17, 360, 665, 543, 310, 730, 203, 829, 781, 273, 219, 38, 166, 799, 15, 27, 514, 14, 19, 41, 306/475, 570, 316, 164, 446, 389, 167, 301, 171, 639, 738, 325, 349, 190, 17, 357, 337, 642, 269, 638, 43, 604, 475/506, 53, 760, 661,
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution provided in Table 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, 6, or 224, or to the reference sequence corresponding to SEQ ID NO: 4, 6, or 224.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution or substitution set provided in Table 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2, wherein the amino acid position(s) are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, 6, or 224, or to the reference sequence corresponding to SEQ ID NO: 4, 6, or 224.
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO. of SEQ ID NOs: 6-796, or to a reference sequence corresponding to an even numbered SEQ ID NO. of SEQ ID NOs: 6-796.
  • the amino acid sequence of the engineered DNA polymerase comprises residues 12 to 844 of an even numbered SEQ ID NO. of SEQ ID NOs: 6-796, or comprises an even- numbered SEQ ID NO. of SEQ ID NOs: 6-796.
  • the amino acid sequence of the engineered DNA polymerase comprises residues 12 to 844 of SEQ ID NO: 6, 224, 258, or 656, or comprises SEQ ID NO: 6, 224, 258, or 656.
  • the engineered DNA polymerase is characterized by at least one improved property as compared to a reference DNA polymerase.
  • the improved property of the engineered DNA polymerase is selected from increased activity, increased stability, increased thermostability, increased processivity, increased fidelity, increased product yield, and increased resistance or tolerance to inhibitor, or any combinations thereof, compared to a reference DNA polymerase having a sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 4, 6, or 224, or to the reference DNA polymerase having the sequence corresponding to SEQ ID NO: 2, 4, 6, or 224.
  • the improved property of the engineered DNA polymerase is in comparison to the reference DNA polymerase having the sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or to the reference DNA polymerase having the sequence corresponding to SEQ ID NO: 2.
  • the engineered DNA polymerase is purified. In some embodiments, the engineered DNA polymerase is provided in solution, or is immobilized on a substrate, such as on solid substrates or membranes or particles.
  • the present disclosure provides a recombinant polynucleotide comprising a polynucleotide sequence encoding an engineered DNA polymerases disclosed herein.
  • the recombinant polynucleotide comprises a polynucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 2532 of an odd numbered SEQ ID NO. of SEQ ID NOS: 5-795, or to a reference polynucleotide sequence corresponding an odd numbered SEQ ID NO. of SEQ ID NOs: 5-795, wherein the recombinant polynucleotide encodes a DNA polymerase.
  • the recombinant polynucleotide comprises a polynucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 2532 of SEQ ID NO: 5, 223, 257, or 655, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 5, 223, 257, or 655, wherein the recombinant polynucleotide encodes a DNA polymerase.
  • the polynucleotide sequence of the recombinant polynucleotide is codon- optimized. In some embodiments, the polynucleotide sequence is codon-optimized for expression in a bacterial cell, fungal cell, insect cell, or mammalian cell.
  • the recombinant polynucleotide comprises a polynucleotide sequence comprising residues 34 to 2532 of an odd numbered SEQ ID NO. of SEQ ID NOs: 5-795, or a polynucleotide sequence comprising an odd numbered SEQ ID NO. of SEQ ID NOs: 5-795.
  • the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 2532 of SEQ ID NO: 5, 223, 257, or 655, or a polynucleotide sequence comprising SEQ ID NO: 5, 223, 257, or 655.
  • the recombinant polynucleotide is operably linked to a control sequence.
  • the control sequence comprises a promoter, particularly a heterologous promoter.
  • the present disclosure provides an expression vector comprising a recombinant polynucleotide provided herein encoding an engineered DNA polymerase.
  • the present disclosure also provides a host cell comprising an expression vector provided herein.
  • the present disclosure provides methods of producing an engineered DNA polymerase polypeptide, the method comprising culturing a host cell described herein under suitable culture conditions such that at least one engineered DNA polymerase is produced. In some embodiments, the method further comprises recovering the engineered DNA polymerase from the culture and/or host cells. In some embodiments, the method further comprises purifying the engineered DNA polymerase.
  • compositions comprising at least one engineered DNA polymerase disclosed herein.
  • the composition comprises at least a buffer.
  • the composition further comprises one or more DNA polymerase substrates, for example, nucleotide substrates and oligonucleotide primer substrate.
  • the composition comprises a DNA template, particularly a heterologous DNA template.
  • the present disclosure provides use of the engineered DNA polymerase in methods of preparing a complementary DNA copy of a target DNA, whole or in part.
  • the present disclosure provides a method of preparing a complementary DNA of a target DNA, whole or in part, comprising contacting a target DNA with an engineered DNA polymerase described herein in presence of appropriate substrates under conditions suitable for DNA polymerase mediated production of a DNA complementary to the target DNA.
  • the engineered DNA polymerase is used to detect a target DNA, the method comprising contacting a sample suspected of containing a target DNA with an engineered DNA polymerase of the present disclosure in presence of appropriate substrates under conditions suitable for DNA polymerase mediated production of a DNA complementary to the target DNA, whole or in part, and detecting presence of the complementary DNA.
  • the sample is a biological sample.
  • the sample is an environmental sample.
  • detecting the complementary DNA is by amplifying the complementary DNA, such as by polymerase chain reaction (PCR), including, among others, qPCR (e.g., TaqMan®), Hot-start PCR, touchdown PCR, asymmetric PCR, multiplex PCR, long or long range PCR, assembly PCR, and inverse PCR; or by isothermal amplification reactions, including, among others, LAMP, whole genome amplification (WGA), and multiple displacement amplification.
  • PCR polymerase chain reaction
  • qPCR e.g., TaqMan®
  • Hot-start PCR Hot-start PCR
  • touchdown PCR asymmetric PCR
  • multiplex PCR long or long range PCR
  • assembly PCR assembly PCR
  • inverse PCR inverse PCR
  • isothermal amplification reactions including, among others, LAMP, whole genome amplification (WGA), and multiple displacement amplification.
  • WGA whole genome amplification
  • the engineered DNA polymerase can be used with a reverse transcriptas
  • the present disclosure also provides a kit comprising at least one engineered DNA polymerase disclosed herein.
  • the kit further comprises one or more of a buffer, nucleotide substrate, and/or oligonucleotide primer substrate.
  • the kit comprises a template DNA, particularly a control DNA template.
  • the present disclosure provides engineered DNA polymerase polypeptides and compositions thereof, as well as polynucleotides encoding the engineered DNA polymerase polypeptides.
  • the engineered DNA polymerase polypeptides display, among others, increased thermal stability, increased polymerization activity, increased fidelity, increased high processivity, increased inhibitor tolerance or resistance, increased input DNA template sensitivity, and/or increased product yield, or any combinations thereof, particularly under conditions involving low concentrations of DNA input, high-throughput analysis, and/or sequencing reaction conditions.
  • the engineered DNA polymerases find use in diagnostic and research applications using minute amounts of DNA or RNA from samples, including cell-free DNA and cell -free RNA, circulating tumor DNA, DNA isolated from circulating tumor cells, circulating fetal DNA, synthetic DNA, DNA isolated from forensic samples, DNA isolated from virally infected cells, fine- needle aspirates, or single cells isolated by FACS (fluorescence activated cell sorting), laser-capture microscopy, or microfluidic devices.
  • FACS fluorescence activated cell sorting
  • laser-capture microscopy or microfluidic devices.
  • numeric ranges are inclusive of the numbers defining the range. Thus, every numerical range disclosed herein is intended to encompass every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. It is also intended that every maximum (or minimum) numerical limitation disclosed herein includes every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein. [0056] As used herein, the term “about” means an acceptable error for a particular value. In some instances “about” means within 0.05%, 0.5%, 1.0%, or 2.0%, of a given value range. In some instances, “about” means within 1, 2, 3, or 4 standard deviations of a given value.
  • ‘EC” number refers to the Enzyme Nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB).
  • the IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze.
  • ATCC refers to the American Type Culture Collection whose biorepository collection includes genes and strains.
  • NCBI refers to National Center for Biological Information and the sequence databases provided therein.
  • Protein “Protein,” “polypeptide,” and “peptide” are used interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).
  • amino acids are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single letter codes.
  • alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C), glutamate (Glu or E), glutamine (Gin or Q), glycine (Gly or G), histidine (His or H), isoleucine (He 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 (Vai or V).
  • the amino acid may be in either the L- or D- configuration about a-carbon (Ca).
  • “Ala” designates alanine without specifying the configuration about the a-carbon
  • “D-Ala” and “L-Ala” designate D-alanine and L-alanine, respectively.
  • upper case letters designate amino acids in the L-configuration about the a-carbon
  • lower case letters designate amino acids in the D-configuration about the a-carbon.
  • A designates L-alanine and “a” designates D-alanine.
  • a designates D-alanine.
  • polypeptide sequences are presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequences are presented in the amino (N) to carboxy (C) direction in accordance with common convention.
  • Fusion protein refers to hybrid proteins created through the joining of two or more polynucleotides that originally encode separate proteins.
  • fusion proteins are created by recombinant technology (e.g., molecular biology techniques known in the art).
  • 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 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 poll, T. aquaticus DNA polymerase (Taq)), DNA-dependent RNA polymerases, and reverse transcriptases.
  • the polymerase is a polypeptide or protein containing sufficient amino acids to carry out a desired enzymatic function of the polymerase.
  • the polymerase does not contain all of the amino acids found in the native enzyme, but only those which are sufficient to allow the polymerase to carry out a desired catalytic activity, including but not limited to 5 ’-3’ polymerization, 5’-3’ exonuclease, and 3’-5’ exonuclease activities.
  • the polymerase includes a DNA polymerase in classification EC 2.7.7.7.
  • DNA polymerase activity refers to the ability of an enzyme to synthesize new DNA strands by the incorporation of deoxynucleoside triphosphates or analogs thereof.
  • Polynucleotide is used herein to denote a polymer comprising at least two nucleotides where the nucleotides are either deoxyribonucleotides or ribonucleotides or mixtures of deoxyribonucleotides and ribonucleotides.
  • the abbreviations used for the genetically encoding nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine
  • nucleosides may be either ribonucleosides or 2 ’-deoxyribonucleosides.
  • the nucleosides may be specified as being either ribonucleosides or 2 ’-deoxyribonucleosides on an individual basis or on an aggregate basis.
  • nucleic acid sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5’ to 3’ direction in accordance with common convention, and the phosphates are not indicated.
  • DNA refers to deoxyribonucleic acid.
  • RNA refers to ribonucleic acid.
  • Duplex and “ds” refer to a double-stranded nucleic acid (e.g., DNA or RNA) molecule comprised of two single-stranded polynucleotides that are complementary in their sequence (e.g., A pairs to T or U, C pairs to G), arranged in an antiparallel 5’ to 3’ orientation, and held together by hydrogen bonds between the nucleobases (e.g., adenine [A], guanine [G], cytosine [C], thymine [T] and uridine
  • a polynucleotide or a polypeptide refer to a material or a material corresponding to the natural or native form of the material that has been modified in a manner that would not otherwise exist in nature or is identical thereto but produced or derived from synthetic materials and/or by manipulation using recombinant techniques.
  • Wild-type and “naturally-occurring” refer to the form found in nature.
  • a wildtype polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.
  • Coding sequence refers to that part of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.
  • Percent (%) sequence identity refers to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (z. e. , gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J.
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters “M” (reward score for a pair of matching residues; always >0) and “N” (penalty score for mismatching residues; always ⁇ 0).
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity “X” from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program can use as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89: 10915).
  • Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided.
  • reference sequence refers to a defined sequence used as a basis for a sequence comparison.
  • a reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence.
  • a reference sequence is at least 20 nucleotide 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 the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e.
  • a “reference sequence” can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes in the primary sequence.
  • a reference sequence corresponding to SEQ ID NO: 2, having a leucine at the residue corresponding to X478 refers to a reference sequence in which the corresponding residue at position X478 in SEQ ID NO: 2 (e.g., a methionine), has been changed to leucine.
  • Comparison window refers to a conceptual segment of contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence.
  • the comparison window is at least 15 to 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the comparison window can be longer than 15-20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows.
  • “Corresponding to”, “reference to,” and “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.
  • the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence.
  • 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 matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned.
  • ‘Mutation” refers to the alteration of a nucleic acid sequence.
  • mutations result in changes to the encoded polypeptide sequence (i.e., as compared to the original sequence without the mutation).
  • the mutation comprises a substitution, such that a different amino acid is produced.
  • the mutation comprises an addition, such that an amino acid is added (e.g., insertion) to the original polypeptide sequence.
  • the mutation comprises a deletion, such that an amino acid is deleted from the original polypeptide sequence. Any number of mutations may be present in a given sequence.
  • amino acid difference and “residue difference” refer to a difference in the amino acid residue at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in a reference sequence.
  • the positions of amino acid differences generally are referred to herein as “Xn,” where n refers to the corresponding position in the reference sequence upon which the residue difference is based.
  • a “residue difference at position X478 as compared to SEQ ID NO: 2” refers to a difference of the amino acid residue at the polypeptide position corresponding to position 478 of SEQ ID NO: 2.
  • a “residue difference at position X478 as compared to SEQ ID NO: 2” refers to an amino acid substitution of any residue other than methionine at the position of the polypeptide corresponding to position 478 of SEQ ID NO: 2.
  • the specific amino acid residue difference at a position is indicated as “XnY” where “Xn” specified the corresponding residue and position of the reference polypeptide (as described above), and “Y” is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the different residue than in the reference polypeptide).
  • the present disclosure also provides specific amino acid differences denoted by the conventional notation “AnB”, where A is the single letter identifier of the residue in the reference sequence, “n” is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide.
  • the “substitution” comprises the deletion of an amino acid, and can be denoted by symbol.
  • the phrase “an amino acid residue nB” denotes the presence of the amino residue in the engineered polypeptide at the defined amino acid position, which may or may not be a substitution in context of a reference polypeptide or amino acid sequence.
  • a polypeptide of the present disclosure can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of the specified positions where residue differences are present relative to the reference sequence.
  • the various amino acid residues that can be used are separated by a (e.g., X478L/X478I, X478L/I, or 478L/I).
  • substitution set and “substitution set” refers to a group of amino acid substitutions within a polypeptide sequence.
  • substitution sets comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions.
  • a substitution set refers to the set of amino acid substitutions that is present in any of the variant DNA polymerase polypeptides listed in any of the Tables in the Examples.
  • the individual substitutions are separated by a semicolon e.g., M478L;R516K;E541D) or slash e.g., M478L/R 16K/E541D or 478L/516K/541D).
  • the phrase “mutation set” is used.
  • Constant amino acid substitution refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids.
  • an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain (e.g.
  • an amino acids having aromatic side chains is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basis side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.
  • an amino acids having aromatic side chains is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basis side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another
  • Non-conservative substitution refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affect: (a) the structure of the peptide backbone in the area of the substitution (e.g. , proline for glycine); (b) the charge or hydrophobicity; and/or (c) the bulk of the side chain.
  • 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.
  • ‘Deletion” refers to modification to the polypeptide by removal of one or more amino acids from the reference polypeptide.
  • Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference enzyme while retaining enzymatic activity and/or retaining the improved properties of an engineered polymerase enzyme.
  • Deletions can be directed to the internal portions and/or terminal portions of the polypeptide.
  • the deletion can comprise a continuous segment or can be discontinuous. As noted above, in some embodiments, deletions are indicated by and may be present in a substitution or substitution set.
  • Insertions refers to modification to the polypeptide by addition of one or more amino acids from the reference polypeptide. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.
  • ‘Combinations” or “combination” in the context of any list of mutations (e.g., substitutions), or amino acid positions of the mutations refers to any combination of the listed mutations and/or amino acid positions. In some embodiments, “combinations” or “combination” refers to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more of the listed mutations and/or amino acid positions.
  • ‘Functional fragment” and “biologically active fragment” are used interchangeably herein, to refer to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion(s) and/or internal deletions, 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 present invention) and that retains substantially all of the activity of the full-length polypeptide.
  • isolated polypeptide refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it (e.g., protein, lipids, and polynucleotides).
  • the term embraces polypeptides which have been removed or purified from their naturally-occurring environment or expression system (e.g., host cell or in vitro synthesis).
  • the recombinant DNA polymerase polypeptides may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations.
  • the recombinant DNA polymerase polypeptides provided herein are isolated polypeptides.
  • substantially pure polypeptide or “purified polypeptide” refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight.
  • a substantially pure DNA polymerase composition will comprise 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 by mole or % weight present in the composition.
  • the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules ( ⁇ 500 Daltons), and elemental ion species are not considered macromolecular species.
  • the isolated recombinant DNA polymerase polypeptides are substantially pure polypeptide compositions.
  • “Improved enzyme property” refers to an engineered DNA polymerase polypeptide that exhibits an improvement in any enzyme property as compared to a reference DNA polymerase polypeptide, such as a wild-type DNA polymerase polypeptide (e.g., the DNA polymerase polypeptide sequence of SEQ ID NO: 2) or another engineered DNA polymerase polypeptide.
  • Improved properties include but are not limited to such properties as increased protein expression, increased thermoactivity, 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 solvent stability, increased solubility, increased fidelity, increased processivity, increased inhibitor resistance or tolerance, and altered temperature profile.
  • ‘Increased enzymatic activity” and “enhanced catalytic activity” refer to an improved property of the engineered DNA polymerase polypeptides, which can be represented by an increase in specific activity (e.g., product produced/time/weight protein) and/or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of DNA polymerase) as compared to the reference DNA polymerase enzyme (e.g., wild-type DNA polymerase and/or another engineered DNA polymerase). Exemplary methods to determine enzyme activity are provided in the Examples.
  • hybridization reactions are performed under conditions of lower stringency, followed by washes of varying but higher stringency (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 2001; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, 2003).
  • the term “moderately stringent hybridization” refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, about 85% identity to the target DNA, with greater than about 90% identity to target-polynucleotide.
  • Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5x Denhart's solution, 5xSSPE, 0.2% SDS at 42 °C, followed by washing in 0.2xSSPE, 0.2% SDS, at 42 °C.
  • “High stringency hybridization” refers generally to conditions that are about 10 °C or less from the thermal melting temperature T m as determined under the solution condition for a defined polynucleotide sequence.
  • a high stringency condition refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65 °C (i.e., if a hybrid is not stable in 0.018M NaCl at 65 °C, it will not be stable under high stringency conditions, as contemplated herein).
  • High stringency conditions can be provided, for example, by hybridization in conditions equivalent to 50% formamide, 5x Denhart's solution, 5xSSPE, 0.2% SDS at 42 °C, followed by washing in 0. 1 xSSPE, and 0.1% SDS at 65 °C.
  • Another high stringency condition comprises hybridizing in conditions equivalent to hybridizing in 5X SSC containing 0.1% (w:v) SDS at 65 °C and washing in O. lx 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.
  • Codon optimized refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is more efficiently expressed in that organism.
  • the genetic code is degenerate, in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome.
  • the polynucleotides encoding the DNA polymerase enzymes are codon optimized for optimal production from the host organism selected for expression.
  • Control sequence refers herein to include all components that are necessary or advantageous for the expression of a polynucleotide and/or polypeptide of the present disclosure.
  • Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide.
  • control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoter sequences, signal peptide sequences, initiation sequences, and transcription terminators.
  • the control sequences include a promoter, and transcriptional and translational stop signals.
  • operably linked refers to a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide, and where appropriate the encoded polypeptide of interest.
  • promoter refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence.
  • the promoter sequence contains transcriptional control sequences that mediate the expression of a polynucleotide of interest.
  • the promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • Suitable reaction conditions or “suitable conditions” refers to those conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffers, co-solvents, etc.) under which a DNA polymerase polypeptide of the present disclosure is capable of converting a substrate to the desired product compound.
  • exemplary “suitable reaction conditions” are provided herein (see, the Examples).
  • Process in the context of an enzymatic conversion process refers to the compound or molecule resulting from the action of the DNA polymerase polypeptide on the substrate.
  • “Culturing” refers to the growing of a population of cells, e.g., host cells, under suitable conditions using any suitable medium (e.g., liquid, gel, or solid).
  • suitable medium e.g., liquid, gel, or solid.
  • Vector is a polynucleotide construct for introducing a polynucleotide sequence into a cell.
  • the vector is an expression vector that is operably linked to a suitable control sequence capable of effecting the expression in a suitable host of a polynucleotide of interest, and where appropriate, an encoded polypeptide.
  • an “expression vector” has a promoter sequence operably linked to the DNA sequence (e.g., transgene) to drive expression in a host cell, and in some embodiments, also comprises a transcription terminator sequence.
  • “Expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.
  • ‘Produces” refers to the production of proteins and/or other compounds by cells. It is intended that the term encompass any step involved in the production of polypeptides including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.
  • “Heterologous” or “recombinant” refers to the relationship between two or more nucleic acid or polypeptide sequences (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) that are derived from different sources and are not associated in nature.
  • “Host cell” and “host strain” refer to suitable hosts for expression vectors comprising DNA provided herein (e.g., a polynucleotide sequences encoding at least one DNA polymerase variant).
  • the host cells are prokaryotic or eukaryotic cells that have been transformed or transfected with vectors constructed using recombinant DNA techniques as known in the art.
  • analogue means a polypeptide having more than 70 % sequence identity but less than 100% sequence identity (e.g., more than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) with a reference polypeptide.
  • analogues include non-naturally occurring amino acid residues including, but not limited, to homoarginine, ornithine and norvaline, as well as naturally occurring amino acids.
  • analogues also include one or more D-amino acid residues and non-peptide linkages between two or more amino acid residues.
  • Effective amount means an amount sufficient to produce the desired result.
  • One of general skill in the art may determine what the effective amount by using routine experimentation.
  • Cell-free DNA refers to DNA circulating freely in the bloodstream and is not contained by or associated with cells.
  • cell-free DNA comprises DNA originally derived and released from normal somatic or germ line cells, cancer cells, fetal cells, microbial cells, or viruses.
  • cell-free RNA refers to RNA circulating freely in the bloodstream and is not contained by or associated with cells.
  • cell free RNA comprises RNA originally derived and released from normal somatic or germ line cells, cancer cells, fetal cells, microbial cells, or viruses.
  • Amplification refers to nucleic acid replication. In some embodiments, the term refers to replication of specific template nucleic acid.
  • PCR Polymerase chain reaction
  • PCR refers to methods of generating multiple copies of a nucleic acid template of interest in presence of nucleic acid primers by repeated cycles of denaturation, annealing, and primer extension with a polymerase, such as described in “PCR: Methods and Protocols” Methods in Molecular Biology,” Springer Protocols (2017) and “Quantitative Real-Time PCR: Methods and Protocols,” Methods in Molecular Biology, Springer Protocols (2014), hereby incorporated by reference.
  • the sequence of denaturation, annealing and extension constitute a “cycle,”
  • the steps of denaturing, primer annealing, and polymerase extension can be repeated (i.e., multiple cycles are used), to amplify a nucleic acid template.
  • the process of annealing and extension occur in one step, sometimes referred to as 2-step PCR.
  • the term “PCR” includes many variations of the method, including, among others, qPCR, Hot-start PCR, touchdown PCR, asymmetric PCR, multiplex PCR, long or long range PCR, assembly PCR, and inverse PCR.
  • Target when used in reference to a method employing a DNA polymerase, refers to the region of nucleic acid for preparation of a complementary DNA.
  • the “target” is sorted out from other nucleic acids present in the methods using a DNA polymerase.
  • a “segment” is a region of nucleic acid within the target sequence.
  • Target DNA when used in context of a DNA polymerase refers to the DNA, all or a portion thereof, that is the object for preparation of a complementary DNA copy.
  • the target DNA can be the whole of the DNA sequence or a portion thereof, such as a segment of the DNA sequence.
  • Target RNA refers to the RNA, all or a portion thereof, that is the object for preparation of a complementary DNA copy.
  • the target RNA can be the whole of the RNA sequence or a portion thereof, such as a segment of the RNA sequence.
  • sample template refers to nucleic acid originating from a sample which is analyzed for the presence of target nucleic acid.
  • background template refers to nucleic acid other than sample template that may or may not be present within a sample. Background template may be inadvertently included in the sample, it may result from carryover, or may be due to the presence of nucleic acid contaminants from which the target nucleic acid is purified. For example, in some embodiments, nucleic acids 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 specific nucleic acid samples or templates.
  • Amplifiable nucleic acid is used in reference to nucleic acids which may be amplified by any amplification method, including but not limited to PCR. In most embodiments, amplifiable nucleic acids comprise sample templates.
  • PCR product refers to the resultant compounds obtained after two or more cycles of PCR amplification (or other amplification method, as indicated by the context), typically comprising the steps of denaturation, annealing, and extension.
  • the terms encompass the situation wherein there has been amplification of one or more segments of one or more target sequences.
  • Amplification reagents and “PCR reagents” refer to those reagents (e.g., deoxyribonucleotide triphosphates, buffer, etc.), needed for amplification except for the primers, nucleic acid template, and the amplification enzyme.
  • 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 specific amplification reagents, as any suitable reagents find use in the present invention.
  • Primer refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally or produced synthetically, recombinantly, or by amplification, which is capable of acting as a point of initiation of nucleic acid synthesis, when placed under conditions in which synthesis of a primer extension product that is complementary to a nucleic acid strand is induced (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase, and at a suitable temperature and pH).
  • primers are single-stranded, but in some embodiments, primers are double -stranded.
  • the primers are of sufficient length to prime the synthesis of extension products in the presence of a nucleic acid polymerase. The exact primer length depends upon many factors, as known to those skilled in the art.
  • Probe refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally or produced synthetically, recombinantly, or by amplification, which is capable of hybridizing to another oligonucleotide of interest. Probes find use in the detection, identification, and/or isolation of particular gene sequences of interest. In some embodiments, probes are labeled with a “reporter molecule” (also referred to as a “label”) that aids in the detection of the probe in a suitable detection system (e.g., fluorescent, radioactive, luminescent, enzymatic, and other systems). It is not intended that the present invention be limited to any particular detection system or label.
  • a suitable detection system e.g., fluorescent, radioactive, luminescent, enzymatic, and other systems. It is not intended that the present invention be limited to any particular detection system or label.
  • Primers, deoxyribonucleotides, and deoxyribonucleosides may contain labels. Indeed, it is not intended that the labeled composition of the present invention be limited to any particular component.
  • Illustrative labels include, but are not limited to 32 P, 35 S, and fluorescent molecules (e.g., fluorescent dyes, including but not limited to green fluorescent protein).
  • “Fidelity,” when used in reference to a polymerase is intended to refer to the accuracy of template-directed incorporation of complementary bases in a synthesized DNA strand relative to the template strand. Typically, fidelity is measured based on the frequency of incorporation of incorrect bases in the newly synthesized nucleic acid strand. The incorporation of incorrect bases can result in point mutations, insertions, or deletions. Fidelity can be calculated according to any method known in the art (see, e.g., Tindall and Kunkel, Biochem., 1988, 27:6008-6013; and Barnes, Gene, 1992, 112:29- 35). A polymerase or polymerase variant can exhibit either high fidelity or low fidelity.
  • high fidelity refers to polymerases with a frequency of accurate base incorporation that exceeds a predetermined value.
  • low fidelity refers to polymerases with a frequency of accurate base incorporation that is lower than a predetermined value.
  • the predetermined value is a desired frequency of accurate base incorporation or the fidelity of a known polymerase (i.e., a reference polymerase).
  • Altered fidelity refers to the fidelity of a polymerase variant that differs from the fidelity of the parent polymerase from which the polymerase variant was derived or a reference polymerase.
  • the altered fidelity is higher than the fidelity of the parent or reference polymerase, while in some other embodiments, the altered fidelity is lower than the fidelity of the parent or reference polymerase.
  • Altered fidelity can be determined by assaying the parent and variant polymerases and comparing their activities using any suitable assay known in the art.
  • Processivity refers to the ability of a nucleic acid modifying enzyme, such as a DNA polymerase, to remain bound to the template or substrate and perform multiple modification reactions. Processivity is generally measured by the number of catalytic events that take place per binding event.
  • Altered processivity refers to the processivity of polymerase, or variants thereof, that differ from the processivity of the parent polymerase from which the variant was derived or a reference polymerase. In some embodiments, the altered processivity is higher than the processivity of the parent or reference enzyme, while in some other embodiments, the altered processivity is lower than the processivity of the parent or reference enzyme. Altered processivity can be determined by assaying the parent/reference and variant enzymes and comparing their activities using any suitable assay known in the art.
  • Subject encompasses mammals such as humans, non-human primates, livestock, companion animals, and laboratory animals (e.g., rodents and lagamorphs). It is intended that the term encompass females as well as males.
  • ‘Patient” means any subject that is being assessed for, treated for, or is experiencing disease.
  • a “sample” for reaction with a DNA polymerase is obtained from a patient.
  • sample refers to a material or substance for reaction with a nucleic acid polymerase, for example, such as for detecting presence of a target nucleic acid or preparing a DNA copy of a target nucleic acid for sequencing or generation of cDNA libraries.
  • the sample is a “biological sample,” which refers to sample of biological tissue or fluid.
  • Such samples are typically from humans, but include tissues isolated from non-human primates, or rodents, e.g., mice, and rats, and includes sections of tissues such as biopsy and autopsy samples, frozen sections taken for histological purposes, blood, plasma, serum, sputum, stool, tears, mucus, hair, skin, etc.
  • a “biological sample” also refers to a cell or population of cells or a quantity of tissue or fluid from organisms.
  • the biological sample has been removed from an animal, but the term “biological sample” can also refer to cells or tissue analyzed in vivo, i.e., without removal from the animal, including cell cultures.
  • a “biological sample” will contain cells from the animal or of organisms, but the term can also refer to non-cellular biological material, such as non-cellular fractions of blood, saliva, or urine.
  • tissue biopsy refers to an amount of tissue removed from an animal, preferably a human, for diagnostic analysis. In a patient with cancer, tissue may be removed from a tumor, allowing the analysis of cells within the tumor.
  • tissue biopsy can refer to any type of biopsy, such as needle biopsy, fine needle biopsy, surgical biopsy, etc.
  • a sample can be from environmental sources, by way of example and not limitation, water (e.g., ocean, river, refuse/sewer, etc.), soil, air, vents, or surfaces (e.g., floors, machinery, counters, etc.).
  • water e.g., ocean, river, refuse/sewer, etc.
  • soil e.g., dirt, river, refuse/sewer, etc.
  • air e.g., dirt, dirt, etc.
  • vents e.g., dirt, dirt, etc.
  • surfaces e.g., floors, machinery, counters, etc.
  • the present disclosure provides DNA polymerases, including engineered DNA polymerase polypeptide variants.
  • the DNA polymerase and engineered DNA polymerase polypeptide variants are useful in performing polymerase reactions, including preparing a complementary DNA of a target DNA target/template.
  • the engineered DNA polymerase variants of the present disclosure find use in the efficient creation of complementary DNA from DNA templates, whole or in part, such as in sequencing (e.g., NGS sequencing), amplification (e.g., PCR), and diagnostic methods, such as for detecting a target nucleic acid.
  • sequencing e.g., NGS sequencing
  • amplification e.g., PCR
  • diagnostic methods such as for detecting a target nucleic acid.
  • engineered DNA polymerase variants can be used in solution, as well as in immobilized embodiments.
  • the engineered DNA polymerase can be prepared and used as non-fiision polypeptides or as fusion polypeptides.
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 4, 6, or 224, or to a reference sequence corresponding to SEQ ID NO: 2, 4, 6, or 224, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 4, 6, or 224, or relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 6, or 224.
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or to the reference sequence corresponding to SEQ ID NO: 2 or 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6 or 224, or to the reference sequence corresponding to SEQ ID NO: 6 or 224, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO. of SEQ ID NOs: 6-796, or to the reference sequence corresponding to an even numbered SEQ ID NO. of SEQ ID NOs: 6-796, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution at amino acid position 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 25, 26, 27, 30, 37, 38, 39, 40, 41, 43, 50, 51, 53, 60, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 78, 79, 80, 85, 86, 87, 89, 90, 94, 97, 99, 102, 103, 107, 108, 109, 111, 113, 118, 119, 121, 123, 124, 125, 131, 132, 138, 139, 142, 144, 147, 151, 155, 164, 166, 167, 171, 172, 178, 179, 181, 185, 190, 191, 193, 196, 199, 203, 207, 213, 216,
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution or amino acid residue 2-, 3N, 13G/W, 14R/S/W, 15R/S, 16R, 17G/R/S, 18G/R/T/W, 19L/S, 20G, 21AG/N, 23T, 25L, 26V, 271, 30A, 37F/W, 38W, 39H/F, 40S, 411, 43M, 50S, 51T, 53S, 60C/L, 62C, 64L/Q/S/V. 66R, 67D/F/I/K/M/R, 68E/G/I/K/Q/R/T, 69V/Y.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution at amino acid position 25, 147, 257, 291, 302, 395, 402, 474, 475, 478, 514, 515, 526, 527, 528, 536, 559, 604, 660, 662, 672, 687, 745, or 760, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 4, 6, or 224, or relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 6, or 224.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution or amino acid residue 25L, 147A/C/H/M/Q, 257A/V, 291F/L, 302P, 395G/H/S, 402G/R/Y, 474A/C/G, 475A/E/S/T/V/Y, 478I/L, 514F/I/L/R/V.
  • amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 4, 6, or 224, or relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 6, or 224.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution at amino acid position 13, 14, 15, 16, 17, 18, 19, 20, 23, 25, 26, 27, 30, 37, 38, 40, 41, 43, 50, 53, 60, 62, 64, 67, 69, 70, 71, 80, 85, 86, 89, 90, 94, 97, 99, 102, 103, 107, 108, 109, 111, 113, 118, 119, 121, 123, 125, 131, 132, 138, 139, 142, 144, 151, 155, 164, 166, 167, 171, 172, 179, 190, 191, 193, 196, 203, 207, 213, 219, 223, 238, 240, 242, 244, 246, 248, 250, 252, 253, 260, 263, 269, 273, 280, 284, 292, 301, 302, 305, 306, 307, 310, 320, 323, 325
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution or amino acid residue 13G/W, 14R/S/W, 15R/S, 16R, 17G/R/S, 18G/R/T/W, 19L/S, 20G, 23T, 25L, 26V, 271, 30A, 37F/W, 38W, 39H, 40S, 411, 43M, 50S, 53S, 60C/L, 62C, 64L/Q/S/V, 67D/F/I/K/M/R, 69V/Y. 70C/F, 71 L/V. 80G/I/K/L/M/V. 85M, 86A/G/N/S/V. 89F/S/V/Y.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution or amino acid residue L13G/W, P14R/S/W, L15R/S, F16R, D17G/R/S, L18G/R/T/W, E19L/S, E20G, K23T, V25L, L26V, L27I, G30A, T37F/W, F38W, Y39H, A40S, L4II, L43M, P50S, M53S, S60C/L, L62C, A64L/Q/S/V, E67D/F/I/K/M/R, G69V/Y, Q70C/F, A71L/V, A80G/I/K/L/M/V.
  • H85M E86A/G/N/S/V, E89F/S/V/Y, A90E/I/L/R/S/V, G94K/R/T, P97C, P99S, F102L, P I 03I/V/W.
  • P306S/V. R307E/G/K/L/M/Q/V. A310E/M/W, E3 I 6L/V. V320L, L323T, S325T, E333D, L337I, T349R, S350A/R, V352G, A354D, L355M, D357S, E360S/T, A361G/S/V, V389L, L392F, N399L, L421F, K436I/M/N/S, R446L/T/V, V451A, V460I, L470M, F472G/R/W, E475T, V485I, F486R, H491E, N496A, V504I, F506Y, I514F/L/R, K519L, T520A/L/R/S, G521R, A526R, A528L/P, E531A, V540
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution at amino acid position 25, 291, 302, 402, 604, 660, or 672, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution 25L, 291F, 302P, 402G, 604R, 660L, or 672L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution set at amino acid positions 291/402/604/660, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution set 291F/402G/604R/660L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution set at amino acid positions 25/302/672, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution set 25L/302P/672L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the amino acid sequence of the engineered DNA polymerase further comprises at least a substitution at amino acid position 2, 3, 21, 39, 51, 66, 68, 72, 73, 78, 79, 87, 124, 147, 178, 181, 185, 199, 216, 224, 233, 239, 241, 251, 257, 262, 271, 275, 288, 291, 303, 316, 317, 327, 328, 336, 338, 341, 342, 351, 356, 359, 362, 365, 368, 370, 371, 372, 373, 374, 376, 377,
  • amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the amino acid sequence of the engineered DNA polymerase further comprises at least a substitution or amino acid residue 2-, 3N, 2IAG/N, 39F, 5 IT, 66R, 68E/G/I/K/Q/R/T, 72A, 731, 78N/S/T/V, 79A/M/R/T, 87V, 124A, 147A/H/M/Q, 178L, 181Q/R, 185T, 199P, 216K, 224L, 233H/K/L, 239V, 241E, 251A/I/L, 257V, 262N, 271G, 275A/T, 288R, 291L, 303T, 316G, 317R, 327L/E, 328L/V, 336E, 338V, 341H/T/Y, 342G, 351H, 356I/S, 359G/
  • the amino acid sequence of the engineered DNA polymerase comprises one or more of substitutions at amino acid positions 147, 257, 395, 474, 475, 478, 515, 526, 527, 528, 536, 559, 662, 687, 745, and 760, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the amino acid sequence of the engineered DNA polymerase comprises one or more of substitutions or amino acid residues 147H, 257V, 395S, 474A, 475A, 478L, 515T, 526S, 527A, 528A, 536V, 559R, 662S, 687A, 745T, and 760W, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the amino acid sequence of the engineered DNA polymerase comprises at least the substitutions or amino acid residues 147H, 257V, 395S, 474A, 475 A, 478L, 515T, 526S, 527A, 528A, 536V, 559R, 662S, 687A, 745T, and 760W, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the amino acid sequence of the engineered DNA polymerase comprises one or more of substitutions at amino acid positions 291, 402, 604, and 660, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 4.
  • the amino acid sequence of the engineered DNA polymerase comprises one or more of substitutions or amino acid residues 29 IF, 402G, 604R, and 660L wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 4.
  • the amino acid sequence of the engineered DNA polymerase comprises at least the substitutions or amino acid residues 29 IF, 402G, 604R, and 660L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 4.
  • the amino acid sequence of the engineered DNA polymerase comprises one or more of substitutions at amino acid positions 121, 147, 242, 257, 395, 474, 475, 478, 515, 526,
  • amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the amino acid sequence of the engineered DNA polymerase comprises one or more substitutions or amino acid residues 121Y, 147H, 242R, 257V, 395S, 474A, 475A, 478L, 515T, 526S, 527A, 528A, 536V, 559R, 662S, 672L, 687A, 745T, and 760W, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the amino acid sequence of the engineered DNA polymerase comprises at least the substitutions or amino acid residues 121Y, 147H, 242R, 257V, 395S, 474A, 475A, 478L, 515T, 526S, 527A, 528A, 536V, 559R, 662S, 672L, 687A, 745T, and 760W, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the amino acid sequence of the engineered DNA polymerase comprises one or more of substitutions at amino acid positions 121, 242, 291, 402, 604, 660, and 672, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 4.
  • the amino acid sequence of the engineered DNA polymerase comprises one or more of substitutions or amino acid residues 121Y, 242R, 291F, 402G, 604R, 660L, and 672L wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 4.
  • the amino acid sequence of the engineered DNA polymerase comprises at least the substitutions or amino acid residues 121Y, 242R, 29 IF, 402G, 604R, 660L, and 672L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 4.
  • the amino acid sequence of the engineered DNA polymerase comprises one or more of substitutions at amino acid positions 25, 147, 257, 302, 395, 474, 475, 478, 515, 526, 527,
  • amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the amino acid sequence of the engineered DNA polymerase comprises one or more of substitutions or amino acid residues 25L, 147H, 257V, 302P, 395S, 474A, 475 A, 478L, 515T, 526S, 527A, 528A, 536V, 559R, 662S, 672L, 687A, 745T and 760W, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the amino acid sequence of the engineered DNA polymerase comprises at least the substitutions or amino acid residues 25L, 147H, 257V, 302P, 395S, 474A, 475A, 478L, 515T, 526S, 527A, 528A, 536V, 559R, 662S, 672L, 687A, 745T and 760W, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the amino acid sequence of the engineered DNA polymerase comprises one or more of substitutions at amino acid positions 25, 291, 302, 402, 604, 660, and 672, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 4.
  • the amino acid sequence of the engineered DNA polymerase comprises one or more of substitutions or amino acid residues 25L, 29 IF, 302P, 402G, 604R, 660L, and 672L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 4.
  • the amino acid sequence of the engineered DNA polymerase comprises at least the substitutions or amino acid residues 25L, 29 IF, 302P, 402G, 604R, 660L, and 672L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 4.
  • the amino acid sequence of the engineered DNA polymerase comprises one or more of substitutions at amino acid positions 25, 121, 147, 257, 302, 395, 427, 474, 475, 478, 515, 526, 527, 528, 536, 559, 662, 672, 687, 745, and 760, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the amino acid sequence of the engineered DNA polymerase comprises one or more of substitutions or amino acid residues 25L, 121V, 147H, 257V, 302P, 395S contour 427L, 474A, 475 A, 478L, 515T, 526S, 527A, 528A, 536V, 559R, 662S, 672L, 687A, 745T, and 760W, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the amino acid sequence of the engineered DNA polymerase comprises at least the substitutions or amino acid residues 25L, 121V, 147H, 257V, 302P, 395S contour 427L, 474A, 475A, 478L, 515T, 526S, 527A, 528A, 536V, 559R, 662S, 672L, 687A, 745T, and 760W, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the amino acid sequence of the engineered DNA polymerase comprises one or more of substitutions at amino acid positions 25, 121, 291, 302, 402, 427, 604, 660, and 672, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 4.
  • the amino acid sequence of the engineered DNA polymerase comprises one or more of substitutions or amino acid residues 25L, 121V, 291F, 302P, 402G, 427L, 604R, 660L, and 672L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 4.
  • the amino acid sequence of the engineered DNA polymerase comprises at least the substitutions or amino acid residues 25L, 121V, 291F, 302P, 402G, 427L, 604R, 660L, and 672L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 4.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution or substitution set at amino acid position(s) 291/402/604/660, 147/291/402/475/514/604/660/687/760, 191/402, 147/257/402/660/687/760, 196/402/475/515/604/760, 147/402/604/660/687/760, 147/402/475/660/760, 661, 829, 514/604/660/662/687/760, 37, 67, 86, 80, 333, 26, 121, 171/242, 672, 695, 119, 341, 660, 223, 71, 123, 25, 64, 138, 94, 263, 90, 89, 144, 302, 151, 107, 23, 109, 248, 103, 139, 70, 118, 155, 113, 253, 250, 85, 69, 260, 252, 142, 102, 62,
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution or substitution set, or amino acid residue(s) 291F/402G/604R/660L, 147C/291F/402G/475E/514I/604R/660L/687H/760F, 191G/402G, 147C/257A/402G/660L/687H/760F, 196A/402G/475E/515G/604R/760F, 147C/402G/604R/660L/687H/760F, 147C/402G/475E/660L/760F, 66 IE, 829K, 514I/604R/660L/662P/687H/760F, 37W, 67R, 86V, 67K, 80V, 333D, 801, 26V, 121V, 171P/242R, 829V, 672L, 695V, 119C, 341T, 660L, 223T, 71
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution or substitution set, or amino acid residue(s) L291F/R402G/H604R/V660L, H147C/L291F/R402G/A475E/V514I/H604R/V660L/A687H/W760F, D 191G/R402G, H147C/V257A/R402G/V660L/A687H/W760F, T196A/R402G/A475E/T515G/H604R/W760F, H147C/R402G/H604R/V660L/A687H/W760F, H147C/R402G/A475E/V660L/W760F, D661E, E829K, V514I/H604R/V660L/S662P/A687H/W760F, T37W,
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution set at amino acid positions 147/257/395/474/475/478/515/526/527/528/536/559/662/687/745/760, 257/395/474/478/514/515/526/527/528/536/559/662/745, 147/191/257/291/395/474/475/478/515/526/527/528/536/559/604/660/662/687/745/760, 291/395/474/475/478/515/526/527/528/536/559/604/662/745, 147/196/257/291/395/474/478/526/527/528/536/559/660/662/687/745, 257/291/395/474/475/478/515/526/527/528/536/559/662/745, 257/291/395/474/475/478/515/526/527/528/536/559/662/745, 257/291/395/474/475/4
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution set or amino acid residues
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution set at amino acid positions 147/257/395/474/475/478/515/526/527/528/536/554/559/662/687/745/760, 147/257/395/474/475/478/515/526/527/528/536/559/580/662/687/745/760, 147/257/395/474/475/478/515/526/527/528/536/559/623/662/687/745/760, 147/257/395/474/475/478/515/521/526/527/528/536/559/662/687/745/760, 147/257/395/474/475/478/515/526/527/528/536/559/662/687/688/745/760, 147/257/395/474/475/478/515/526/527/528/536/559/662/687/688/745/760, 147/257/395/474/475/478/515/526/527/528/5
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution set or amino acid residues 147H/257V/395S/474A/475A/478L/515T/526S/527A/528A/536V/554Y/559R/662S/687A/745T/760W, 147H/257V/395S/474A/475A/478L/515T/526S/527A/528A/536V/559R/580S/662S/687A/745T/760W, 147H/257V/395S/474A/475A/478L/515T/526S/527A/528A/536V/559R/623T/662S/687A/745T/760W, 147H/257V/395S/474A/475A/478L/515T/521R/526S/527A/528A/536V/559R/662S/687A/745T/760W, 147H/257V/395S/474A/475A/478L/515T/5
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution set at amino acid positions
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution set or amino acid residues 13G/25L/147H/257V/302P/395S/474A/475A/478L/515T/526S/527A/528A/536V/559R/662S/672L/687 A/745T/760W, 25L/147H/257V/302P/307G/395S/474A/475A/478L/515T/526S/527A/528A/536V/559R/662S/672L/687 A/745T/760W, 18T/25L/147H/257V/302P/395S/474A/475A/478L/515T/526S/527A/528A/536V/559R/662S/672L/687 A/745T/760W, 25L/147H/257V/302P/395S/474A/475A/478L/515T/526S/527A/528A/536V/559R/662S/672L/687 A
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution at an amino acid position provided in Table 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution provided in Table 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution or substitution set at the amino acid positions provided in Table 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2, wherein the amino acid position(s) are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution or substitution set provided in Table 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2, wherein the amino acid position(s) are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the amino acid sequence of the engineered DNA polymerase comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence having a substitution or substitution set provided in Table 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2, wherein the amino acid position(s) are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6 or 224, or to the reference sequence corresponding to SEQ ID NO: 6 or 224, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6 or 224, or relative to the reference sequence corresponding to SEQ ID NO: 6 or 224.
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO. of SEQ ID NOs: 212-796, or to a reference sequence corresponding to an even numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6 or 224, or relative to the reference sequence corresponding to SEQ ID NO: 6 or 224.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution at amino acid position 13, 14, 15, 16, 17, 18, 19, 20, 23, 25, 26, 27, 30, 37, 38, 40, 41, 43, 50, 53, 60, 62, 64, 67, 69, 70, 71, 80, 85, 86, 89, 90, 94, 97, 99, 102, 103, 107, 108, 109, 111, 113, 118, 119, 121, 123, 125, 131, 132, 138, 139, 142, 144, 151, 155, 164, 166, 167, 171, 172, 179, 190, 191, 193, 196, 203, 207, 213, 219, 223, 238, 240, 242, 244, 246, 248, 250, 252, 253, 260, 263, 269, 273, 280, 284, 292, 301, 302, 305, 306, 307, 310, 320, 323, 325
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution or an amino acid residue 13G/W, 14R/S/W, 15R/S, 16R, 17G/R/S, 18G/R/T/W, 19L/S, 20G, 23T, 25L, 26V, 271, 30A, 37F/W, 38W, 39H, 40S, 411, 43M, 50S, 53S, 60C/L, 62C, 64L/Q/S/V. 67D/F/I/K/M/R, 69V/Y. 70C/F, 7 I L/V. 80G/I/K/L/M/V.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution at amino acid position 25, 291, 302, 402, 604, 660, or 672, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6 or 224, or relative to the reference sequence corresponding to SEQ ID NO: 6 or 224.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution or amino acid residue 25L, 29 IF, 302P, 402G, 604R, 660L, or 672L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6 or 224, or relative to the reference sequence corresponding to SEQ ID NO: 6 or 224.
  • the amino acid sequence of the engineered DNA polymerase further comprises at least a substitution at amino acid position 2, 3, 21, 39, 51, 66, 68, 72, 73, 78, 79, 87, 124, 147, 178, 181, 185, 199, 216, 224, 233, 239, 241, 251, 257, 262, 271, 275, 288, 291, 303, 316, 317, 327, 328, 336, 338, 341, 342, 351, 356, 359, 362, 365, 368, 370, 371, 372, 373, 374, 376, 377,
  • the amino acid sequence of the engineered DNA polymerase further comprises at least a substitution or amino acid residue 2-, 3N, 21AG/N, 39F, 5 IT, 66R, 68E/G/I/K/Q/R/T, 72A, 731, 78N/S/T/V, 79A/M/R/T, 87V, 124A, 147A/H/M/Q, 178L, 181Q/R, 185T, 199P, 216K, 224L, 233H/K/L, 239V, 241E, 251A/I/L, 257V, 262N, 271G, 275A/T, 288R, 291L, 303T, 316G, 317R, 327L/E, 328L/V, 336E, 338V, 341H/T/Y, 342G, 351H, 356I/S, 359G/V, 362
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6, or to the reference sequence corresponding to SEQ ID NO: 6, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 844 of an even-numbered SEQ ID NO of SEQ ID NOs: 212-314, or to the reference sequence corresponding to an even-numbered SEQ ID NO of SEQ ID NOs: 212-314, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution or substitution set at amino acid position(s) 554, 580, 623, 521, 688, 672, 25/302/672, 520, 526, 25/121/242/672, 597, 586, 589, 284, 551, 675, 25/86/121/333/672/829, 25/121, 25/86/90/121/829, 581, 399, 576, 121/242/672, 99, 302/672/829, 756, 121/333, 528, 555, 121/333/672/829, 556, 280, 519, 99/263/661, 748/749, 496, 25/86/90/263/672, 754, 121, 121/302/672, 660/829, 223/660/661/829, or 684, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6, or relative to the reference sequence corresponding to
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution or substitution set, or amino acid residue(s) 554Y, 580S, 623T, 521R, 688V, 672L, 25L/G302P/A672L, 520S, 526R, 25L/121Y/242R/672L, 520L, 597A, 586V, 589C, 284L, 551R, 6751, 25L/E86S/L121V/E333D/A672L/E829K, 25L/L121Y, 25L/E86S/A90V/L121M/E829K, 581L, 399L, 576H, 121Y/K242R/A672L, 520R, 586A, 99S, 302P/672L/829V, 756L, 121Y/E333D, 528L, 581V, 555V, 528P, 121V/333
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution or substitution set, or amino acid residue(s) S554Y, T580S, S623T, G521R, R688V, A672L, V25L/G302P/A672L, T520S, S526R, V25L/L121Y/K242R/A672L, T520L, V597A, S586V, D589C, A284L, K551R, T675I, V25L/E86S/L121V/E333D/A672L/E829K, V25L/L121Y, V25L/E86S/A90V/L121M/E829K, A581L, N399L, N576H, L121Y/K242R/A672L, T520R, S586A, P99S, G302P/A672L/E829V,
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 224, or to the reference sequence corresponding to SEQ ID NO: 224, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 224, or relative to the reference sequence corresponding to SEQ ID NO: 224.
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 844 of an even-numbered SEQ ID NO of SEQ ID NOs: 316-796, or to the reference sequence corresponding to SEQ ID NO: 316-796, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 224, or relative to the reference sequence corresponding to SEQ ID NO: 224.
  • the amino acid sequence of the engineered DNA polymerase comprises a substitution or substitution set at amino acid position(s) 13, 307, 18, 830, 16, 60, 472, 40, 791, 567, 771, 306, 460, 179, 783, 238, 323, 320, 616, 446, 20, 436, 305, 132, 451, 446, 843, 350, 504, 131, 486, 485, 352, 355, 647, 240, 780, 354, 39, 361, 338, 17, 360, 665, 543, 310, 730, 203, 829, 781, 273, 219, 38, 166, 799, 15, 27, 514, 14, 19, 41, 306/475, 570, 316, 164, 446, 389, 167, 301, 171, 639, 738, 325, 349, 190, 17, 357, 337, 642, 269, 638, 43, 604, 475/506, 53, 760, 661,
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution or substitution set, or amino acid residue(s) 13G, 307G, 18T, 830A, 16R, 60L, 307V, 472G, 40S, 791V, 567K, 307K, 771V, 306S, 4601, 179L, 7831, 238R, 323T, 320L, 616S, 446L, 20G, 18G, 436S, 305V, 132V, 305C, 451A, 446T, 843D, 350R, 307Q, 5041, 13 IL, 486R, 4851, 843 A, 352G, 355M, 647S, 240K, 780R, 179E, 354D, 472R, 306V, 472W, 39H, 361S, 338V, 436M, 17S, 361V, 360T, 305R, 6651, 360S,
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution or substitution set, or amino acid residue(s) L13G, R307G, L18T, V830A, F16R, S60L, R307V, F472G, A40S, L791V, R567K, R307K, L771V, P306S, V460I, Q179L, L783I, S238R, L323T, V320L, L616S, R446L, E20G, L18G, K436S, K305V, L132V, K305C, V451A, R446T, Q843D, S350R, R307Q, V504I, V131L, F486R, V485I, Q843A, V352G, L355M, K647S, R240K, F780R, Q179E, A354D, F472R, P306V
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution at an amino acid position provided in Table 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, 6, or 224, or to the reference sequence corresponding to SEQ ID NO: 4, 6, or 224.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution provided in Table 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, 6, or 224, or to the reference sequence corresponding to SEQ ID NO: 4, 6, or 224.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution or substitution set at the amino acid positions provided in Table 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2, wherein the amino acid position(s) are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, 6, or 224, or to the reference sequence corresponding to SEQ ID NO: 4, 6, or 224.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution or substitution set provided in Table 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2, wherein the amino acid position(s) are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, 6, or 224, or to the reference sequence corresponding to SEQ ID NO: 4, 6, or 224.
  • the amino acid sequence of the engineered DNA polymerase comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence having a substitution or substitution set provided in Table 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2, wherein the amino acid position(s) are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, 6, or 224, or to the reference sequence corresponding to SEQ ID NO: 4, 6, or 224.
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO. of SEQ ID NOs: 6-796, or to a reference sequence corresponding to an even numbered SEQ ID NO. of SEQ ID NOs: 6-796.
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152
  • the engineered DNA polymerase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO. 6, 224, 258, or 656, or to a reference sequence corresponding to SEQ ID NO: 6, 224, 258, or 656.
  • the engineered DNA polymerase comprises an amino acid sequence comprising residues 12 to 844 of an even numbered SEQ ID NO.
  • the amino acid sequence of the engineered DNA polymerase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and/or deletions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally includes 1, 2, 3, 4, or 5 substitutions, insertions, and/or deletions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally includes 1, 2, 3, 4, or 5 substitutions.
  • the substitutions comprises non-conservative and/or conservative substitutions. In some embodiments, the substitutions comprises conservative substitutions. In some embodiments, the substitutions comprises non-conservative substitutions. In some embodiments, guidance on nonconservative and conservative substitutions are provided by the variants disclosed herein.
  • the engineered DNA polymerase comprises an amino acid sequence comprising residues 12 to 844 of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 172, 174, 176, 178, 180,
  • the amino acid sequence of the engineered DNA polymerase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and/or deletions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally includes 1, 2, 3, 4, or 5 substitutions, insertions, and/or deletions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally includes 1, 2, 3, 4, or 5 substitutions.
  • the substitutions comprises non-conservative and/or conservative substitutions. In some embodiments, the substitutions comprises conservative substitutions. In some embodiments, the substitutions comprises non-conservative substitutions. In some embodiments, guidance on non-conservative and conservative substitutions are provided by the variants disclosed herein.
  • the engineered DNA polymerase comprises an amino acid sequence comprising SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,
  • the amino acid sequence of the engineered DNA polymerase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and/or deletions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally includes 1, 2, 3, 4, or 5 substitutions, insertions, and/or deletions. In some embodiments, the amino acid sequence of the engineered DNA polymerase optionally includes 1, 2, 3, 4, or 5 substitutions. In some embodiments, the substitutions comprises non- conservative and/or conservative substitutions. In some embodiments, the substitutions comprises conservative substitutions. In some embodiments, the substitutions comprises non-conservative substitutions. In some embodiments, guidance on non-conservative and conservative substitutions are provided by the variants disclosed herein.
  • the engineered DNA polymerase comprises an amino acid sequence comprising residues 12 to 844 of SEQ ID NO: 6, 224, 258, or 656, or an amino acid sequence comprising SEQ ID NO: 6, 224, 258, or 656.
  • the amino acid sequence of the engineered DNA polymerase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and/or deletions.
  • the amino acid sequence of the engineered DNA polymerase includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions.
  • the amino acid sequence of the engineered DNA polymerase optionally includes 1, 2, 3, 4, or 5 substitutions, insertions, and/or deletions.
  • the amino acid sequence of the engineered DNA polymerase optionally includes 1, 2, 3, 4, or 5 substitutions.
  • the engineered DNA polymerase includes 1, 2, 3, or 4 substitutions in the amino acid sequence.
  • the engineered DNA polymerase is provided as a fusion protein.
  • the engineered DNA polymerase described herein can be fused to a variety of polypeptide sequences, such as, by way of example and not limitation, polypeptide tags that can be used for detection, purification, and/or conjugation.
  • the fusion protein of the engineered DNA polymerase comprises a glycine-histidine or histidine-tag (His-tag).
  • the fusion protein of the engineered DNA polymerase comprises an epitope tag, such as c-myc, FLAG, V5, or hemagglutinin (HA).
  • the fusion protein of the engineered DNA polymerase comprises a GST, SUMO, Strep, MBP, or GFP tag.
  • the fusion protein of the engineered DNA polymerase comprises a polylysine, for example 2-12 lysine units.
  • the polylysine is for conjugation to a support medium.
  • the fusion is to the amino (N-) terminus of engineered DNA polymerase polypeptide.
  • the fusion is to the carboxy (C-) terminus of the engineered DNA polymerase polypeptide.
  • the engineered DNA polymerase of the present disclosure has DNA polymerase activity. In some embodiments, the engineered DNA polymerase has at least one improved or enhanced property as compared to a reference DNA polymerase.
  • the engineered DNA polymerase has increased activity as compared to the reference DNA polymerase. In some embodiments, the engineered DNA polymerase has at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, or 20- or more increase in activity compared to the reference DNA polymerase.
  • the engineered DNA polymerase has increased stability as compared to the reference DNA polymerase. In some embodiments, the engineered DNA polymerase has increased thermostability as compared to the reference DNA polymerase. [0198] In some embodiments, the engineered DNA polymerase has increased processivity as compared to the reference DNA polymerase. In some embodiments, the engineered DNA polymerase has increased fidelity as compared to the reference DNA polymerase.
  • the engineered DNA polymerase has increased input DNA template sensitivity as compared to the reference DNA polymerase. In some embodiments, the engineered DNA polymerase has increased product yield, for example in a PCR reaction, as compared to the reference DNA polymerase. In some embodiments, the engineered DNA polymerase has increased product yield with about 25, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 copies of input target DNA as compared to the reference DNA polymerase (e.g., as provided in the Examples).
  • the engineered DNA polymerase has increased resistance or tolerance to inhibitor(s), for example guanidine isothiocyanate (GITC), as compared to the reference DNA polymerase.
  • inhibitor(s) for example guanidine isothiocyanate (GITC)
  • the reference DNA polymerase comprises a sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 4, 6, or 224. In some embodiments, the reference DNA polymerase has an amino acid sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or the amino acid sequence corresponding to SEQ ID NO: 2.
  • the engineered DNA polymerase has one or more improved properties selected from i) increased activity, ii) increased stability, iii) increased thermostability, iv) increased processivity, v) increased fidelity, vi) increased input DNA template sensitivity, vii) increased product yield, and viii) increased resistance or tolerance to inhibitor(s), or any combination of i), ii), iii), vi), v), vi), vii), and viii) as compared to a reference DNA polymerase.
  • the reference DNA polymerase has the amino acid sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 4, 6, or 224, or the amino acid sequence corresponding to SEQ ID NO: 2, 4, 6, or 224. In some embodiments, the reference DNA polymerase has the amino acid sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or the amino acid sequence corresponding to SEQ ID NO: 2. In some embodiments, specific improvements in DNA polymerase properties are provided in the Examples.
  • the engineered DNA polymerase polypeptide described herein is an isolated composition. In some embodiments, the engineered DNA polymerase polypeptide is purified, as further discussed herein.
  • the present disclosure further provides functional fragments or biologically active fragments of engineered DNA polymerase polypeptides described herein.
  • a functional fragment or biologically active fragment of the engineered DNA polymerase is provided herewith.
  • a functional fragment or biologically active fragments of an engineered DNA polymerase comprises at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the activity of the DNA polymerase polypeptide from which it was derived (i.e., the parent DNA polymerase).
  • functional fragments or biologically active fragments comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the DNA polymerase.
  • the functional fragment will be truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, less than 50 amino acids, less than 55 amino acids, less than 60 amino acids, less than 65 amino acids, or less than 70 amino acids.
  • the functional fragments or biologically active fragments of the engineered DNA polymerase polypeptide described herein include at least a mutation or mutation set in the amino acid sequence of the engineered DNA polymerase described herein. Accordingly, in some embodiments, the functional fragments or biologically active fragments of the engineered DNA polymerase display the enhanced or improved property associated with the mutation or mutation set in the parent DNA polymerase.
  • the present disclosure provides recombinant polynucleotides encoding the engineered DNA polymerases described herein.
  • the recombinant polynucleotides are operatively linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide construct capable of expressing the engineered DNA polymerase.
  • an expression construct containing at least one heterologous polynucleotide encoding the engineered DNA polymerase polypeptide(s) is introduced into appropriate host cells to express the corresponding DNA polymerase polypeptide(s).
  • the present disclosure provides methods and compositions for the production of each and every possible variation of polynucleotides that could be made that encode the engineered DNA polymerase polypeptides described herein by selecting combinations based on the possible codon choices, and all such polynucleotide variations are to be considered specifically disclosed for any engineered DNA polymerase polypeptide described herein, including the amino acid sequences presented in the Examples (e.g., in Tables 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2) and in the Sequence Listing.
  • the codons are preferably optimized for the chosen host cell for protein production.
  • preferred codons in bacteria are used for expression in bacteria.
  • preferred codons in fungal cells are used for expression in fungal cells.
  • preferred codons in insect cells are used for expression in insect cells.
  • preferred codons in mammalian cells are used for expression in mammalian cells.
  • codon optimized polynucleotides encoding an engineered DNA polymerase polypeptide described herein contain preferred codons at about 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90% of the codon positions in the full length coding region.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 4, 6, or 224, or to a reference sequence corresponding to SEQ ID NO: 2, 4, 6, or 224, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 4, 6, or 224, or relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 6, or 224.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or to the reference sequence corresponding to SEQ ID NO: 2 or 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6 or 224, or to the reference sequence corresponding to SEQ ID NO: 6 or 224, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO. of SEQ ID NOs: 6-796, or to the reference sequence corresponding to an even numbered SEQ ID NO. of SEQ ID NOs: 6-796, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a substitution at amino acid position 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 25, 26, 27, 30, 37, 38, 39, 40, 41, 43, 50, 51, 53, 60, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 78, 79, 80, 85, 86, 87, 89, 90, 94, 97, 99, 102, 103, 107, 108, 109, 111, 113, 118, 119, 121, 123, 124, 125, 131, 132, 138, 139, 142, 144, 147, 151,
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a substitution at amino acid position 25, 147, 257, 291, 302, 395, 402, 474, 475, 478, 514, 515, 526, 527, 528, 536, 559, 604, 660, 662, 672, 687, 745, or 760, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, 4, 6, or 224, or relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 6, or 224.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a substitution at amino acid position 13, 14, 15, 16, 17, 18, 19, 20, 23, 25, 26, 27, 30, 37, 38, 40, 41, 43, 50, 53, 60, 62, 64, 67, 69, 70, 71, 80, 85, 86, 89, 90, 94, 97, 99, 102, 103, 107, 108, 109, 111, 113, 118, 119, 121, 123, 125, 131, 132, 138, 139, 142, 144, 151, 155, 164, 166, 167, 171, 172, 179, 190, 191, 193, 196, 203, 207, 213, 219, 223, 238, 240, 242, 244, 246, 248, 250, 252, 253, 260, 263, 269, 273,
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered DNA polymerase comprising an amino acid sequence comprising at least a substitution at amino acid position 25, 291, 302, 402, 604, 660, or 672, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution set at amino acid positions 291/402/604/660, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the amino acid sequence of the engineered DNA polymerase comprises at least a substitution set at amino acid positions 25/302/672, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence further comprising at least a substitution at amino acid position 2, 3, 21, 39, 51, 66, 68, 72, 73, 78, 79, 87, 124, 147, 178, 181, 185, 199, 216, 224, 233, 239, 241, 251, 257, 262, 271, 275, 288, 291, 303, 316, 317, 327, 328, 336, 338, 341,
  • amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 291/402/604/660, 147/291/402/475/514/604/660/687/760, 191/402, 147/257/402/660/687/760, 196/402/475/515/604/760, 147/402/604/660/687/760, 147/402/475/660/760, 661, 829, 514/604/660/662/687/760, 37, 67, 86, 80, 333, 26, 121, 171/242, 672, 695, 119, 341, 660, 223, 71, 123, 25, 64, 138, 94, 263, 90, 89, 144, 302, 151, 107, 23, 109, 248, 103, 139, 70, 118,
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence comprising at least a substitution or substitution set provided in Tables 4.1 and 4.2, relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered DNA polymerase comprising an amino acid sequence comprising at least a substitution or substitution set provided in Tables 5.1 and 5.2, relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered DNA polymerase comprising an amino acid sequence comprising at least a substitution or substitution set provided in Tables 6.1 and 6.2, relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the amino acid sequence of the engineered DNA polymerase comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence having a substitution or substitution set provided in Table 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2, wherein the amino acid position(s) are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6 or 224, or to the reference sequence corresponding to SEQ ID NO: 6 or 224, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6 or 224, or relative to the reference sequence corresponding to SEQ ID NO: 6 or 224.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO. of SEQ ID NOs: 212-796, or to a reference sequence corresponding to an even numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6 or 224, or relative to the reference sequence corresponding to SEQ ID NO: 6 or 224.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered DNA polymerase comprising an amino acid sequence comprising at least a substitution at amino acid position 13, 14, 15, 16, 17, 18, 19, 20, 23, 25, 26, 27, 30, 37, 38, 40, 41, 43, 50, 53, 60, 62, 64, 67, 69, 70, 71, 80, 85, 86, 89, 90, 94, 97, 99, 102, 103, 107, 108, 109, 111, 113, 118, 119, 121, 123, 125, 131, 132, 138, 139, 142, 144, 151, 155, 164, 166, 167, 171, 172, 179, 190, 191, 193, 196, 203, 207, 213, 219, 223, 238, 240, 242, 244, 246, 248, 250, 252, 253, 260, 263, 269, 273,
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered DNA polymerase comprising an amino acid sequence comprising at least a substitution at amino acid position 25, 291, 302, 402, 604, 660, or 672, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6 or 224, or relative to the reference sequence corresponding to SEQ ID NO: 6 or 224.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered DNA polymerase comprising an amino acid sequence further comprising at least a substitution at amino acid position 2, 3, 21, 39, 51, 66, 68, 72, 73, 78, 79, 87, 124, 147, 178, 181, 185, 199, 216, 224, 233, 239, 241, 251, 257, 262, 271, 275, 288, 291, 303, 316, 317, 327,
  • amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6 or 224, or relative to the reference sequence corresponding to SEQ ID NO: 6 or 224.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6, or to the reference sequence corresponding to SEQ ID NO: 6, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 844 of an even-numbered SEQ ID NO of SEQ ID NOs: 212-314, or to the reference sequence corresponding to an even-numbered SEQ ID NO of SEQ ID NOs: 212-314, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered DNA polymerase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 554, 580, 623, 521, 688, 672, 25/302/672, 520, 526, 25/121/242/672, 597, 586, 589, 284, 551, 675, 25/86/121/333/672/829, 25/121, 25/86/90/121/829, 581, 399, 576, 121/242/672, 99, 302/672/829, 756, 121/333, 528, 555, 121/333/672/829, 556, 280, 519, 99/263/661, 748/749, 496, 25/86/90/263/672, 754, 121, 121/302/672, 660/829, 223/660/661/829, or 684, wherein the amino acid positions are relative
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 224, or to the reference sequence corresponding to SEQ ID NO: 224, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 224, or relative to the reference sequence corresponding to SEQ ID NO: 224.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 844 of an even-numbered SEQ ID NO of SEQ ID NOs: 316-796, or to the reference sequence corresponding to SEQ ID NO: 316-796, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 224, or relative to the reference sequence corresponding to SEQ ID NO: 224.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered DNA polymerase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 13, 307, 18, 830, 16, 60, 472, 40, 791, 567, 771, 306, 460, 179, 783, 238, 323, 320, 616, 446, 20, 436, 305, 132, 451, 446, 843, 350, 504, 131, 486, 485, 352, 355, 647, 240, 780, 354, 39, 361, 338, 17, 360, 665, 543, 310, 730, 203, 829, 781, 273, 219, 38, 166, 799, 15, 27, 514, 14, 19, 41, 306/475, 570, 316, 164, 446, 389, 167, 301, 171, 639, 738, 325, 349, 190, 17,
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered DNA polymerase comprising an amino acid sequence comprising at least a substitution at an amino acid position provided in Table 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, 6, or 224, or relative to the reference sequence corresponding to SEQ ID NO: 4, 6, or 224.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered DNA polymerase comprising an amino acid sequence comprising at least a substitution provided in Table 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, 6, or 224, or relative to the reference sequence corresponding to SEQ ID NO: 4, 6, or 224.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered DNA polymerase comprising an amino acid sequence comprising at least a substitution or substitution set at the amino acid positions provided in Tables 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2, wherein the amino acid position(s) are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, 6, or 224, or relative to the reference sequence corresponding to SEQ ID NO: 4, 6, or 224.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered DNA polymerase comprising an amino acid sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence having a substitution or substitution set provided in Tables 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2, wherein the amino acid position(s) are relative to the reference sequence corresponding to residues 12 to 844 of SEQ ID NO: 4, 6, or 224, or relative to the reference sequence corresponding to SEQ ID NO: 4, 6, or 224.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered DNA polymerase comprising an amino acid sequence having least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence comprising residues 12 to 844 of an engineered DNA polymerase set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2, or to the sequence comprising an engineered DNA polymerase set forth in Tables 4.1, 4.2, 5.1, 5.2, 6.1, and 6.2.
  • the recombinant polynucleotide comprising a polynucleotide sequence encoding the engineered DNA polymerase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to residues 12 to 844 of an even numbered SEQ ID NO. of SEQ ID NOs: 6-796, or to a reference sequence corresponding to an even numbered SEQ ID NO. of SEQ ID NOs: 6-796.
  • the recombinant polynucleotide comprising a polynucleotide sequence encoding the engineered DNA polymerase comprising an amino acid sequence comprising residues 12 to 844 of an even numbered SEQ ID NO. of SEQ ID NO. of SEQ ID NOs: 6-796, or the amino acid sequence comprising an even numbered SEQ ID NO. of SEQ ID NOs: 6-796.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding the engineered DNA polymerase comprising an amino acid sequence comprising residues 12 to 844 of SEQ ID NO: 6, 224, 258, or 656, or an amino acid sequence comprising SEQ ID NO: 6, 224, 258, or 656.
  • the recombinant polynucleotide comprises a polynucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 2532 of an odd numbered SEQ ID NO. of SEQ ID NOS: 5-795, or to a reference polynucleotide sequence corresponding an odd numbered SEQ ID NO. of SEQ ID NOs: 5-795, wherein the recombinant polynucleotide encodes a DNA polymerase.
  • the recombinant polynucleotide comprises a polynucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 2532 of SEQ ID NO: 5, 223, 257, or 655, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 5, 223, 257, or 655, wherein the recombinant polynucleotide encodes a DNA polymerase.
  • the recombinant polynucleotide comprises a polynucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 2532 of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131
  • polynucleotide sequence encodes a DNA polymerase
  • the recombinant polynucleotide comprises a polynucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,
  • the recombinant polynucleotide encoding an engineered DNA polymerase comprises a polynucleotide sequence comprising nucleotide residues 34 to 2532 of an odd numbered SEQ ID NO. of SEQ ID NOs: 5-795, or a polynucleotide sequence comprising an odd numbered SEQ ID NO. of SEQ ID NOs: 5-795.
  • the recombinant polynucleotide encoding an engineered DNA polymerase comprises a polynucleotide sequence comprising nucleotide residues 34 to 2532 of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,

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Abstract

La présente divulgation concerne des polypeptides d'ADN polymérase modifiés et des compositions de ceux-ci, ainsi que des polynucléotides codant pour les polypeptides d'ADN polymérase modifiés. La présente divulgation concerne également des procédés d'utilisation des polypeptides d'ADN polymérase modifiés ou des compositions de ceux-ci pour des applications biologiques diagnostiques et moléculaires.
PCT/US2025/028112 2024-05-08 2025-05-07 Variants d'adn polymérase Pending WO2025235589A1 (fr)

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