Mutant DNA polymerase with improved strand displacement ability

Abstract

The present invention discloses a DNA polymerase having increased 5′-3′ strand displacement activity and significantly reduced 5′-3′ exonuclease and endonuclease activities relative to the corresponding unmodified polymerase. The polymerase can be used in a variety of disclosed primer extension methods. Also disclosed is a related composition, which includes a recombinant nucleic acid, a vector and a host cell, which can be used, for example, for the production of a DNA polymerase.

Description

[0001] Priority information

[0002] This patent application claims priority to U.S. Provisional Patent Application No. 62 / 730,908, filed on September 13, 2018, the contents of which are incorporated herein by reference in their entirety for all purposes. The sequence listing written in the file SequenceListing_31934-US.txt, a 150,900-byte machine format IBM-PC, MS-Windows operating system, created on August 29, 2018, is incorporated herein by reference in its entirety for all purposes. Technical Field

[0003] The present invention provides DNA polymerases with improved activities, including increased 5'-3' strand displacement activity and significantly reduced 5'-3' exonuclease / endonuclease activity, and the use of such polymerases in various applications, including nucleic acid polynucleotide extension and amplification. Background Art

[0004] DNA polymerases are responsible for the replication and maintenance of the genome, a role that is essential for accurately transmitting genetic information from one generation to the next. DNA polymerases act as enzymes responsible for DNA synthesis in cells. They are activated in the presence of metal activators (such as Mg 2+ ), deoxyribonucleoside triphosphates are polymerized in the order indicated by the replicated DNA template or polynucleotide template. In vivo, DNA polymerase participates in a series of DNA synthesis processes, which include DNA replication, DNA repair, recombination and gene amplification. During each DNA synthesis process, the DNA template is replicated once or at most several times to produce identical replicas. On the contrary, in vitro, DNA replication can be repeated many times such as during the polymerase chain reaction (see, e.g., U.S. Patent No. 4,683,202).

[0005] In the preliminary study with polymerase chain reaction (PCR), DNA polymerase is added at the beginning of each round of DNA replication (referring to U.S. Patent number 4,683,202). Subsequently, it is determined that heat-stable DNA polymerase can be obtained from bacteria grown at high temperatures, and it is determined that these enzymes only need to be added once (referring to U.S. Patent number 4,889,818 and U.S. Patent number 4,965,188). Under the high temperature used during PCR, these enzymes will not be irreversibly inactivated. As a result, people can carry out repeated polymerase chain reaction cycles without adding fresh enzyme at the beginning of each synthetic addition process. DNA polymerase, particularly heat-stable polymerase is the key to numerous technologies in recombinant DNA research and the medical diagnosis of disease. Especially for diagnostic applications, the target nucleic acid sequence may be only a small part of the DNA or RNA in question, so it may be difficult to detect the presence of the target nucleic acid sequence when not amplifying.

[0006] The overall folding pattern of DNA polymerases resembles that of the human right hand and contains three unique subdomains: the palm, fingers, and thumb. (See Beese et al., Science 260:352-355, 1993); Patel et al., Biochemistry 34:5351-5363, 1995). Although the structures of the finger and thumb subdomains vary greatly between polymerases of different sizes and cellular functions, the catalytic palm subdomains can overlap. For example, the motif A, which interacts with the incoming dNTP and stabilizes the transition state during chemical catalysis, can overlap, with an average deviation of about one between mammalian pol α and prokaryotic pol I family DNA polymerases. (Wang et al., Cell 89:1087-1099, 1997). Structurally, motif A starts with an antiparallel β-strand containing mainly hydrophobic residues and continues to an α-helix. The primary amino acid sequence of the active site of DNA polymerases is extremely conserved. In the case of motif A, for example, the sequence DYSQIELR (SEQ ID NO:22) is retained in polymerases from organisms separated by millions of years of evolution, including, for example, Thermus aquaticus, Chlamydia trachomatis, and Escherichia coli.

[0007] In addition to being highly conserved, the active site of DNA polymerases has also been shown to be relatively mutagenic, being able to accommodate certain amino acid substitutions without significantly reducing DNA polymerase activity (see, e.g., U.S. Patel et al., U.S. Pat. No. 6,602,695). Such mutant DNA polymerases can provide various selective advantages in, for example, diagnostic and research applications involving nucleic acid synthesis reactions.

[0008] Strand displacement refers to the ability of a polymerase to displace rather than degrade downstream DNA encountered during DNA synthesis. During strand displacement replication, only one DNA strand is replicated at a time. Strand displacement synthesis releases single-stranded DNA, which is then replicated as double-stranded DNA. Many thermostable DNA polymerases exhibit rapid and sustained primer extension DNA synthesis, but strand displacement DNA synthesis efficiency is low. The present disclosure provides a thermostable DNA polymerase with improved strand displacement activity at high temperatures, which results in improved 5'-3' strand displacement activity and significantly reduced 5'-3' exonuclease / endonuclease activity. Summary of the invention

[0009] Provided herein are thermostable DNA polymerases with improved strand displacement activity at high temperatures when other DNA polymerases with significant strand displacement activity, such as Phi 29 DNA polymerase or Bst DNA polymerase, are ineffective. The disclosure describes mutations in the polymerase domain of DNA polymerases, which result in improved 5'-3' strand displacement activity and significantly reduced exonuclease and / or endonuclease activity. In certain embodiments, the DNA polymerase has reduced 5'-3' exonuclease and / or endonuclease activity. Mutations described herein provide unexpected advantages that are unforeseen based on the existing three-dimensional structures of closely related DNA polymerases, and mutant polymerases described herein will also show enhanced strand displacement activity, which is unpredictable even in retrospect.

[0010] On the one hand, provided herein is a DNA polymerase having improved activity, including increased 5'-3' strand displacement activity and significantly reduced 5'-3' exonuclease / endonuclease activity relative to a corresponding unmodified control polymerase, and a method for preparing and using such DNA polymerase. In some embodiments, the improved DNA polymerase has increased 5'-3' strand displacement activity compared to the control DNA polymerase. In some embodiments, the improved DNA polymerase has significantly reduced 5'-3' exonuclease / endonuclease activity compared to the control DNA polymerase. In some embodiments, the control DNA polymerase comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the control DNA polymerase comprises the amino acid sequence of SEQ ID NO:1, except that the control DNA polymerase has a mutation at the amino acid corresponding to position 46 of SEQ ID NO:1. In some embodiments, the control DNA polymerase comprises a glycine (Gly / G) to glutamic acid (Glu / E) mutation at the amino acid corresponding to position 46 of SEQ ID NO:1. The G46E mutation weakens the activity of 5'-3' exonucleases and endonucleases, which is undesirable for a chain displacement enzyme. In some embodiments, the control DNA polymerase comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the control DNA polymerase is encoded by the nucleic acid sequence of SEQ ID NO: 41. In some embodiments, the improved or mutant DNA polymerase described herein is encoded by a nucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 41. In some embodiments, the control DNA polymerase comprises the amino acid sequence of SEQ ID NO: 42.

[0011] In some embodiments, the improved DNA polymerase comprises one or more mutations, or a combination of mutations, at positions corresponding to the following positions of SEQ ID NO: 1 or SEQ ID NO: 40:

[0012] 686, 693, 516, 633, 415, 420, 636, 752, 768, 525, 694, 491, 516, 515, 666, 402, 555, 582, 737, 759, 521, 546, 668, 456, 507, 571, 652, 832, 498, 524, 598, 616, 444, 498, 660, 673, 493, 511, 648, 749 and 635.

[0013] In some embodiments, the improved DNA polymerase comprises one or more mutations, or a combination of mutations, at positions corresponding to the following positions of SEQ ID NO: 1 or SEQ ID NO: 40:

[0014] 686V, A693V, T516I, V633I, Q415H, E420D, E636G, N752S, V768M, R525G, F694S, Q491H, T516S, S515F, T666M, E402V, V555A, N582D, A737T, A759T, L521Q, T546A, N668S, A456T, K507M, T571A, S652F, A832V, D498E, L524V, R598G, M616I, A444T, M660K, Y673N, E493D, T511S, M648I, M749L, and / or Q635K.

[0015] In some embodiments, the improved DNA polymerase comprises an amino acid sequence substantially identical to SEQ ID NO: 1 (e.g., having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity), wherein the amino acid sequence of the DNA polymerase comprises one or more mutations, or a combination of mutations, at positions corresponding to the following positions of SEQ ID NO: 1 or SEQ ID NO: 40:

[0016] 686, 693, 516, 633, 415, 420, 636, 752, 768, 525, 694, 491, 516, 515, 666, 402, 555, 582, 737, 759, 521, 546, 668, 456, 507, 571, 652, 832, 498, 524, 598, 616, 444, 498, 660, 673, 493, 511, 648, 749 and 635.

[0017] In some embodiments, the improved DNA polymerase comprises an amino acid sequence substantially identical to SEQ ID NO: 1 (e.g., having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity), wherein the amino acid sequence of the DNA polymerase comprises one or more mutations, or a combination of mutations, at positions corresponding to the following positions of SEQ ID NO: 1 or SEQ ID NO: 40:

[0018] 660, Y673, E493, T511, M648, M749, and / or Q635.

[0019] In some embodiments, the improved DNA polymerase comprises an amino acid sequence substantially identical to SEQ ID NO: 1 (e.g., having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity), wherein:

[0020] i. the amino acid of the DNA polymerase corresponding to position 686 of SEQ ID NO: 1 is any amino acid except I;

[0021] ii. the amino acid of the DNA polymerase corresponding to position 693 of SEQ ID NO: 1 is any amino acid except A;

[0022] iii. the amino acid of the DNA polymerase corresponding to position 516 of SEQ ID NO: 1 is any amino acid except T;

[0023] iv. the amino acid of the DNA polymerase corresponding to position 633 of SEQ ID NO: 1 is any amino acid except V;

[0024] v. the amino acid of the DNA polymerase corresponding to position 415 of SEQ ID NO: 1 is any amino acid except Q;

[0025] vi. the amino acid of the DNA polymerase corresponding to position 420 of SEQ ID NO: 1 is any amino acid except E;

[0026] vii. The amino acid of the DNA polymerase corresponding to position 636 of SEQ ID NO: 1 is any amino acid except E;

[0027] viii. The amino acid of the DNA polymerase corresponding to position 752 of SEQ ID NO: 1 is any amino acid except N;

[0028] ix. the amino acid of the DNA polymerase corresponding to position 768 of SEQ ID NO: 1 is any amino acid except V;

[0029] x. the amino acid of the DNA polymerase corresponding to position 525 of SEQ ID NO: 1 is any amino acid except R;

[0030] xi. the amino acid of the DNA polymerase corresponding to position 694 of SEQ ID NO: 1 is any amino acid except F;

[0031] xii. The amino acid of the DNA polymerase corresponding to position 491 of SEQ ID NO: 1 is any amino acid other than Q;

[0032] xiii. The amino acid of the DNA polymerase corresponding to position 516 of SEQ ID NO: 1 is any amino acid other than T;

[0033] xiv. The amino acid of the DNA polymerase corresponding to position 515 of SEQ ID NO: 1 is any amino acid except S;

[0034] xv. The amino acid of the DNA polymerase corresponding to position 666 of SEQ ID NO: 1 is any amino acid except T;

[0035] xvi. The amino acid of the DNA polymerase corresponding to position 402 of SEQ ID NO: 1 is any amino acid except E;

[0036] xvii. The amino acid of the DNA polymerase corresponding to position 555 of SEQ ID NO: 1 is any amino acid other than V;

[0037] xviii. The amino acid of the DNA polymerase corresponding to position 582 of SEQ ID NO: 1 is any amino acid except N;

[0038] xix. The amino acid of the DNA polymerase corresponding to position 737 of SEQ ID NO: 1 is any amino acid except A;

[0039] xx. the amino acid of the DNA polymerase corresponding to position 759 of SEQ ID NO: 1 is any amino acid except A;

[0040] xxi. The amino acid of the DNA polymerase corresponding to position 521 of SEQ ID NO: 1 is any amino acid except L;

[0041] xxii. The amino acid of the DNA polymerase corresponding to position 546 of SEQ ID NO: 1 is any amino acid except T;

[0042] xxiii. The amino acid of the DNA polymerase corresponding to position 668 of SEQ ID NO: 1 is any amino acid except N;

[0043] xxiv. The amino acid of the DNA polymerase corresponding to position 456 of SEQ ID NO: 1 is any amino acid except A;

[0044] xxv. The amino acid of the DNA polymerase corresponding to position 507 of SEQ ID NO: 1 is any amino acid except K;

[0045] xxvi. The amino acid of the DNA polymerase corresponding to position 571 of SEQ ID NO: 1 is any amino acid except T;

[0046] xxvii. The amino acid of the DNA polymerase corresponding to position 652 of SEQ ID NO: 1 is any amino acid except S;

[0047] xxviii. The amino acid of the DNA polymerase corresponding to position 832 of SEQ ID NO: 1 is any amino acid except A;

[0048] xxix. The amino acid of the DNA polymerase corresponding to position 498 of SEQ ID NO: 1 is any amino acid except D;

[0049] xxx. The amino acid of the DNA polymerase corresponding to position 524 of SEQ ID NO: 1 is any amino acid except L;

[0050] xxxi. The amino acid of the DNA polymerase corresponding to position 598 of SEQ ID NO: 1 is any amino acid except R;

[0051] xxxii. The amino acid of the DNA polymerase corresponding to position 616 of SEQ ID NO: 1 is any amino acid except M;

[0052] xxxiii. The amino acid of the DNA polymerase corresponding to position 444 of SEQ ID NO: 1 is any amino acid except A;

[0053] xxxiv. The amino acid of the DNA polymerase corresponding to position 660 of SEQ ID NO: 1 is any amino acid except M;

[0054] xxxv. The amino acid of the DNA polymerase corresponding to position 673 of SEQ ID NO: 1 is any amino acid except Y;

[0055] xxxvi. The amino acid of the DNA polymerase corresponding to position 493 of SEQ ID NO: 1 is any amino acid except E;

[0056] xxxvii. The amino acid of the DNA polymerase corresponding to position 511 of SEQ ID NO: 1 is any amino acid except T;

[0057] xxxviii. The amino acid of the DNA polymerase corresponding to position 648 of SEQ ID NO: 1 is any amino acid except M;

[0058] xxxix. The amino acid of the DNA polymerase corresponding to position 749 of SEQ ID NO: 1 is any amino acid except M; and / or

[0059] xl. The amino acid of the DNA polymerase corresponding to position 635 of SEQ ID NO: 1 is any amino acid except Q.

[0060] In some embodiments, the improved DNA polymerase comprises an amino acid sequence substantially identical to SEQ ID NO: 1 (e.g., having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity), wherein:

[0061] i. the amino acid of the DNA polymerase corresponding to position 686 of SEQ ID NO: 1 is V;

[0062] ii. the amino acid of the DNA polymerase corresponding to position 693 of SEQ ID NO: 1 is V;

[0063] iii. the amino acid of the DNA polymerase corresponding to position 516 of SEQ ID NO: 1 is 1;

[0064] iv. the amino acid of the DNA polymerase corresponding to position 633 of SEQ ID NO: 1 is 1;

[0065] v. the amino acid of the DNA polymerase corresponding to position 415 of SEQ ID NO: 1 is H;

[0066] vi. the amino acid of the DNA polymerase corresponding to position 420 of SEQ ID NO: 1 is D;

[0067] vii. The amino acid of the DNA polymerase corresponding to position 636 of SEQ ID NO: 1 is G;

[0068] viii. The amino acid of the DNA polymerase corresponding to position 752 of SEQ ID NO: 1 is S;

[0069] ix. the amino acid of the DNA polymerase corresponding to position 768 of SEQ ID NO: 1 is M;

[0070] x. the amino acid of the DNA polymerase corresponding to position 525 of SEQ ID NO: 1 is G;

[0071] xi. the amino acid of the DNA polymerase corresponding to position 694 of SEQ ID NO: 1 is S;

[0072] xii. The amino acid of the DNA polymerase corresponding to position 491 of SEQ ID NO: 1 is H;

[0073] xiii. The amino acid of the DNA polymerase corresponding to position 516 of SEQ ID NO: 1 is S;

[0074] xiv. The amino acid of the DNA polymerase corresponding to position 515 of SEQ ID NO: 1 is F;

[0075] xv. The amino acid of the DNA polymerase corresponding to position 666 of SEQ ID NO: 1 is M;

[0076] xvi. The amino acid of the DNA polymerase corresponding to position 402 of SEQ ID NO: 1 is V;

[0077] xvii. The amino acid of the DNA polymerase corresponding to position 555 of SEQ ID NO: 1 is A;

[0078] xviii. The amino acid of the DNA polymerase corresponding to position 582 of SEQ ID NO: 1 is D;

[0079] xix. The amino acid of the DNA polymerase corresponding to position 737 of SEQ ID NO: 1 is T;

[0080] xx. the amino acid of the DNA polymerase corresponding to position 759 of SEQ ID NO: 1 is T;

[0081] xxi. The amino acid of the DNA polymerase corresponding to position 521 of SEQ ID NO: 1 is Q;

[0082] xxii. The amino acid of the DNA polymerase corresponding to position 546 of SEQ ID NO: 1 is A;

[0083] xxiii. The amino acid of the DNA polymerase corresponding to position 668 of SEQ ID NO: 1 is S;

[0084] xxiv. The amino acid of the DNA polymerase corresponding to position 456 of SEQ ID NO: 1 is T;

[0085] xxv. The amino acid of the DNA polymerase corresponding to position 507 of SEQ ID NO: 1 is M;

[0086] xxvi. The amino acid of the DNA polymerase corresponding to position 571 of SEQ ID NO: 1 is A;

[0087] xxvii. The amino acid of the DNA polymerase corresponding to position 652 of SEQ ID NO: 1 is F

[0088] xxviii. The amino acid of the DNA polymerase corresponding to position 832 of SEQ ID NO: 1 is V;

[0089] xxix. The amino acid of the DNA polymerase corresponding to position 498 of SEQ ID NO: 1 is E;

[0090] xxx. The amino acid of the DNA polymerase corresponding to position 524 of SEQ ID NO: 1 is V;

[0091] xxxi. The amino acid of the DNA polymerase corresponding to position 598 of SEQ ID NO: 1 is G;

[0092] xxxii. The amino acid of the DNA polymerase corresponding to position 616 of SEQ ID NO: 1 is 1;

[0093] xxxiii. The amino acid of the DNA polymerase corresponding to position 444 of SEQ ID NO: 1 is T;

[0094] xxxiv. The amino acid of the DNA polymerase corresponding to position 660 of SEQ ID NO: 1 is K;

[0095] xxxv. The amino acid of the DNA polymerase corresponding to position 673 of SEQ ID NO: 1 is any amino acid except Y;

[0096] xxxvi. The amino acid of the DNA polymerase corresponding to position 493 of SEQ ID NO: 1 is D;

[0097] xxxvii. The amino acid of the DNA polymerase corresponding to position 511 of SEQ ID NO: 1 is S;

[0098] xxxviii. The amino acid of the DNA polymerase corresponding to position 648 of SEQ ID NO: 1 is 1;

[0099] xxxix. The amino acid of the DNA polymerase corresponding to position 749 of SEQ ID NO: 1 is L; and / or

[0100] xl. The amino acid of the DNA polymerase corresponding to position 635 of SEQ ID NO: 1 is K.

[0101] In some embodiments, the improved DNA polymerase comprises an amino acid sequence substantially identical to SEQ ID NO: 1 (e.g., having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity), wherein the amino acid sequence of the DNA polymerase comprises one or more mutations, or a combination of mutations, at positions corresponding to the following positions of SEQ ID NO: 1 or SEQ ID NO: 40:

[0102] I686V, A693V, T516I, V633I, Q415H, E420D, E636G, N752S, V768M, R525G, F694S, Q491H, T516S, S515F, T666M, E402V, V555A, N582D A737T, A759T, L521Q, T546A, N668S, A456T, K507M, T571A, S652F, S515F, A832V, D498E, L524V, R598G, M616I, A444T, D498E, M660K, Y673N, E493D, T511S, M648I, M749L, and / or Q635K.

[0103] In some embodiments, the improved DNA polymerase comprises one or more mutations, or a combination of mutations, at the following positions of SEQ ID NO: 1 or SEQ ID NO: 40:

[0104] 686, 693, 516, 633, 415, 420, 636, 752, 768, 525, 694, 491, 516, 515, 666, 402, 555, 582, 737, 759, 521, 546, 668, 456, 507, 571, 652, 515, 832, 498, 524, 598, 616, 444, 498, 660, 673, 493, 511, 648, 749 and 635.

[0105] In some embodiments, the improved DNA polymerase comprises one or more mutations, or a combination of mutations, at the following positions of SEQ ID NO: 1 or SEQ ID NO: 40:

[0106] I686V, A693V, T516I, V633I, Q415H, E420D, E636G, N752S, V768M, R525G, F6 94S, Q491H, T516S, S515F, T666M, E402V, V555A, N582D, A737T, A759T, L521 Q, T546A, N668S, A456T, K507M, T571A, S652F, S515F, A832V, D498E, L524V, R598G, M616I, A444T, M660K, Y673N, E493D, T511S, M648I, M749L, and / or Q635K.

[0107] In some embodiments, the amino acid sequence of the DNA polymerase comprises a single mutation and / or a combination of mutations at a position corresponding to a position of SEQ ID NO: 1 or SEQ ID NO: 40:

[0108] i.I686V and A693V;

[0109] ii.T516I and V633I;

[0110] iii.Q415H, E420D, E636G, N752S and V768M;

[0111] iv.R525G and F694S;

[0112] v.Q491H and T516S;

[0113] vi.S515F and T666M;

[0114] vii. E402V, V555A and N582D;

[0115] viii.A737T and A759T;

[0116] ix.L521Q and T546A;

[0117] x.N668S;

[0118] xi.A456T;

[0119] xii.K507M, T571A and S652F;

[0120] xiii.S515F and A832V;

[0121] xiv.D498E, L524V, R598G and M616I;

[0122] xv.A444T, D498E, M660K and Y673N;

[0123] xvi. E493D, T511S, M648I and M749L; and / or

[0124] xvii.Q635K.

[0125] In some embodiments, the mutations are located in the following "hot spots" or segments in the primary amino acid sequence corresponding to amino acids 515-516, 521-525, 633-636, 666-668, and 693-694 of SEQ ID NO: 1 or SEQ ID NO:40.

[0126] In some embodiments, the improved DNA polymerase further comprises an amino acid sequence substantially identical to SEQ ID NO:40 (e.g., having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity), wherein the amino acid of the DNA polymerase corresponding to position 46 of SEQ ID NO:40 is Glu (E).

[0127] In some embodiments, the improved DNA polymerase further comprises an amino acid sequence substantially identical to SEQ ID NO: 1 (e.g., having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity), wherein the amino acid of the DNA polymerase corresponding to position 580 of SEQ ID NO: 1 is any amino acid except D or E. In some embodiments, the amino acid of the DNA polymerase corresponding to position 580 of SEQ ID NO: 1 is any amino acid except D. In some embodiments, the amino acid of the DNA polymerase corresponding to position 580 of SEQ ID NO: 1 is selected from the group consisting of L, G, T, Q, A, S, N, R and K. In some embodiments, the amino acid of the DNA polymerase corresponding to position 580 of SEQ ID NO: 1 is G.

[0128] In some embodiments, the improved DNA polymerase further comprises an amino acid sequence substantially identical to SEQ ID NO: 1 (e.g., having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity), wherein the amino acid of the DNA polymerase corresponding to position 709 of SEQ ID NO: 1 is any amino acid except I. In some embodiments, the amino acid of the DNA polymerase corresponding to position 709 of SEQ ID NO: 1 is selected from the group consisting of K, R, S, G, and A. In some embodiments, the amino acid of the DNA polymerase corresponding to position 709 of SEQ ID NO: 1 is K.

[0129] A variety of DNA polymerases are suitable for mutations according to the present invention. Particularly suitable are thermostable polymerases, which include wild-type or naturally occurring thermostable polymerases from species of various thermophiles, and synthetic thermostable polymerases derived from such wild-type or naturally occurring enzymes by amino acid replacement, insertion or deletion or other modifications. Exemplary unmodified forms of the polymerase include, for example, CS5, CS6 or Z05 DNA polymerases, or functional DNA polymerases with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity. Other unmodified polymerases include, for example, a DNA polymerase from any of the following species of thermophiles (or a functional DNA polymerase having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to such a polymerase): Thermotoga maritima; Thermus aquaticus; Thermus thermophilus; Thermus flavus; Thermus filamentosus; Thermus sp. sps17; Thermus sp. Z05; Thermus napolinum; Thermus africanus; Thermus caldophilus, Deinococcus radiodurans, Bacillus stearothermophilus, or Bacillus thermotenes. Suitable polymerases also include those that have reverse transcriptase (RT) activity and / or are capable of incorporating unconventional nucleotides, such as ribonucleotides or other 2'-modified nucleotides.

[0130] Although thermostable DNA polymerases with effective reverse transcription activity are particularly suitable for performing RT-PCR, especially single enzyme RT-PCR, thermoactive but not thermostable DNA polymerases with effective reverse transcription activity are also suitable for mutations according to the present invention. For example, the properties of increased reverse transcriptase efficiency, mismatch tolerance, extension rate and / or RT inhibitor tolerance can be used for the RT step in RT-PCR, and this step does not need to be performed at a temperature that will inactivate thermoactive but not thermostable DNA polymerases. After the RT step, a thermostable DNA polymerase can be added, or it may have been included in the reaction mixture to perform the PCR amplification step. For example, as described in the examples, before the RT step, the improved DNA polymerase described herein can be combined with a second thermostable DNA polymerase in a buffer suitable for extension and amplification of RNA and DNA templates. Examples of suitable thermostable DNA polymerases are described in U.S. Patent Nos. 4,889,818 and 5,773,258 and 5,677,152 to Gelfand et al., which are expressly incorporated herein by reference in their entirety. In some embodiments, the second thermostable DNA polymerase is DNA polymerase (deoxynucleoside triphosphate: DNA deoxynucleotidyl transferase, EC 2.7.7.7). In some embodiments, the second thermostable DNA polymerase is a reversibly inactivated thermostable polymerase, as described below. In one embodiment, the reversibly inactivated thermostable polymerase is AmpliTaq DNA polymerase (Roche Applied Science, Indianapolis, IN, USA). This second approach will particularly benefit from the use of chemically modified thermostable DNA polymerases (or other HotStart technologies that inactivate thermostable DNA polymerases) so that they are not fully activated during the RT step. An example of a thermoactive but not thermostable DNA polymerase with efficient reverse transcription activity is the DNA polymerase from Carboxydothermus hydrogenoformans (Chy; SEQ ID NO: 39). See, e.g., U.S. Pat. Nos. 6,468,775 and 6,399,320.

[0131] In some embodiments, the DNA polymerase has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence identity to a polymerase selected from the group consisting of:

[0132] (a) Thermus sp. Z05 DNA polymerase (Z05) (SEQ ID NO: 1);

[0133] (b) Thermus aquaticus DNA polymerase (Taq) (SEQ ID NO: 2);

[0134] (c) Thermus filamentosa DNA polymerase (Tfi) (SEQ ID NO: 3);

[0135] (d) Thermus flavivirida DNA polymerase (Tfl) (SEQ ID NO: 4);

[0136] (e) Thermus sps17 DNA polymerase (Sps17) (SEQ ID NO: 5);

[0137] (f) Thermus thermophilus DNA polymerase (Tth) (SEQ ID NO: 6); and

[0138] (g) Thermus caldophilus DNA polymerase (Tca) (SEQ ID NO: 7)

[0139] (h) Carbonophilus hydrogenoformis DNA polymerase (Chy) (SEQ ID NO: 39)

[0140] In some embodiments, the DNA polymerase is a Thermotoga DNA polymerase. For example, in some embodiments, the DNA polymerase has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence identity with a polymerase selected from the group consisting of:

[0141] (a) Thermotoga maritima DNA polymerase (Tma) (SEQ ID NO: 34);

[0142] (b) Thermotoga naples DNA polymerase (Tne) (SEQ ID NO: 35).

[0143] In some embodiments, the DNA polymerase has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence identity with SEQ ID NO: 1, SEQ ID NO: 40, or SEQ ID NO: 42. In some embodiments, the DNA polymerase is a Z05 DNA polymerase, further comprising a substitution at position 580, and the amino acid at position 580 is any amino acid except D or E. In some embodiments, the DNA polymerase is a Z05 DNA polymerase, and the amino acid at position 580 may be any amino acid except D. In some embodiments, the DNA polymerase is a Z05 DNA polymerase, and the amino acid at position 580 is selected from the group consisting of L, G, T, Q, A, S, N, R, and K. In some embodiments, the DNA polymerase is a Z05 DNA polymerase, and the amino acid at position 580 is G. In some embodiments, the DNA polymerase is a Z05 DNA polymerase further comprising a substitution at position 709, and the amino acid at position 709 is any amino acid except I. In some embodiments, the DNA polymerase is a Z05 DNA polymerase, and the amino acid at position 709 is selected from the group consisting of K, R, S, G, and A. In some embodiments, the DNA polymerase is a Z05 DNA polymerase, and the amino acid at position 709 is K.

[0144] In some embodiments, the control DNA polymerase is Z05, Z05 D580G, or Z05D580G I709K polymerase. In some embodiments, the control DNA polymerase is C21 or C21G46E polymerase (SEQ ID NO: 40). In some embodiments, the control DNA polymerase comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 40, or SEQ ID NO: 42.

[0145] Mutant or improved polymerases may include other non-substitution modifications. One such modification is a heat-reversible covalent modification that inactivates the enzyme, but when incubated at high temperatures (such as temperatures typically used for polynucleotide extension), it reverses to activate the enzyme. Exemplary reagents for such heat-reversible modifications are described in U.S. Patent Nos. 5,773,258 and 5,677,152, which are expressly incorporated herein by reference in their entirety.

[0146] In various other aspects, the disclosure provides recombinant nucleic acids encoding mutant or improved DNA polymerases as described herein, vectors comprising recombinant nucleic acids, and host cells transformed using vectors. In certain embodiments, the vector is an expression vector. Host cells comprising such expression vectors can be used in the methods of the present invention to produce mutant or improved polymerases by culturing host cells under conditions suitable for expressing recombinant nucleic acids. Polysaccharases of the present invention may be included in reaction mixtures and / or test kits. Embodiments of recombinant nucleic acids, host cells, vectors, expression vectors, reaction mixtures, and test kits are as described above and as described herein.

[0147] On the other hand, a method for carrying out polynucleotide extension is provided. The method generally includes contacting the DNA polymerase with the strand displacement activity with the 5'-3' exonuclease / endonuclease activity with the increase as described herein and the significantly reduced nucleotide triphosphate, thereby producing the primer extended. The polynucleotide template can be, for example, RNA or DNA template. The nucleotide triphosphate can include unconventional nucleotides, such as ribonucleotides and / or labeled nucleotides. Further, primers or templates can include one or more nucleotide analogs. In some variants, the polynucleotide extension method is a method for polynucleotide amplification, which includes contacting mutant or improved DNA polymerase with primer pairs, polynucleotide templates and nucleotide triphosphate under conditions suitable for polynucleotide amplification. The polynucleotide extension reaction can be, for example, PCR, isothermal extension or sequencing (for example, 454 sequencing reactions). The polynucleotide template can be from any type of biological sample.

[0148] In some embodiments, the primer extension method comprises a strand displacement reaction, a polymerase chain reaction (PCR), isothermal amplification, or an amplification reaction selected from loop-mediated amplification (LAMP), cross primer amplification (CSA) and polymerase chain displacement reaction (PCDR). In some embodiments, conditions suitable for primer extension include high temperature.

[0149] Optionally, the primer extension reaction may include an actual or potential inhibitor of a reference or unmodified polymerase. The inhibitor may inhibit the nucleic acid extension rate and / or strand displacement activity of a reference or unmodified (control) polymerase. In some embodiments, the inhibitor is hemoglobin or a degradation product thereof. For example, in some embodiments, the hemoglobin degradation product is a heme decomposition product such as heme, hematoporphyrin, or bilirubin. In some embodiments, the inhibitor is an iron chelator or a purple pigment. In some embodiments, the inhibitor is heparin or melanin. In certain embodiments, the inhibitor is an intercalating dye. In some embodiments, the intercalating dye is [2-[N-bis-(3-dimethylaminopropyl)-amino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylene]-1-phenyl-quinolinium]+ In some embodiments, the intercalating dye is [2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylene]-1-phenyl-quinolinium] + In some embodiments, the intercalating dye is not [2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylene]-1-phenyl-quinolinium] + In some embodiments, conditions suitable for extension include Mg ++ In some embodiments, conditions suitable for extension include Mn ++ .

[0150] The present disclosure also provides a kit that can be used for such polynucleotide extension methods. Typically, the kit includes at least one container that provides a mutant or improved DNA polymerase as described herein. In certain embodiments, the kit further includes one or more other containers that provide one or more other reagents. For example, in a specific variant, one or more other containers provide nucleoside triphosphates; a buffer suitable for polynucleotide extension; and / or one or more primers or probe polynucleotides that can hybridize with a predetermined polynucleotide template under polynucleotide extension conditions. The polynucleotide template can be from any type of biological sample.

[0151] Further provided is a reaction mixture comprising a polymerase described herein.The reaction mixture can also include template nucleic acid (DNA and / or RNA), one or more primers or probe polynucleotides, nucleoside triphosphates (including, for example, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, labeled nucleoside triphosphates, unconventional nucleoside triphosphates), buffers, salts, labels (for example, fluorophores). In certain embodiments, the reaction mixture includes an iron chelator or a purple dye. In certain embodiments, the reaction mixture includes hemoglobin or hemoglobin degradation products. For example, in certain embodiments, hemoglobin degradation products include heme decomposition products, such as heme, hemoglobin, hematoporphyrin and bilirubin. In other embodiments, the reaction mixture includes heparin or its salt. Optionally, the reaction mixture includes an intercalating dye (including but not limited to those described above or elsewhere herein). In certain embodiments, the reaction mixture includes a template nucleic acid separated from blood. In other embodiments, the template nucleic acid is RNA, and the reaction mixture includes heparin or its salt.

[0152] In some embodiments, the reaction mixture comprises two or more polymerases. For example, in some embodiments, the reaction mixture comprises an improved DNA polymerase with increased strand displacement activity as described herein, and another polymerase with increased reverse transcription efficiency (e.g., increased activity of extending RNA template). In one embodiment, the reaction mixture comprises a blend of an improved DNA polymerase with increased strand displacement activity as described herein and a second thermostable DNA-dependent polymerase. The second thermostable DNA-dependent polymerase can be a reversibly modified polymerase as described above, so that the enzyme is inactivated at a temperature suitable for the reverse transcription step, and is activated for a period of up to about 12 minutes under suitable conditions, such as at a high temperature of about 90°C to 100°C. Suitable conditions for activating a reversibly inactivated thermostable polymerase are provided, for example, in a hot start PCR reaction. Examples of suitable second thermostable DNA-dependent polymerases are described in U.S. Patents 5,773, 258, and 5,677,152.

[0153] Further embodiments are described herein.

[0154] definition

[0155] Unless otherwise indicated, all scientific and technical terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs. Although substantially any method and material similar to the method and material described herein can be used in the practice or testing of the present invention, only exemplary methods and materials are described. For purposes of the present invention, the following terms are defined as follows.

[0156] The terms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0157] "Amino acid" refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. As used herein, the term "amino acid" includes the following twenty natural or genetically encoded α-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). In the event that "X" residues are not defined, they should be defined as "any amino acid". The structures of these twenty natural amino acids are described, for example, in Stryer et al., Biochemistry, 5th edition, Freeman and Company (2002), which is incorporated by reference. Other amino acids such as selenocysteine ​​and pyrrolysine may also be genetically encoded (Stadtman (1996) "Selenocysteine," Annu Rev Biochem. 65:83-100 and Ibba et al. (2002) “Genetic code: introducing pyrrolysine,” Curr Biol. 12(13):R464-R466, all of which are incorporated by reference). The term "amino acid" also includes unnatural amino acids, modified amino acids (e.g., with modified side chains and / or backbones), and amino acid analogs. See, e.g., Zhang et al. (2004) "Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells," Proc.Natl.Acad.Sci.USA 101(24):8882-8887, Anderson et al. (2004) "An expanded genetic code with a functional quadrupletcodon" Proc.Natl.Acad.Sci.USA 101(20):7566-7571, Ikeda et al. (2003) "Synthesis of a novel histidine analogue and its efficient incorporation into a protein invivo," Protein Eng.Des.Sel. 16(9):699-706, Chin et al. (2003) “An Expanded Eukaryotic Genetic Code,” Science 301(5635):964-967, James et al. (2001) "Kinetic characterization of ribonuclease S mutants containing photoisomerizablephenylazophenylalanine residues," Protein Eng.Des.Sel.14(12):983-991, Kohrer et al. (2001) “Import ofamber and ochre suppressor tRNAs into mammalian cells: A general approach to site-specific insertion of amino acid analogues into proteins,” Proc.Natl.Acad.Sci.USA 98(25):14310-14315, Bacher et al. (2001) "Selection and Characterization of Escherichia coli Variants Capable ofGrowth on an Otherwise Toxic Tryptophan Analogue," J. Bacteriol. 183(18):5414-5425, Hamano-Takaku et al. (2000) "A Mutant Escherichia coli Tyrosyl-tRNASynthetase Utilizes the Unnatural Amino Acid Azatyrosine More Efficientlythan Tyrosine," J.Biol.Chem. 275(51):40324-40328, and Budisa et al. (2001) “Proteins with {beta}-(thienopyrrolyl)alanines as alternative chromophores and pharmaceutically active amino acids,” Protein Sci. 10(7):1281-1292, each of which is incorporated by reference.

[0158] For further illustration, amino acids are generally organic acids that include substituted or unsubstituted amino, substituted or unsubstituted carboxyl, and one or more side chains or groups, or analogs of any of these groups. Exemplary side chains include, for example, sulfhydryl, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynyl, ether, borate, borate, phospho, phosphino, phosphine, heterocycle, enone, imine, aldehyde, ester, thioacid, hydroxylamine, or any combination of these groups. Other representative amino acids include, but are not limited to, amino acids comprising photoactivated cross-linkers, metal binding amino acids, spin-labeled amino acids, fluorescent amino acids, amino acids comprising metals, amino acids with novel functional groups, amino acids that covalently or non-covalently interact with other molecules, photosensitive photocaged and / or photoisomerizable amino acids, radioactive amino acids, amino acids comprising biotin or biotin analogs, glycosylated amino acids, other carbohydrate-modified amino acids, amino acids comprising polyethylene glycol or polyethers, heavy atom substituted amino acids, chemically cleavable and / or photocleavable amino acids, amino acids comprising carbon-linked sugars, redox-active amino acids, amino thioacid-containing amino acids, and amino acids comprising one or more toxic moieties.

[0159] The term "biological sample" covers a variety of sample types obtained from an organism and can be used in a diagnostic or monitoring assay. The term covers urine, urine sediment, blood, saliva and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom and their progeny. The term covers samples that have been processed in any way after purchase, such as by using reagents, solubilization, precipitation or enrichment of certain components. The term covers clinical samples and also includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, biological fluids and tissue samples.

[0160] The term "mutant" in the context of the DNA polymerase of the present invention means a polypeptide, typically a recombinant polypeptide, comprising one or more amino acid substitutions relative to the corresponding functional DNA polymerase.

[0161] The term "unmodified form" in the context of a mutant polymerase is a term used herein to define a mutant DNA polymerase of the invention: the term "unmodified form" refers to a functional DNA polymerase having a mutant polymerase amino acid sequence other than one or more amino acid positions designated as characterizing a mutant polymerase. Thus, reference to a mutant DNA polymerase in terms of (a) its unmodified form and (b) one or more specific amino acid substitutions means that, except for the specific amino acid substitutions, the mutant polymerase otherwise has the same amino acid sequence as the unmodified form in a specific motif. "Unmodified polymerases" (and thus modified forms with increased reverse transcriptase efficiency, mismatch tolerance, extension rate, and / or RT and polymerase inhibitor tolerance) may contain other mutations to provide desired functionality, such as improved dideoxyribonucleotides, ribonucleotides, ribonucleotide analogs, incorporation of dye-labeled nucleotides, regulation of 5'-nuclease activity, regulation of 3'-nuclease (or proofreading) activity, etc. Thus, in carrying out the invention described herein, an unmodified form of DNA polymerase is predetermined. The DNA polymerase of unmodified form can be, for example, wild-type and / or naturally occurring DNA polymerase, or the DNA polymerase that has been intentionally modified. The polymerase of unmodified form is preferably a thermostable DNA polymerase, such as DNA polymerase from various thermophilic bacteria and functional variants thereof, which have basic sequence identity with wild-type or naturally occurring thermostable polymerase. Such variants can include, for example, chimeric DNA polymerases, such as chimeric DNA polymerases described in U.S. Patent Nos. 6,228,628 and 7,148,049, which are incorporated herein by reference in their entirety. In certain embodiments, the polymerase of unmodified form has reverse transcriptase (RT) activity.

[0162] The term "thermostable polymerase" refers to an enzyme that is stable to heat, is heat-resistant, retains sufficient activity to achieve subsequent polynucleotide extension reactions after the time required to achieve double-stranded nucleic acid denaturation at high temperature, and does not irreversibly denature (inactivate). The heating conditions required for nucleic acid denaturation are well known in the art and are illustrated in, for example, U.S. Patent Nos. 4,683,202, 4,683,195 and 4,965,188, which are incorporated herein by reference. As used herein, thermostable polymerases are suitable for temperature cycling reactions, such as polymerase chain reaction ("PCR"). Irreversible denaturation for the purposes of this article refers to permanent and complete loss of enzyme activity. For thermostable polymerases, enzyme activity refers to the catalytic action of combining nucleotides in an appropriate manner to form polynucleotide extension products complementary to the template nucleic acid chain. Thermostable DNA polymerases from thermophiles include, for example, DNA polymerases from Thermotoga maritima, Thermus aquaticus, Thermus thermophilus, Thermus flavivirus, Thermus filamentosus, Thermus sp. sp17, Thermus sp. Z05, Thermus caldophilus, Bacillus thermotenicus, Thermotoga naples, and Thermus africanus.

[0163] The term "thermoactive" refers to an enzyme that retains catalytic properties at temperatures typically used for reverse transcription or annealing / extension steps in RT-PCR and / or PCR reactions (i.e., 45-80°C). A thermostable enzyme is one that does not irreversibly inactivate or denature when subjected to the elevated temperatures required for nucleic acid denaturation. A thermoactive enzyme may or may not be thermostable. A thermoactive DNA polymerase may be DNA or RNA dependent on thermophilic or mesophilic species, including but not limited to Escherichia coli, Moloney murine leukemia virus, and avian myeloblastosis virus.

[0164] As used herein, a "chimeric" protein refers to a protein whose amino acid sequence represents a fusion product of subsequences of amino acid sequences from at least two different proteins. Chimeric proteins are generally not produced by direct manipulation of the amino acid sequence, but are expressed from a "chimeric" gene encoding a chimeric amino acid sequence. In certain embodiments, for example, the unmodified form of the mutant DNA polymerase of the present invention is a chimeric protein composed of an amino terminal (N-terminal) region derived from a Thermus species DNA polymerase and a carboxyl terminal (C-terminal) region derived from a Tma DNA polymerase. The N-terminal region refers to a region extending from the N-terminus (amino acid position 1) to an internal amino acid. Similarly, the C-terminal region refers to a region extending from an internal amino acid to the C-terminus.

[0165] The term "aptamer" refers to a single-stranded DNA that recognizes and binds to a DNA polymerase and effectively inhibits polymerase activity, as described in U.S. Patent No. 5,693,502, which is hereby expressly incorporated herein by reference in its entirety. The purposes of aptamers and dUTP / UNG in RT-PCR are, for example, in Smith, ES et al., (Amplification of RNA: High-temperature Reverse Transcription and DNA Amplification with a Magnesium-activated Thermostable DNA Polymerase, in PCR Primer: A Laboratory Manual, 2nd edition, Dieffenbach, CW and Dveksler, GS, eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 211-219, (2003)) and are also mentioned in the work for discussion.

[0166] In the context of mutant DNA polymerases, "correspondence" to another sequence (e.g., region, fragment, nucleotide or amino acid position, etc.) is based on a numbering convention according to the position number of the nucleotide or amino acid, and then the sequences are aligned in a manner that maximizes the percentage of sequence identity. The amino acid "corresponding to position [X] of [specific sequence]" refers to the amino acid in the target polypeptide that is aligned with the equivalent amino acid of the specified sequence. Typically, as described herein, an alignment algorithm, such as BLAST as described below, can be used to determine the amino acid corresponding to the polymerase position. Because not all positions within a given "corresponding region" need to be the same, non-matching positions within the corresponding region may be considered "corresponding positions." Therefore, as used herein, the "amino acid position corresponding to amino acid position [X]" of a specific DNA polymerase refers to equivalent positions based on alignments in other DNA polymerases and structural homologs and families. In some embodiments of the invention, the "correspondence" of amino acid positions is determined relative to a polymerase region comprising one or more motifs of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:39. When the polymerase polypeptide sequence differs from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 39 (e.g., by amino acid changes or additions or deletions of amino acids), the specific mutations that may be associated with the improved activity discussed herein will not be in the same position number as in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 39. For example, this is illustrated in Table 1.

[0167] As used herein, "recombinant" refers to an amino acid sequence or nucleotide sequence that has been intentionally modified by a recombinant method. The term "recombinant nucleic acid" herein means a nucleic acid that is initially formed in vitro, usually by the manipulation of nucleic acids by restriction endonucleases in a form that does not normally exist in nature. Therefore, for the purposes of the present invention, an isolated linear form of a mutant DNA polymerase nucleic acid or an expression vector formed in vitro by connecting DNA molecules that are not normally joined are considered to be recombinant. It should be understood that once a recombinant nucleic acid is prepared and reintroduced into a host cell, it will replicate non-recombinantly using the in vivo cellular machinery of the host cell rather than in vitro manipulation; however, such a nucleic acid, once produced by recombinant means, is still considered to be recombinant for the purposes of the present invention, although it is subsequently replicated non-recombinantly. A "recombinant protein" is a protein made using recombinant technology, i.e., by the expression of the above-mentioned recombinant nucleic acids.

[0168] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.

[0169] The term "host cell" refers to unicellular prokaryotic and eukaryotic organisms (eg, bacteria, yeast, and actinomycetes), as well as individual cells from higher plants or animals grown in cell culture.

[0170] The term "vector" refers to a DNA, usually double-stranded, into which a foreign DNA may have been inserted. The vector may be, for example, of plasmid origin. The vector comprises a "replicon" polynucleotide sequence that promotes autonomous replication of the vector in a host cell. Foreign DNA is defined as heterologous DNA, which is a DNA that does not naturally exist in a host cell, such as a replication vector molecule, an encoding selectable marker or a screenable marker, or an encoding transgenic. The vector is used to transport foreign or heterologous DNA into a suitable host cell. Once entering the host cell, the vector may be independent of the host chromosome DNA replication or integration with the host chromosome DNA, and multiple copies of the vector and its inserted DNA may be generated. In addition, the vector may also comprise an essential element that allows the inserted DNA to be transcribed into an mRNA molecule or otherwise causes the inserted DNA to be replicated into multiple copies of RNA. Some expression vectors additionally comprise sequence elements adjacent to the inserted DNA, which increase the half-life of the expressed mRNA and / or allow mRNA to be translated into protein molecules. Therefore, many mRNA and polypeptide molecules encoded by the inserted DNA may be rapidly synthesized.

[0171] The term "nucleotide" refers, in addition to naturally occurring ribonucleotide or deoxyribonucleotide monomers, to related structural variants thereof, including derivatives and analogs, which are functionally equivalent with respect to the specific context in which the nucleotide is used (e.g., hybridizing with a complementary base), unless the context clearly indicates otherwise.

[0172] The term "nucleic acid" or "polynucleotide" refers to a polymer that can correspond to a ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) polymer or an analog thereof. This includes polymers of nucleotides, such as RNA and DNA, as well as synthetic forms, modified (e.g., chemically or biochemically modified) forms thereof, and mixed polymers (e.g., including both RNA and DNA subunits). Exemplary modifications include methylation, substitution of one or more of the naturally occurring nucleotides with analogs, internucleotide modifications such as uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), pendant moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylating agents, and modified bonds (e.g., alpha anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a specified sequence via hydrogen bonds and other chemical interactions. Typically the nucleotide monomers are linked via phosphodiester bonds, although synthetic forms of nucleic acids may include other bonds (e.g., peptide nucleic acids as described by Nielsen et al. (Science 254: 1497-1500, 1991). A nucleic acid can be or include, for example, a chromosome or chromosome fragment, a vector (e.g., an expression vector), an expression cassette, a naked DNA or RNA polymer, a product of the polymerase chain reaction (PCR), an oligonucleotide, a probe, and a primer. A nucleic acid can be, for example, single-stranded, double-stranded, or triple-stranded, and is not limited to any particular length. Unless otherwise indicated, a particular nucleic acid sequence optionally includes or encodes a complementary sequence in addition to any explicitly indicated sequence.

[0173] The term "oligonucleotide" refers to a nucleic acid comprising at least two nucleic acid monomer units (e.g., nucleotides). Oligonucleotides typically include about six to about 175 nucleic acid monomer units, more typically about eight to about 100 nucleic acid monomer units, and more typically about 10 to about 50 nucleic acid monomer units (e.g., about 15, about 20, about 25, about 30, about 35, or more nucleic acid monomer units). The exact size of an oligonucleotide depends on many factors, including the ultimate function or purpose of the oligonucleotide. Oligonucleotides are optionally prepared by any suitable method, including but not limited to isolation of existing or natural sequences, DNA replication or amplification, reverse transcription, cloning of appropriate sequences and restriction enzyme cleavage, or by direct chemical synthesis such as the phosphotriester method of Narang et al. (Meth. Enzymol. 68: 90-99, 1979); the phosphodiester method of Brown et al. (Meth. Enzymol. 68: 109-151, 1979); the diethylphosphoramidite method of Beaucage et al. (Tetrahedron Lett. 22: 1859-1862, 1981); the triester method of Matteucci et al. (J. Am. Chem. Soc. 103: 3185-3191, 1981); automated synthesis; or the solid support method of U.S. Pat. No. 4,458,066, or other methods known to those skilled in the art. All of these references are incorporated by reference.

[0174] The term "primer" as used herein refers to a polynucleotide that can serve as a starting point for template-directed nucleic acid synthesis under conditions that initiate polynucleotide extension (e.g., in the presence of required nucleoside triphosphates (determined by the template to be replicated) and a polymerase in an appropriate buffer and at a suitable temperature or temperature cycle (e.g., in a polymerase chain reaction). To further illustrate, primers can also be used in other multiple oligonucleotide-mediated synthesis processes, including initiators for processes related to RNA de novo synthesis and in vitro transcription (e.g., nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), etc.). Primers are typically single-stranded oligonucleotides (e.g., oligodeoxyribonucleotides). The appropriate length of a primer depends on the intended use of the primer, but it typically ranges from 6 to 40 nucleotides, more typically from 15 to 35 nucleotides. Short primer molecules typically require lower temperatures to form sufficiently stable hybrid complexes with the template. Primers do not need to reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with the template for primer extension to occur. In certain embodiments, the term "primer pair" means a set of primers that includes a 5' sense primer (sometimes referred to as "forward") that hybridizes to the complementary sequence at the 5' end of the nucleic acid sequence to be amplified and a 3' antisense primer (sometimes referred to as "reverse") that hybridizes to the 3' end of the sequence to be amplified (e.g., if the target sequence is expressed as RNA or is RNA). If desired, the primers can be labeled by combining with a label detectable by spectroscopic, photochemical, biochemical, immunochemical or chemical methods. For example, useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (commonly used in ELISA assays), biotin, or haptens and proteins for which antisera or monoclonal antibodies are available.

[0175] When referring to nucleic acid bases, nucleoside triphosphates or nucleotides, the term "conventional" or "natural" refers to those naturally occurring in the polynucleotide (i.e., for DNA these are dATP, dGTP, dCTP and dTTP). In addition, in in vitro DNA synthesis reactions such as sequencing, dITP and 7-deaza-dGTP are often used in place of dGTP, while 7-deaza-dATP can be used in place of dATP. These may be collectively referred to as dNTPs.

[0176] When it comes to nucleic acid bases, nucleosides or nucleotides, the term "unconventional" or "modified" includes conventional bases, nucleosides or nucleotides naturally present in a particular polynucleotide, modified, derivatives or analogs. Compared with conventional dNTPs, some unconventional nucleotides are modified at the 2' position of ribose. Therefore, although the naturally occurring nucleotides for RNA are ribonucleotides (i.e., ATP, GTP, CTP, UTP, collectively referred to as rNTPs), because these nucleotides have a hydroxyl group at the 2' position of sugar, compared to dNTPs, as used herein, ribonucleotides as substrates for DNA polymerases are unconventional nucleotides. As used herein, unconventional nucleotides include, but are not limited to, compounds used as terminators for nucleic acid sequencing. Exemplary terminator compounds include, but are not limited to, those compounds called dideoxynucleoside triphosphates with a 2', 3' dideoxy structure. Dideoxynucleoside triphosphates ddATP, ddTTP, ddCTP and ddGTP are collectively referred to as ddNTPs. Other examples of terminator compounds include 2'-PO4 analogs of ribonucleotides (see, e.g., U.S. Publication Nos. 2005 / 0037991 and 2005 / 0037398, both of which are incorporated by reference). Other unconventional nucleotides include phosphorothioate dNTPs ([α-S]dNTPs), 5'-[α-borane]-dNTPs, [α]-methyl-phosphate dNTPs, and ribonucleoside triphosphates (rNTPs). Unconventional bases can be labeled with radioactive isotopes, such as 32 P. 33 P or 35S; fluorescent label; chemiluminescent label; bioluminescent label; hapten label, such as biotin; or enzyme label, such as streptavidin or avidin. Fluorescent label can include negatively charged dyes, such as dyes of the fluorescein family, or electrically neutral dyes, such as dyes of the rhodamine family, or positively charged dyes, such as dyes of the cyanine family. Dyes of the fluorescein family include, for example, FAM, HEX, TET, JOE, NAN, and ZOE. Dyes of the rhodamine family include Texas Red, ROX, R110, R6G, and TAMRA. Various dyes or nucleotides labeled with FAM, HEX, TET, JOE, NAN, ZOE, ROX, R110, R6G, Texas Red, and TAMRA are sold by Perkin-Elmer (Boston, MA), Applied Biosystems (Foster City, CA), or Invitrogen / Molecular Probes (Eugene, OR). The cyanine family of dyes includes Cy2, Cy3, Cy5, and Cy7, and are sold by GE Healthcare UK Limited (Amersham Place, Little Chalfont, Buckinghamshire, England).

[0177] As used herein, "sequence identity percentage" is determined by comparing two optimally aligned sequences in a comparison window, wherein for optimal alignment of the two sequences, the sequence portion in the comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence (which does not include additions or deletions). The percentage is calculated by determining the number of positions of identical nucleic acid bases or amino acid residues present in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window and multiplying the result by 100 to obtain the sequence identity percentage.

[0178] In the context of two or more nucleic acid or peptide sequences, the term "identical" or "identity" percentage refers to two or more identical sequences or subsequences. When using one of the following sequence comparison algorithms or using manual comparison and eye detection in a comparison window or a specified region for maximum correspondence and comparison and comparison, if the sequence has a specific percentage of identical nucleotides or amino acid residues (e.g., at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity in a specific region), then they are "substantially identical" to each other. If the sequence has at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% identity, then they are "substantially identical". These definitions also refer to the complementary sequence of the test sequence. Optionally, the identity exists over a region that is at least about 50 nucleotides in length, or more usually over a region that is 100 to 500 or 1000 or more nucleotides in length.

[0179] In the context of two or more polypeptide sequences, the term "similarity" or "similarity percentage" refers to two or more sequences or subsequences that have a specified percentage of identical or similar (e.g., 60% similarity, optionally 65%, 70%, 75%, 80%, 85%, 90% or 95% similarity in a particular region) amino acid residues defined by conservative amino acid substitutions when compared and aligned for maximum correspondence over a comparison window or specified region using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Sequences are "substantially similar" to each other if they have at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% similarity to each other. Optionally, this similarity exists over a region of at least about 50 amino acids in length, or more typically over a region of at least about 100 to 500 or 1000 or more amino acids in length.

[0180] For sequence comparison, one sequence is usually used as a reference sequence to which a test sequence is compared. When a sequence comparison algorithm is used, the test and reference sequences are input into a computer, subsequence coordinates are specified, and, if necessary, sequence algorithm program parameters are specified. Default program parameters are usually used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the percent sequence identity or similarity of the test sequence relative to the reference sequence based on the program parameters.

[0181] As used herein, a "comparison window" includes a segment with reference to any number of consecutive positions selected from the group consisting of: 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, wherein a sequence can be compared to a reference sequence of the same number of consecutive positions after the two sequences are optimally aligned. Methods of sequence alignment for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482, 1970, by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970, by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0182] Examples of algorithms suitable for determining sequence identity and sequence similarity percentages are BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (Nuc. Acids Res. 25: 3389-402, 1977) and Altschul et al. (J. Mol. Biol. 215: 403-10, 1990). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (see the Internet www.ncbi.nlm.nih.gov / ). The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short fields of length W in the query sequence that match or meet a certain positive threshold score T when aligned with the same length field of the database sequence. T is called the adjacent field score threshold (Altschul et al., supra). These initial adjacent field hits serve as seeds for initiating searches to find longer HSPs containing them. Field hits are extended bidirectionally along each sequence as long as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for matching residue pairs; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction stops when the cumulative alignment score decreases by the amount X from its maximum achieved value; when the cumulative score reaches or falls below zero due to the accumulation of one or more negative-scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses the default word length (W) of 11, expectation (E) of 10, M=5, N=-4, and compares two chains. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989), alignments (B) 50, and an expectation (E) of 10, M=5, N=-4.

[0183] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-87, 1993). One similarity metric provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences occurs by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of a test nucleic acid to a reference nucleic acid is less than about 0.2, typically less than about 0.01, and more typically less than about 0.001.

[0184] The term "reverse transcription efficiency" refers to the ratio of RNA molecules that are reverse transcribed as cDNA in a given reverse transcription reaction. In certain embodiments, relative to the DNA polymerases of these unmodified forms, the mutant DNA polymerases of the present invention have improved reverse transcription efficiency. That is, under a specific set of reaction conditions, these mutant DNA polymerases reverse transcribe a higher proportion of RNA templates than their unmodified forms. Not limited by theory, the ability of the RNA templates of the reverse transcription of mutant DNA polymerases as herein described to be higher in proportion can be attributed to the reverse transcription activity of increase, for example, the nucleotide incorporation rate of increase and / or the enzyme continuous synthesis ability of increase. Reverse transcription efficiency can be measured, for example, by using RNA templates to measure the intersection point (Cp) of PCR reactions, and by comparing the Cp value with the Cp value of the control reaction, wherein the DNA templates of the same sequence (except that U is replaced by T) are amplified, wherein RNA and DNA amplification use a common primer set and identical polymerase, for example, as described in the example. The test polymerase has an improved RT efficiency: when RNA is used as a template, the test polymerase has a reduced Cp value compared to the control polymerase, while when DNA is used as a template, it has a substantially unchanged Cp value relative to the control polymerase. In some embodiments, the polymerase of the invention has an improved RT efficiency such that the Cp is at least one, two, three, four, five, six, seven, eight, nine, ten or more units less than the corresponding control polymerase on the RNA template.

[0185] The term "mismatch tolerance" refers to the ability of a polymerase to tolerate sequences containing mismatches when extending a nucleic acid (e.g., a primer or other oligonucleotide) in a template-dependent manner by attaching one or more nucleotides (e.g., covalently) to the nucleic acid. The term "3' mismatch tolerance" refers to the ability of a polymerase to tolerate sequences containing mismatches (almost complementary) where the nucleic acid to be extended (e.g., a primer or other oligonucleotide) has a mismatch with its template at the 3' terminal nucleotide of the primer. The mismatch with the template may also be at the 3' penultimate nucleotide of the primer, or at another position within the primer sequence.

[0186] The term "mismatch discrimination" refers to the ability of a polymerase to distinguish between a fully complementary sequence and a sequence containing mismatches when extending a nucleic acid (e.g., a primer or other oligonucleotide) in a template-dependent manner by attaching one or more nucleotides (e.g., covalently) to the nucleic acid. The term "3'-mismatch discrimination" refers to the ability of a polymerase to distinguish between a fully complementary sequence and a sequence containing mismatches (almost complementary) wherein the nucleic acid to be extended (e.g., a primer or other oligonucleotide) has a mismatch at the 3' end of the nucleic acid compared to the template to which the nucleic acid is hybridized. The term "mismatch" refers to the presence of one or more base mismatches (or "non-complementary base pairs") within a complementary duplex-forming (or potential duplex-forming) sequence.

[0187] The term "Cp value" or "crossing point" value refers to a value that allows the input target nucleic acid to be quantified. The Cp value can be determined according to the second-derivative maximum method (Van Luu-The, et al., "Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction," BioTechniques, Vol. 38, No. 2, February 2005, pp. 287-293). In the second-derivative method, Cp corresponds to the first peak of the second-derivative curve. This peak corresponds to the beginning of the logarithmic linear phase. The second-derivative method calculates the second-derivative value of the real-time fluorescence intensity curve and only obtains one value. The initial Cp method is based on a locally defined differentiable approximation of the intensity value, for example, by a polynomial function. The third-order derivative is then calculated. The Cp value is the smallest root of the third-order derivative. Cp can also be determined using the fit point method, where Cp is determined by the intersection of parallel lines with a threshold line in the log-linear region (Van Luu-The, et al., BioTechniques, Vol. 38, No. 2, February 2005, pp. 287-293). The LightCycler instrument provided by Roche provides Cp values ​​by calculation according to the second derivative maximum method.

[0188] The term "PCR efficiency" refers to an indicator of cycle-to-cycle amplification efficiency. The PCR efficiency for each condition was calculated using the following equation: % PCR efficiency = (10 (-斜率) -1) x 100, where the slope is calculated by linear regression of the log copy number plotted on the y-axis and the Cp plotted on the x-axis. PCR efficiency can be measured using perfectly matched or mismatched primer templates.

[0189] The term "nucleic acid extension rate" refers to the rate at which a biocatalyst (e.g., an enzyme such as a polymerase, a ligase, etc.) extends a nucleic acid (e.g., a primer or other oligonucleotide) by attaching one or more nucleotides (e.g., covalently) to the nucleic acid in a template-dependent or template-independent manner. For illustration, certain mutant DNA polymerases described herein have improved nucleic acid extension rates relative to the unmodified forms of the DNA polymerases, such that under a given set of reaction conditions they can extend primers at a higher rate than the unmodified forms.

[0190] The term "tolerance to RT and polymerase inhibitors" refers to the ability of a polymerase to maintain activity (polymerase or reverse transcription activity) in the presence of an amount of an inhibitor that inhibits the polymerase activity or reverse transcription activity of a control polymerase. In some embodiments, the improved polymerase is capable of having polymerase or reverse transcription activity in the presence of an amount of an inhibitor that substantially eliminates the control polymerase activity.

[0191] The term "5'-nuclease probe" refers to an oligonucleotide comprising at least one luminescent labeling portion and used for a 5'-nuclease reaction to achieve target nucleic acid detection. In some embodiments, for example, a 5'-nuclease probe includes only a single luminescent portion (e.g., a fluorescent dye, etc.). In certain embodiments, a 5'-nuclease probe includes a self-complementary region so that the probe can form a hairpin structure under selected conditions. To further illustrate, in some embodiments, a 5'-nuclease probe includes at least two labeling portions, and after cutting or otherwise separating one of the two labels from an oligonucleotide, the emission intensity increases. In certain embodiments, a 5'-nuclease probe is labeled with two different fluorescent dyes, such as a 5' terminal reporter dye and a 3' terminal quencher dye or portion. In some embodiments, a 5'-nuclease probe is labeled at one or more positions different from or in addition to the terminal position. When the probe is intact, energy transfer generally occurs between two fluorophores, so that the fluorescence emission from the reporter dye is at least partially quenched. During the extension step of the polymerase chain reaction, for example, a 5'-nuclease probe bound to a template nucleic acid is cleaved by the 5' to 3' nuclease activity of, for example, Taq polymerase or another polymerase having such activity, so that the fluorescence emission of the reporter dye is no longer quenched. Exemplary 5'-nuclease probes are also described in, for example, U.S. Patent No. 5,210,015, U.S. Patent No. 5,994,056, and U.S. Patent No. 6,171,785, each of which is incorporated herein by reference. In other embodiments, the 5' nuclease probe can be labeled with two or more different reporter dyes and a 3' terminal quenching dye or moiety.

[0192] The term "FRET" or "Fluorescence Resonance Energy Transfer" or "Foerster Resonance Energy Transfer" refers to the transfer of energy between at least two chromophores, a donor chromophore and an acceptor chromophore (called a quencher). When the donor is excited by light radiation having a suitable wavelength, the donor typically transfers energy to the acceptor. The acceptor typically re-emits the transferred energy in the form of light radiation having a different wavelength. When the acceptor is a "dark" quencher, it dissipates the transferred energy in a form other than light. Whether a particular fluorophore acts as a donor or an acceptor depends on the properties of the other members of the FRET pair. Commonly used donor-acceptor pairs include the FAM-TAMRA pair. Commonly used quenchers are DABCYL and TAMRA. Commonly used dark quenchers include BlackHoleQuenchers TM (BHQ) (Biosearch Technologies, Inc., Novato, Cal.), Iowa Black TM (Integrated DNA Tech., Inc., Coralville, Iowa) and BlackBerry TM Quencher 650 (BBQ-650) (Berry & Assoc., Dexter, Mich.).

[0193] The term "strand displacement activity" refers to the ability of a polymerase to displace downstream DNA encountered during synthesis. The Royal Society of Chemistry defines the term "strand displacement" as "the expulsion of a broken 3' single-stranded DNA molecule that forms a heteroduplex DNA with its complementary strand in a complete double-stranded DNA. The Watson-Crick base pairs in the original duplex are restored. The repelled 3' single-stranded DNA molecule reanneals with its original complementary sequence to re-form two complete double-stranded molecules. " In some embodiments, the term includes an isothermal amplification method called strand displacement amplification (SDA).

[0194] The term "high temperature" refers to a temperature above the melting temperature of a double-stranded DNA molecule. The melting temperature (Tm) is defined as the temperature at which half of the DNA strands are in a random coil or single-stranded (ssDNA) state. As is well known in the art, Tm depends on the length of the DNA molecule and its specific nucleotide sequence. Typical examples of high temperatures encountered in PCR denaturation, annealing and extension reactions are 60°C or higher, for example, from about 60°C to about 95°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0195] Figure 1Sequence identities are provided between the following DNA polymerase I enzymes: Thermus sp. Z05 DNA polymerase (Z05); Thermus aquaticus DNA polymerase (Taq); Thermus filamentosus DNA polymerase (Tfi); Thermus flavus DNA polymerase (Tfl); Thermus sps17 DNA polymerase (Sps17); Thermus thermophilus DNA polymerase (Tth); Thermus caldophilus DNA polymerase (Tca); Deinococcus radiodurans DNA polymerase (Dra); Thermotoga maritima DNA polymerase (Tma); Thermotoga naples DNA polymerase (Tne); Thermus africanus DNA polymerase (Taf); Bacillus stearothermophilus DNA polymerase (Bst); and Bacillus thermotenes DNA polymerase (Bca). (A) Sequence identity across the entire polymerase I enzyme (corresponding to amino acids 1-834 of Z05); and (B) sequence identity across the polymerase subdomain corresponding to amino acids 420-834 of Z05.

[0196] Figure 2 Sequence identities between various Thermus species DNA polymerase I enzymes are provided: Thermus species Z05 DNA polymerase (Z05); Thermus aquaticus DNA polymerase (Taq); Thermus filamentosus DNA polymerase (Tfi); Thermus flavus DNA polymerase (Tfl); Thermus species sps17 DNA polymerase (Sps17); Thermus thermophilus DNA polymerase (Tth); and Thermus caldophilus DNA polymerase (Tca). (A) Sequence identity over the entire polymerase I enzyme (corresponding to amino acids 1-834 of Z05); and (B) Sequence identity over the polymerase subdomain corresponding to amino acids 420-834 of Z05.

[0197] Figure 3 The assay design described in the example is shown. Four complementary oligonucleotides Oligo A (green), Oligo B (light blue), Oligo C (dark blue) and locked nucleic acid (LNA) Oligo D (red) are annealed to each other. Oligo A is complementary to Oligo B and initiates polymerization. Oligo B is the template and has a FAM – fluorescent reporter at the 5'-end. Oligo C is complementary to Oligo B and has a BHQ quencher at the 3'-end. Locked nucleic acid Oligo D is complementary to template Oligo B and acts as a high energy fence. When polymerase, Mg 2+ When nucleotides are added to the annealed mixture, polymerization extends Oligo A and the newly synthesized strand displaces LNA Oligo D and Oligo C, thereby releasing the quencher from the fluorescent probe. A fluorescent signal is generated after the cycle is completed.

[0198] Figure 4A Results are shown for wells with bacterial colonies (clones) expressing mutant polymerases with increased strand displacement activity compared to the control G46E C21 clone. Plate 6 shows results for the G46E C21 clone (all wells of plate 6 contain the original G46E C21 clone). Plate 1 shows increased strand displacement activity for the following clones that were subsequently sequenced: F24 (containing I686V and A693V mutations), L3 (containing T516I and V633I mutations), and P19 (containing Q415H, E420D, E636G, N752S, and V768M mutations).

[0199] Figure 4B The results for plates 2-5 are shown. Plate 6 (above) is used as a control for all these experiments. Plate 2 shows the chain displacement activity of the increase of the following clones, and it has the subsequent sequencing results for clones: M22 (comprising R525G and F694S mutations), G9 (comprising Q491H and T516S mutations), N19 (comprising S515F and T666M mutations) and N7 (comprising E402V, V555A and N582D mutations). Plate 3 shows the chain displacement activity of the increase of the following clones, and it has the subsequent sequencing results for clones: A24 (comprising A737T and A759T mutations), F21 (comprising L521Q and T546A mutations), G10 (comprising N668S mutations) and I14 (comprising A456T mutations). Plate 4 shows the increased strand displacement activity of the following clones, which have subsequent sequencing results for the clones: G23 (comprising K507M, T571A and S652F mutations) and K12 (comprising S515F and A832V mutations). Plate 5 shows the increased strand displacement activity of the following clones, which have subsequent sequencing results for the clones: C6 (comprising D498E, L524V, R598G and M616I mutations), G20 (comprising A444T, D498E, M660K and Y673N mutations), G23 (comprising E493D, T511S, M648I and M749L mutations) and H21 (comprising Q635K mutation).

[0200] Figure 5A and Figure 5B Data are provided showing that the mutant polymerases described herein have significantly reduced endonuclease and exonuclease activities. DETAILED DESCRIPTION

[0201] The present disclosure provides improved DNA polymerase, wherein relative to functional DNA polymerase, one or more amino acids in the polymerase domain have been mutated.DNA polymerase as described herein is the tolerance of mismatch tolerance, extension rate, RT and polymerase inhibitors with increased strand displacement activity and / or increased relative to the polymerase of unmodified form.In certain embodiments, mutant DNA polymerase can be used for superior or equivalent performance compared with parent enzyme at lower concentration.In certain embodiments, mutant DNA polymerase has increased strand displacement activity relative to unmodified or control polymerase, while retaining substantially the same DNA dependent polymerase activity.In certain embodiments, mutant DNA polymerase has significantly reduced exonuclease or endonuclease activity.In certain embodiments, mutant DNA polymerase has reduced 5' to 3' exonuclease activity.

[0202] DNA polymerases with increased strand displacement activity can be used, for example, in polymerase chain reaction (PCR) and isothermal amplification (such as strand displacement amplification (SDA)) at high temperatures, and in DNA amplification techniques such as loop-mediated amplification (LAMP), cross primer amplification (CSA), and polymerase strand displacement reaction (PCDR). Thus, DNA polymerases can be used in a variety of applications involving polynucleotide extension, including, for example, applications in recombinant DNA research and medical diagnosis of disease.

[0203] In some embodiments, the DNA polymerase further comprises a motif of SEQ ID NO:29 and / or SEQ ID NO:38.

[0204] This motif is present in the "finger" domain (Lα helix) of many family A-type DNA-dependent DNA polymerases, especially the thermostable DNA polymerases from thermophilic bacteria (Li et al., EMBO J. 17: 7514-7525, 1998). For example, Figure 1 An amino acid sequence alignment of a region of the "finger" domain of DNA polymerases from several bacteria is shown: Bacillus thermotenes, Bacillus stearothermophilus, Deinococcus radiodurans, Thermus africanus, Thermotoga maritima, Thermotoga napolinum, Thermus aquaticus, Thermus caldophilus, Thermus filamentosus, Thermus flavicum, Thermus sps17, Thermus sp Z05, and Thermus thermophilus. As shown, native sequences corresponding to the motif described above are present in each of these polymerases, indicating a conserved function for this region of the polymerase. Figure 2 Sequence identities between these DNA polymerases are provided.

[0205] In some embodiments, the polymerases described herein with improved activity and / or properties are wild-type or naturally occurring DNA polymerases, such as, polymerases from any of the species of thermophiles listed above, or are substantially identical to such wild-type or naturally occurring DNA polymerases. For example, in some embodiments, the polymerase is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 42. In one variant, the unmodified form of the polymerase is from a species of the genus Thermus. In other embodiments of the invention, the unmodified polymerase is from a thermophilic species other than the Thermus species, e.g., Thermotoga genus. Complete nucleic acid and amino acid sequences for a number of thermostable DNA polymerases are available. The sequences of each of the Thermus aquaticus (Taq) (SEQ ID NO: 2), Thermus thermophilus (Tth) (SEQ ID NO: 6), Thermus sp. Z05 (SEQ ID NO: 1), Thermus sps17 (SEQ ID NO: 5), Thermotoga maritima (Tma) (SEQ ID NO: 34), and Thermus africanus (Taf) (SEQ ID NO: 33) polymerases have been disclosed in PCT International Patent Publication No. WO 92 / 06200, which is incorporated herein by reference. The sequence of the DNA polymerase from Thermus flavus (SEQ ID NO: 4) has been disclosed in Akhmetzjanov and Vakhitov (Nucleic Acids Research 20: 5839, 1992), which is incorporated herein by reference. The sequence of the thermostable DNA polymerase from Thermus caldophilus (SEQ ID NO:7) is found in EMBL / GenBank Accession No. U62584. The sequence of the thermostable DNA polymerase from Thermus filamentosa can be recovered from ATCC Deposit No. 42380 using, for example, the methods provided in U.S. Pat. No. 4,889,818 and the sequence information provided in Table 1. The sequence of the Thermotoga naples DNA polymerase (SEQ ID NO:35) is from GeneSeq Patent Database Accession No. R98144 and PCT WO 97 / 09451, each of which is incorporated herein by reference.The sequence of the thermostable DNA polymerase from Bacillus thermotenes (SEQ ID NO: 37) is described in, for example, Uemori et al. Biochem (Tokyo) 113 (3): 401-410, 1993; see also Swiss-Prot database accession number Q04957 and GenBank accession number D12982 and BAA02361), each of which is incorporated by reference. Examples of unmodified forms of DNA polymerases that can be modified as described herein are also described in, for example, U.S. Patent Nos. 6,228,628; 6,346,379; 7,030,220; 6,881,559; 6,794,177; 6,468,775; and U.S. Patent Nos. 7,148,049; 7,179,590; 7,410,782; 7,378,262, each of which is incorporated by reference. Representative full-length polymerase sequences are also provided in the sequence listing.

[0206] Also suitable for mutations described herein are functional DNA polymerases that have been previously modified (e.g., by amino acid substitutions, additions or deletions). In some embodiments, such functionally modified polymerases comprise the amino sequence of SEQ ID NO:40 or SEQ ID NO:42, or have substantial sequence identity or similarity to SEQ ID NO:40 or SEQ ID NO:42, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:40 or SEQ ID NO:42. In some embodiments, suitable unmodified DNA polymerases also include functional variants of wild-type or naturally occurring polymerases. Such variants will typically have substantial sequence identity or similarity to the wild-type or naturally occurring polymerase, typically having at least 80% sequence identity, and more typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0207] In some embodiments, the polymerases described herein further comprise a nuclease domain (eg, corresponding to positions 1 to 291 of Z05 (SEQ ID NO: 1)).

[0208] In some embodiments, the polymerase described herein is a chimeric polymerase, that is, it comprises polypeptide regions from two or more enzymes. Examples of such chimeric DNA polymerases are described in, for example, U.S. Patent No. 6,228,628, which is incorporated herein by reference in its entirety. Chimeric CS family DNA polymerases are particularly suitable, including CS5 (SEQ ID NO: 27) and CS6 (SEQ ID NO: 28) polymerases and variants thereof, which have amino acid sequence identity or similarity substantially to SEQ ID NO: 27 or SEQ ID NO: 28 (generally having at least 80% amino acid sequence identity, and more generally having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity). CS5 and CS6 DNA polymerases are chimeric enzymes derived from Thermus species Z05 and Thermotoga maritima (Tma) DNA polymerases. They contain the N-terminal 5'-nuclease domain of the Thermus species enzyme and the C-terminal 3'-5' exonuclease and polymerase domains of the Tma enzyme. These enzymes have efficient reverse transcriptase activity, can extend primers containing nucleotide analogs, and can incorporate α-phosphorothioate dNTPs, dUTP, dITP, and dNTPs labeled with fluorescein and the cyanine dye family. CS5 and CS6 polymerases are also efficient Mg 2+ Activating PCR enzymes. CS5 and CS6 chimeric polymerases are further described, for example, in U.S. Pat. No. 7,148,049, which is incorporated herein by reference in its entirety.

[0209] In some embodiments, the amino acid substitution is a single amino acid substitution.Relative to the unmodified polymerase, the DNA polymerase provided herein can comprise one or more amino acid substitutions in the active site.

[0210] In some embodiments, the polymerase described herein further comprises the amino acid motif of SEQ ID NO: 38 (corresponding to the D580X mutation of Z05 (SEQ ID NO: 1)) as follows:

[0211] Thr-Gly-Arg-Leu-Ser-Ser-X7-X8-Pro-Asn-Leu-Gln-Asn

[0212] (Also referred to herein as single-letter codes

[0213] TGRLSS-X7-X8-PNLQN) (SEQ ID NO: 38); wherein

[0214] X7 is Ser(S) or Thr(T); and

[0215] X8 is any amino acid except Asp (D) or Glu (E)

[0216] Mutations characterized by SEQ ID NO: 38 are discussed in more detail in, e.g., U.S. Patent Publication No. 2009 / 0148891. Such functional variant polymerases will typically have substantial sequence identity or similarity to a wild-type or naturally occurring polymerase (e.g., SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 42), typically at least 80% amino acid sequence identity, more typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity.

[0217] In some embodiments, the polymerase described herein further comprises the amino acid motif of SEQ ID NO: 29 (corresponding to the I709X mutation of Z05 (SEQ ID NO: 1)) as follows:

[0218] X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 -X 13 -Gly-Tyr-Val-

[0219] X 14 -Thr-Leu (also referred to herein by the single letter code X1-X2-X3-

[0220] X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 -X 13 -GYVX 14 -TL)

[0221] (SEQ ID NO:29); wherein

[0222] X1 is Ala(A), Asp(D), Ser(S), Glu(E), Arg(R) or

[0223] Gln(Q);

[0224] X2 is Trp (W) or Tyr (Y);

[0225] X3 is any amino acid except Ile (I), Leu (L) or Met (M);

[0226] X4 is Glu(E), Ala(A), Gln(Q), Lys(K), Asn(N) or

[0227] Asp(D);

[0228] X5 is Lys(K), Gly(G), Arg(R), Gln(Q), His(H) or

[0229] Asn(N);

[0230] X6 is Thr (T), Val (V), Met (M) or Ile (I);

[0231] X7 is Leu (L), Val (V) or Lys (K);

[0232] X8 is Glu (E), Ser (S), Ala (A), Asp (D) or Gln (Q);

[0233] X9 is Glu (E) or Phe (F);

[0234] X 10 is Gly(G) or Ala(A);

[0235] X 11 is Arg(R) or Lys(K);

[0236] X 12 is Lys(K), Arg(R), Glu(E), Thr(T) or Gln(Q);

[0237] X 13 is Arg (R), Lys (K) or His (H); and

[0238] X 14 is Glu(E), Arg(R) or Thr(T).

[0239] In some embodiments, such functional variant polymerases will typically have substantial sequence identity or similarity to a wild-type or naturally occurring polymerase (e.g., SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 42), typically having at least 80% amino acid sequence identity, more typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity.

[0240] In some embodiments, the DNA polymerase comprises amino acid substitutions corresponding to SEQ ID NO:38 and SEQ ID NO:29. In some embodiments, the amino acid substitutions include leucine (L), glycine (G), threonine (T), glutamine (Q), alanine (A), serine (S), asparagine (N), arginine (R) and lysine (K) at position X8 of SEQ ID NO:38. In certain embodiments, the amino acid substitutions include glycine (G) at position X8 of SEQ ID NO:38. In some embodiments, the amino acid substitutions include lysine (K), arginine (R), serine (S), glycine (G) or alanine (A) at position X3 of SEQ ID NO:29. In certain embodiments, the amino acid substitutions include lysine (K) at position X3 of SEQ ID NO:29.

[0241] Other suitable amino acid substitutions at one or more of the identified sites can be determined using known methods, such as site-directed mutagenesis and determination of polynucleotide extension properties in assays further described herein or known to those of skill in the art, such as those described in U.S. Patent Application Publication Nos. 2009 / 0148891 and 2009 / 0280539, which are incorporated herein by reference in their entireties.

[0242] Because the precise length of DNA polymerases varies, the precise amino acid positions corresponding to X8 (SEQ ID NO:38) and X3 (SEQ ID NO:29) can vary depending on the specific mutant polymerase used. Amino acid and nucleic acid sequence alignment programs are readily available (see, e.g., those mentioned above), and are used to assist in identifying the exact amino acids (and corresponding codons) for modification according to the present invention in view of the specific motifs identified herein. The positions corresponding to X8 and X3 for representative chimeric thermostable DNA polymerases and thermostable DNA polymerases from exemplary thermophilic species are shown in Table 1.

[0243] Table 1. Amino acid positions corresponding to motif positions X8 (of SEQ ID NO: 38) and X3 (of SEQ ID NO: 29) in exemplary polymerases.

[0244]

[0245]

[0246] In some embodiments, the DNA polymerase of the invention is derived from Thermus sp. Z05 DNA polymerase (SEQ ID NO: 1) or a variant thereof (e.g., with a D580G mutation, etc.). As mentioned above, in the Thermus sp. Z05 DNA polymerase, position X8 corresponds to aspartic acid (D) at position 580, and position X3 corresponds to isoleucine (I) at position 709. Thus, in certain variants of the invention, relative to the Thermus sp. Z05 DNA polymerase (or a DNA polymerase substantially identical to SEQ ID NO: 1, e.g., having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity), the mutant polymerase further comprises at least one amino acid substitution at D580 and / or I709. In certain embodiments, the amino acid residue at position 580 of SEQ ID NO: 1 can be selected from leucine (L), glycine (G), threonine (T), glutamine (Q), alanine (A), serine (S), asparagine (N), arginine (R) and lysine (K). Therefore, in some embodiments, the amino acid residue at position 580 of SEQ ID NO: 1 is glycine (G). Further, in certain embodiments, the amino acid at position 709 of SEQ ID NO: 1 is not I. In some embodiments, the amino acid at position 709 of SEQ ID NO: 1 is selected from G, A, V, R, F, W, P, S, T, C, Y, N, Q, D, E, K, L, M or H. In some embodiments, the amino acid at position 709 of SEQ ID NO: 1 is K, R, S, G or A. In some embodiments, the amino acid at position 709 of SEQ ID NO: 1 is K.

[0247] Exemplary Thermus sp. Z05 DNA polymerase mutants include those comprising the amino acid substitutions I709K (or I709R, I709S, I709G, I709A) and / or D580G.

[0248] The present inventors have shown that substitutions at the amino acid corresponding to position 709 of SEQ ID NO: 1 above can result in a DNA polymerase with improved (i.e., increased) reverse transcription efficiency, increased RT-PCR activity (e.g., more efficient amplification of RNA templates without affecting PCR efficiency on DNA templates), increased activity in the presence of Mg, and improved RT-PCR activity. 2+ In some embodiments, the present invention provides an improved RT-PCR efficiency in the presence of an inhibitor (e.g., a decomposition product of hemoglobin, such as heme and / or heparin), an increased reverse transcriptase activity, an increased extension rate compared to a control polymerase, and improved 3'-mismatch tolerance. See U.S. Patent Publication No. US2012-0258501, the contents of which are incorporated herein by reference in their entirety. Therefore, it is expected that the improved polymerase described herein comprising a substitution at the amino acid corresponding to position 709 of SEQ ID NO: 1 will also have the above-mentioned improved properties.

[0249] In addition to mutations and substitutions described herein, the DNA polymerase of the present invention may also include other non-substitution modifications. Such modifications may include, for example, covalent modifications known in the art to give other advantages in applications including polynucleotide extensions. For example, a type of such modification is a heat-reversible covalent modification that inactivates the enzyme, but when incubated at high temperatures (such as temperatures typically used for polynucleotide extensions), it reverses to activate the enzyme. Exemplary reagents for such heat-reversible modifications are described in U.S. Patent Nos. 5,773,258 and 5,677,152, which are expressly incorporated herein by reference in their entirety.

[0250] The DNA polymerase of the present invention can be constructed by mutating the DNA sequence encoding the corresponding unmodified polymerase (e.g., the corresponding variant from which the wild-type polymerase or the polymerase of the present invention is derived), such as by using a technique commonly referred to as site-directed mutagenesis. The nucleic acid molecules encoding the unmodified form of the polymerase can be mutated by various polymerase chain reaction (PCR) techniques well known to those of ordinary skill in the art. (See, for example, PCR Strategies (MAInnis, DHGelfand, and JJ Sninsky, ed., 1995, Academic Press, San Diego, CA) Chapter 14; PCR Protocols: A Guide to Methods and Applications (MAInnis, DHGelfand, JJ Sninsky and TJ White, ed., Academic Press, NY, 1990) described.

[0251] As a non-limiting example, the dual primer system used in the transformation site-directed mutagenesis kit from Clontech can be used to introduce site-directed mutations into a polynucleotide encoding an unmodified form of a polymerase. After denaturation of the target plasmid in this system, two primers are simultaneously annealed to the plasmid; one of these primers contains the desired site-directed mutation, and the other contains a mutation at another point in the plasmid that causes the elimination of a restriction site. The synthesis of the second chain is then performed, the two mutations are tightly linked, and the resulting plasmid is transformed into an E. coli mutS strain. Plasmid DNA is isolated from the transformed bacteria, restricted with the relevant restriction enzymes (thereby linearizing the unmutated plasmid), and then re-transformed into E. coli. This system allows mutations to be generated directly in the expression plasmid without subcloning or generating single-stranded phagemids. The tight connection of the two mutations and the subsequent linearization of the unmutated plasmid result in high mutation efficiency and allow minimal screening. After the synthesis of the initial restriction site primers, this method only requires the use of a new primer type for each mutation site. A set of "designed degenerate" oligonucleotide primers can be synthesized to introduce all required mutants at a given site simultaneously, rather than preparing mutants at each position separately. Plasmid DNA in the mutagenesis region can be sequenced to screen transformants to identify and sort mutant clones. Each mutant DNA can then be restricted and analyzed by electrophoresis, such as on a mutation detection enhancement gel (MallinckrodtBaker, Inc., Phillipsburg, NJ), to confirm that there are no other changes in the sequence (by comparing with a control that is not mutagenized). Alternatively, the entire DNA region can be sequenced to confirm that no other mutation events occur outside the target region.

[0252] DNA polymerases with more than one amino acid substitution can be generated in various ways. In the case where amino acids are densely located in the polypeptide chain, they can be mutated simultaneously using an oligonucleotide encoding all the required amino acid substitutions. However, if the amino acids are located at a certain distance from each other (e.g., separated by more than ten amino acids), it is more difficult to generate a single oligonucleotide encoding all the required changes. As an alternative, one of two alternative methods can be used. In the first method, isolated oligonucleotides are produced for each amino acid to be substituted. The oligonucleotides are then annealed to the single-stranded template DNA at the same time, and the second strand of DNA synthesized from the template will encode all the required amino acid substitutions. An alternative method involves two or more rounds of mutagenesis to produce the required mutants. The first round is as described for the single mutant: the DNA encoding the unmodified polymerase is used for the template, the oligonucleotide encoding the first amino acid substitution required is annealed to this template, and then a heteroduplex DNA molecule is generated. The second round of mutagenesis uses the mutant DNA produced in the first round of mutagenesis as a template. Therefore, this template already contains one or more mutations. Then the oligonucleotides encoding other required amino acid substitutions are annealed to this template, and the resulting DNA strand now encodes the mutations from both the first and second rounds of mutagenesis. The DNA thus produced can be used as a template in the third round of mutagenesis, etc. Alternatively, the multi-site mutagenesis method of Seyfang and Jin (Anal. Biochem. 324: 285-291. 2004) can be utilized.

[0253] Therefore, a recombinant nucleic acid encoding any one of the DNA polymerases of the present invention is also provided. Various vectors can be made using the nucleic acid encoding the DNA polymerase of the present invention. Any vector comprising a replicon and a control sequence derived from a species compatible with a host cell can be used in the practice of the present invention. Typically, an expression vector includes a transcription and translation regulatory nucleic acid region operably connected to a nucleic acid encoding a DNA polymerase. The term "control sequence" refers to a DNA sequence required for the expression of an operably connected coding sequence in a specific host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. In addition, the vector may include a forward and reverse regulatory element (PRE) to enhance the half-life of the transcribed mRNA (see U.S. Pat. No. 4,666,848 of Gelfand et al.). The transcription and translation regulatory nucleic acid region is generally suitable for host cells for expressing polymerases. A variety of appropriate expression vectors and suitable regulatory sequences for various host cells are known in the art. Typically, transcription and translation regulatory sequences may include, for example, promoter sequences, ribosome binding sites, transcription start and stop sequences, translation start and stop sequences, and enhancer or activator sequences. In a typical embodiment, the regulatory sequence includes a promoter and a transcription start and stop sequence. The vector also generally includes a polylinker region comprising several restriction sites for exogenous DNA insertion. In certain embodiments, a "fusion tag" is used to facilitate purification and the removal of subsequent tag / tag sequences (if necessary), such as a "histidine tag (His-Tag)". However, these are usually unnecessary when purifying heat-active and / or heat-stable proteins from a mesophilic host (e.g., Escherichia coli), wherein a "heat step" can be used. The construction of a suitable vector comprising a DNA encoding a replication sequence, a regulatory sequence, a phenotypic selection gene, and a target polymerase is prepared using standard recombinant DNA procedures. Isolated plasmids, viral vectors, and DNA fragments are cut, clipped, and connected together in a specific order to generate the desired vector, which is well known in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, NY, 2nd edition 1989)).

[0254] In certain embodiments, the expression vector includes a selectable marker gene to allow selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used. Suitable selection genes can include, for example, genes encoding ampicillin and / or tetracycline resistance, which enable cells transformed with these vectors to grow in the presence of these antibiotics.

[0255] In one aspect of the invention, a nucleic acid encoding a DNA polymerase is introduced into a cell alone or in combination with a vector. As used herein, "introduced" or grammatical equivalents mean that the nucleic acid enters the cell in a manner suitable for subsequent nucleic acid integration, amplification and / or expression. The method of introduction is primarily determined by the target cell type. Exemplary methods include CaPO4 precipitation, liposome fusion, Electroporation, viral infection, etc.

[0256] In some embodiments, prokaryotes are often used as host cells for the initial cloning steps of the present invention. They are particularly useful for the rapid production of large amounts of DNA, the production of single-stranded DNA templates for site-directed mutagenesis, the simultaneous screening of many mutants, and DNA sequencing of the generated mutants. Suitable prokaryotic host cells include E. coli K12 strain 94 (ATCC No. 31,446), E. coli strain W3110 (ATCC No. 27,325), E. coli K12 strain DG116 (ATCC No. 53,606), E. coli X1776 (ATCC No. 31,537), and E. coli B; however, many other strains of E. coli such as HB101, JM101, NM522, NM538, NM539, and many other species and genera of prokaryotes including Bacillus such as Bacillus subtilis, other Enterobacteriaceae such as Salmonella typhimurium or Serratia marcescens, and various species of Pseudomonas can all be used as hosts. Prokaryotic host cells or other host cells with rigid cell walls are typically transformed using the calcium chloride method as described in Section 1.82 of Sambrook et al., supra. Alternatively, electroporation can be used to transform these cells. Prokaryotic transformation techniques are described, for example, in Dower, in Genetic Engineering, Principles and Methods 12: 275-296 (Plenum Publishing Corp., 1990); Hanahan et al., Meth. Enzymol., 204: 63, 1991. Plasmids commonly used for E. coli transformation include pBR322, pUCI8, pUCI9, pUCI18, pUC119, and Bluescript M13, all of which are described in Sections 1.12-1.20 of Sambrook et al., supra. However, many other suitable vectors are also available.

[0257] DNA polymerases of the present invention are generally produced by culturing host cells under appropriate conditions for inducing or causing expression of DNA polymerases, and the host cells are transformed with expression vectors comprising nucleic acids encoding DNA polymerases. Methods for culturing transformed host cells under conditions suitable for protein expression are well known in the art (see, e.g., Sambrook et al., supra). Suitable host cells for producing polymerases from plasmid vectors comprising λpL promoters include Escherichia coli strain DG116 (ATCC No. 53606) (see U.S. Patent No. 5,079,352 and Lawyer, FC et al., PCR Methods and Applications 2: 275-87, 1993, both of which are incorporated herein by reference). After expression, polymerases can be harvested and separated. Methods for purifying thermostable DNA polymerases are described in, e.g., Lawyer et al., supra. Once purified, DNA polymerases can be tested for increased chain displacement activity, improved RT efficiency, increased mismatch tolerance, extension rate and / or tolerance to RT and polymerase inhibitors (e.g., as described in the examples).

[0258] The improved DNA polymerase of the present invention can be used for any purpose in which such enzymatic activity is necessary or required. Therefore, in another aspect of the present invention, a polynucleotide extension method (e.g., PCR) using a polymerase is provided. Conditions suitable for polynucleotide extension are known in the art. (See, e.g., Sambrook et al., supra; see also Ausubel et al., Short Protocols in Molecular Biology (4th ed., John Wiley & Sons 1999). Typically, a primer is annealed, i.e., hybridized to a target nucleic acid to form a primer-template complex. The primer-template complex is contacted with a DNA polymerase and a nucleoside triphosphate in a suitable environment to allow one or more nucleotides to be added to the 3'-end of the primer, thereby producing an extended primer complementary to the target nucleic acid. The primer may include, for example, one or more nucleotide analogs. In addition, the nucleoside triphosphate may be a conventional nucleotide, an unconventional nucleotide (e.g., a ribonucleotide or a labeled nucleotide), or a mixture thereof. In some variants, the polynucleotide extension reaction includes amplification of the target nucleic acid. Suitable conditions for nucleic acid amplification using a DNA polymerase and a primer pair are also known in the art (e.g., PCR amplification methods). (See, e.g., Sambrook et al., supra; Ausubel et al., supra; PCR Applications: Protocols for Functional Genomics (Innis et al., ed., Academic Genomics, 2001). Press 1999). In other non-mutually exclusive embodiments, the polynucleotide extension reaction comprises reverse transcription of an RNA template (e.g., RT-PCR). In some embodiments, an improved polymerase is found for 454 sequencing (Margulies, M et al. 2005, Nature, 437, 376-380).

[0259] Optionally, the primer extension reaction may include an actual or potential inhibitor of a reference or unmodified polymerase. The inhibitor may inhibit, for example, the nucleic acid extension rate and / or reverse transcription efficiency of a reference or unmodified (control) polymerase. In some embodiments, the inhibitor is hemoglobin or its degradation product. For example, in some embodiments, the hemoglobin degradation product is a heme decomposition product, such as heme, hematoporphyrin or bilirubin. In some embodiments, the inhibitor is an iron chelator or a purple pigment. In some embodiments, the inhibitor is heparin. In certain embodiments, the inhibitor is an intercalating dye. In certain embodiments, the inhibitor is melanin, which has been described as a polymerase inhibitor. See, for example, Ekhardt, et al., Biochem Biophys Res Commun. 271 (3): 726-30 (2000).

[0260] The DNA polymerase of the present invention can be used to extend the template in the presence of a polynucleotide template separated from a sample (such as blood) containing a polymerase inhibitor. For example, the DNA polymerase of the present invention can be used to extend the template in the presence of hemoglobin, the main component of blood, or in the presence of hemoglobin degradation products. Hemoglobin can be degraded into various heme decomposition products, such as heme, hemoglobin, hematoporphyrin and bilirubin. Therefore, in certain embodiments, the DNA polymerase of the present invention can be used to extend the template in the presence of hemoglobin degradation products (including but not limited to heme, hemoglobin, hematoporphyrin and bilirubin). In certain embodiments, the hemoglobin degradation product is heme. In certain embodiments, the DNA polymerase of the present invention can be used to extend the template in the presence of about 0.5 to 20.0 μM, about 0.5 to 10.0 μM, about 0.5 to 5.0 μM, about 1.0 to 10.0 μM, about 1.0 to 5.0 μM, about 2.0 to 5.0 μM or about 2.0 to 3.0 μM heme. In other embodiments, the DNA polymerase of the present invention can be used to extend the template in the presence of at least about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 10.0, 20.0 or greater than 20 μM heme. The decomposition products of hemoglobin include iron chelators and purple pigments. Therefore, in some embodiments, the DNA polymerase of the present invention can be used to extend the template in the presence of iron chelators and / or purple pigments. In other embodiments, the DNA polymerase of the present invention can be used to extend the template in the presence of a certain amount of hemoglobin degradation products, which can inhibit the extension of the same template by a reference or control DNA polymerase.

[0261] DNA polymerase of the present invention can be used for extending template in the presence of heparin. Heparin is usually present in samples separated from blood as an anticoagulant. In certain embodiments, DNA polymerase of the present invention can be used for extending template in the presence of about 1.0 to 400 ng / μl, 1.0 to 300 ng / μl, 1.0 to 200 ng / μl, 5.0 to 400 ng / μl, 5.0 to 300 ng / μl, 5.0 to 200 ng / μl, 10.0 to 400 ng / μl, 10.0 to 300 ng / μl or 10.0 to 200 ng / μl heparin. In some embodiments, the DNA polymerase of the invention can be used to extend the template in the presence of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400 ng / μ l or more than 400 ng / μ l heparin. In other embodiments, the DNA polymerase of the invention can be used to extend the template in the presence of an amount of heparin that inhibits extension of the same template by a reference or control DNA polymerase.

[0262] In some embodiments, the improved polymerase described herein is used in a strand displacement reaction. In some embodiments, the strand displacement reaction is carried out in a mixture comprising a DNA template, one or more primers and a thermostable DNA polymerase as described herein. The reaction mixture typically comprises all four standard deoxyribonucleoside triphosphates (dNTPs) and a buffer containing a divalent cation and a monovalent cation. Exemplary cations include, for example, Mg, 2+ , although other cations such as Mn 2+ or Co 2+ The DNA polymerase can be activated. In other embodiments, the strand displacement reaction is performed with a thermoactive DNA polymerase of the present invention. In a specific embodiment, the improved polymerase described herein allows for more efficient strand displacement reactions at high temperatures while having reduced exonuclease and / or endonuclease activity.

[0263] In some embodiments, the improved polymerase has increased strand displacement activity compared to a control polymerase.It was not previously appreciated that amino acid substitutions at corresponding positions of SEQ ID NO: 1 or SEQ ID NO: 40 described herein could result in increased strand displacement activity.

[0264] In some embodiments, using RNA template, the improved polymerase has increased reverse transcription efficiency, while using DNA template, the polymerase activity is not significantly reduced. Therefore, in some embodiments, when compared with the control polymerase, the improved DNA polymerase has increased RT efficiency, while the DNA-dependent polymerase activity is not significantly reduced. In some embodiments, the improved DNA polymerase described herein has a DNA-dependent polymerase activity substantially the same as the control polymerase. Therefore, in some embodiments, the DNA-dependent polymerase activity of the improved DNA polymerase described herein is at least about 90% of the control polymerase activity, for example, at least about 90%, 91%, 92%, 93%, 94%, 95% or higher of the control polymerase activity. The DNA-dependent polymerase activity can be measured, for example, by amplifying the DNA template as described herein and determining the Cp value. Therefore, in some embodiments, the DNA polymerase has an improved RT efficiency, which is measured as having a reduced Cp value compared to the control polymerase when RNA is used as a template, and having a substantially unchanged Cp value relative to the control polymerase when DNA is used as a template. For example, when amplifying a DNA template, the improved DNA polymerase can have a Cp value that differs by less than 1.0, less than 0.5, less than 0.4, less than 0.3, less than 0.2, or less than 0.1 compared to a control polymerase. In some embodiments, DNA-dependent polymerase activity is determined as described in the examples.

[0265] In certain embodiments, the improved polymerase of the present invention can increase reverse transcription efficiency by reducing the reaction time required for extending RNA template.For example, compared with the control polymerase, the improved polymerase described herein can significantly shorten the reaction time required for RNA transcription into cDNA, thereby improving reverse transcriptase efficiency.Not limited by theory, the improved polymerase can increase RT efficiency by, for example, increasing the activity of the enzyme on the RNA template, such as increasing the rate of nucleotide incorporation and / or increasing the continuous synthesis ability of the polymerase, thereby effectively shortening the extension time of RNA template or RNA template colony.When using unmodified or control polymerase, the reaction time for the initial RT step is usually about 30 minutes or longer at 65 degrees Celsius.Therefore, in certain embodiments, the improved polymerase can be less than about 30 minutes, less than about 20 minutes, less than about 10 minutes, less than about 8 minutes, less than about 5 minutes, less than about 4 minutes, less than about 3 minutes or less than about 2 minutes at 65 degrees Celsius.The RNA template is transcribed into cDNA. In certain embodiments, the improved polymerase can transcribe into cDNA the RNA template (comprising the first 800 bases of HCV genotype Ib5'NTR) derived from hepatitis C virus (HCV) transcript JP2-5 in a shorter time or faster than the control polymerase. For example, the improved polymerase can transcribe into full-length cDNA 240 bases of HCV JP2-5 RNA template in the same reaction conditions approximately less than 15 seconds, less than 30 seconds, less than one minute, less than two minutes, less than 3 minutes, less than 4 minutes, less than 5 minutes or approximately less than 10 minutes than the control polymerase. In certain embodiments, the improved polymerase can transcribe into full-length cDNA 240 bases of HCV JP2-5 RNA template faster than the control polymerase, for example, faster than the control polymerase by approximately 5 seconds, 10 seconds, 15 seconds, 30 seconds, 45 seconds or 60 seconds or faster under the same reaction conditions. In certain embodiments, the reaction conditions are those described in the examples. In some embodiments, the improved polymerase described herein is contacted with the RNA template in the above reaction mixture at 65 degrees Celsius for about 2 minutes. As described in the examples, the extension step can be followed by PCR amplification of the extended template.

[0266] Use Mn 2+ As divalent metal ion activators, the most efficient RT activity has been achieved in thermostable DNA polymerases. However, it is well known that when Mn is present in the reaction 2+When the fidelity of the DNA polymerase is low, the fidelity of the DNA polymerase is low. Unless one is trying to generate mutations, one generally prefers to keep the fidelity high. Fortunately, most routine sequencing, PCR, and RT-PCR applications do not require high fidelity conditions, as the detection system is usually focused on the product population. With the advent of next-generation sequencing, digital PCR, etc., product fidelity is becoming increasingly important, and methods that allow for higher fidelity DNA synthesis are becoming essential. Using Mg 2+ Using Mg as a divalent metal ion activator to achieve efficient RT activity is an excellent way to significantly improve the fidelity of DNA polymerases and allow for more reliable replication of nucleic acid targets. Therefore, in some embodiments, as described in the examples, the improved polymerases of the present invention allow the use of Mg 2+ Acts as a divalent metal ion activator to efficiently extend and / or amplify RNA templates.

[0267] Because polysaccharase as herein described also can have the mispairing tolerance of increase, so polysaccharase is used for the target template change that may occur and no matter how change at high temperature still expect in the method for amplification template.The example of this type of template can include for example virus, bacterium or other pathogen sequence.In many embodiments, it is desirable to simply determine whether individual (human or non-human animal) has viral infection or other infection, no matter how the definite viral variant of having infected individual.As an example, even if the specific virus of infected individual has the sudden change that causes mispairing at primer hybridization site, primer pair can also be used to use polysaccharase amplification HCV of the present invention and detect the existence of HCV.

[0268] The target nucleic acid can be from a biological or synthetic source. The target can be, for example, DNA or RNA. Typically, where an amplicon is generated, the amplicon will consist of DNA, although ribonucleotides or synthetic nucleotides may also be incorporated into the amplicon. Where it is desired to detect RNA, the amplification process will typically involve the use of reverse transcription, including, for example, reverse transcription PCR (RT-PCR).

[0269] Specific target sequences can include, for example, viral nucleic acids (e.g., human immunodeficiency virus (HIV), hepatitis B virus (HBV), (cytomegalovirus (CMV), parvo B19 virus, Epstein-Barr virus, hepatitis C virus (HCV), human papillomavirus (HPV), Japanese encephalitis virus (JEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV), Murray Valley encephalitis virus, and Kunkin virus), bacterial nucleic acids (e.g., Staphylococcus aureus, Neisseria meningitidis, Plasmodium falciparum, Chlamydia trachomatis, Chlamydia trachomatis), mycobacteria, fungal nucleic acids, or animal or plant nucleic acids. In some embodiments, the target nucleic acid is an animal (e.g., human) nucleic acid or is derived from an animal (e.g., human) sample (i.e., a virus or other pathogenic organism). The nucleic acid may be present in a sample from an animal biopsy, a blood sample, a urine sample, a fecal sample, a saliva, etc.). In some embodiments, the target nucleic acid is, for example, a human genetic region, which may include variants associated with a disease (e.g., cancer, diabetes, etc.). Because in some embodiments, the polymerases of the invention have mismatch tolerance, such enzymes are particularly useful, for example, where multiple related sequences may be present in the target sequence. As an example, the invention may be used to detect viral pathogens where the viral pathogens have sufficient variation in their genomes to make it difficult or impossible to design a single or small set of primers that will amplify most or all possible viral genomes; or where there is sufficient variation in cancer or other disease genetic markers where sequence variation is known or may occur.

[0270] Other methods for detecting extension products or amplification products using improved polymerase as described herein include using fluorescent double-stranded nucleotides in combination with dyes or fluorescent double-stranded nucleotides intercalating dyes. The example of fluorescent double-stranded DNA in combination with dyes includes SYBR-green (molecular probes). The double-stranded DNA in combination with dyes can be used in combination with melting curve analysis to measure primer extension products and / or amplification products. Melting curve analysis can be performed on real-time PCR instruments, such as ABI 5700 / 7000 (96-well format) or ABI 7900 (384-well format) instruments with onboard software (SDS2.1). Alternatively, melting curve analysis can be performed as endpoint analysis. The exemplary method of melting point analysis is described in U.S. Patent Publication No. 2006 / 0172324, the contents of which are clearly incorporated herein by reference in their entirety.

[0271] In another aspect of the present invention, a kit for the primer extension method described herein is provided. In some embodiments, the kit is zoned for ease of use and includes at least one container, which provides a modified DNA polymerase according to the present invention. One or more other containers providing other reagents may also be included. In some embodiments, the kit may also include a blood collection tube, container or unit containing heparin or its salt or releasing heparin into a solution. The blood collection unit may be a heparinized tube. Such other containers may include any reagent or other element known to a skilled person for a primer extension procedure according to the above method, including reagents for, for example, nucleic acid amplification procedures (e.g., PCR, RT-PCR), DNA sequencing procedures, or DNA labeling procedures. For example, in certain embodiments, the kit further includes a container, which provides a 5' sense primer that can be hybridized with a predetermined polynucleotide template under primer extension conditions, or provides a primer pair including a 5' sense primer and a corresponding 3' antisense primer. In other non-mutually exclusive variants, the kit includes one or more (conventional and / or unconventional) containers providing nucleoside triphosphates. In certain embodiments, the kit includes α-phosphorothioate dNTPs, dUTPs, dITPs, and / or labeled dNTPs, such as fluorescein or cyanine dye family dNTPs. In other non-mutually exclusive embodiments, the kit includes one or more containers providing a buffer suitable for a primer extension reaction.

[0272] In another aspect of the invention, a reaction mixture is provided, comprising a polymerase having increased reverse transcriptase efficiency, mismatch tolerance, extension rate and / or RT and polymerase inhibitor tolerance as described herein. The reaction mixture may further comprise reagents for, for example, nucleic acid amplification procedures (e.g., PCR, RT-PCR), DNA sequencing procedures, or DNA labeling procedures. For example, in certain embodiments, the reaction mixture comprises a buffer suitable for primer extension reactions. The reaction mixture may also comprise template nucleic acid (DNA and / or RNA), one or more primers or probe polynucleotides, nucleoside triphosphates (including, for example, deoxyribonucleotides, ribonucleotides, labeled nucleotides, unconventional nucleotides), salts (e.g., Mn 2+ Mg 2+), a label (e.g., a fluorophore). In some embodiments, the reaction mixture comprises a 5'-sense primer that can hybridize with a predetermined polynucleotide template under primer extension conditions, or a primer pair comprising a 5'-sense primer and a corresponding 3' antisense primer. In some embodiments, the reaction mixture comprises α-phosphorothioate dNTPs, dUTPs, dITPs and / or labeled dNTPs, such as fluorescein or cyanine dye family dNTPs. In some embodiments, the reaction mixture comprises an iron chelator or a violet dye. In certain embodiments, the reaction mixture comprises hemoglobin or a degradation product of hemoglobin. For example, in certain embodiments, the degradation product of hemoglobin includes heme decomposition products, such as heme, hemoglobin, hematoporphyrin and bilirubin. In other embodiments, the reaction mixture comprises heparin or a salt thereof. In certain embodiments, the reaction mixture comprises a template nucleic acid separated from blood. In other embodiments, the template nucleic acid is RNA, and the reaction mixture comprises heparin or a salt thereof.

[0273] In certain embodiments, reaction mixture comprises two or more polymerases.For example, in certain embodiments, reaction mixture comprises the first DNA polymerase with increased reverse transcriptase efficiency compared with control polymerase, and the second DNA polymerase with DNA dependent polymerase activity.The second DNA polymerase can be a wild-type or unmodified polymerase, or can be a modified polymerase with increased DNA dependent polymerase activity.By providing a polymerase with increased reverse transcriptase activity and a polymerase with DNA dependent polymerase activity, such reaction mixture can be used for amplifying RNA templates (for example, RT-PCR).

[0274] Examples

[0275] The following examples are provided to illustrate but not to limit the claimed invention.

[0276] Example 1: Library generation

[0277] In brief, the steps in this screening process include library generation, expression and partial purification of mutant enzymes, screening of enzymes for desired properties, DNA sequencing, clone purification, and further characterization of selected candidate mutants. Each of these steps is further described below.

[0278] Clone library generation: Nucleic acid encoding the C21 DNA polymerase G46E mutation was cloned using GeneMorph II for error-prone PCR TM Random mutagenesis kit (Agilent Technologies) was used to perform error-prone (mutagenic) PCR. TMThe PCR fragments were cloned using the Takara Bio USA, Inc cloning system to create a mutagenesis library. The cloned inserts were transformed into chemically competent LK4 cells. The library was then screened for elevated strand displacement activity of the expressed mutant polymerase.

[0279] Assay Design: Assay design is Figure 3 Four complementary oligonucleotides Oligo A (green), Oligo B (light blue), Oligo C (dark blue) and locked nucleic acid (LNA) Oligo D (red) are annealed to each other. Oligo A is complementary to Oligo B and initiates polymerization. Oligo B is the template and has a FAM – fluorescent reporter at the 5'-end. Oligo C is complementary to Oligo B and has a BHQ quencher at the 3'-end. Locked nucleic acid Oligo D is complementary to template Oligo B and acts as a high energy fence. When polymerase, Mg 2+ When nucleotides are added to the annealed mixture, polymerization extends Oligo A and the newly synthesized strand displaces LNA Oligo D and Oligo C, thereby releasing the quencher from the fluorescent probe. A fluorescent signal is generated after the cycle is completed. The thermal cycling conditions are:

[0280] Denaturation 95℃ 3”

[0281] Annealing 60℃ 5”

[0282] Extension 65°C 30”

[0283] 30 cycles

[0284] Cloned PCR fragments were sequenced to determine the presence of mutations in any individual clone.

[0285] Nuclease activity To determine nuclease activity, extension reactions are performed without the addition of dNTPs. Any increase in fluorescence signal when no dNTPs are present in the reaction is attributed to the nuclease activity of the enzyme. If no increase in fluorescence signal is observed, the enzyme has little or significantly reduced endonuclease and exonuclease activity.

[0286] Results: A number of clones were identified that had increased strand displacement activity compared to the parental G46E C21 polymerase. Representative plates containing individual bacterial colonies (clones) were Figure 4A and 4B In summary, the following clones with increased strand displacement activity were sequenced and mutations are indicated (where the first number corresponds to Figure 4A and 4B The board number is shown in the figure, and the letter-number (e.g., F24) corresponds to the hole on the board):

[0287] Plate 1_Clone F24: Mutations: I686V and A693V

[0288] Plate 1_Clone L3: Mutations: T516I and V633I

[0289] Plate 1_Clone P19: Mutations: Q415H, E420D, E636G, N752S, V768M

[0290] Plate 2_Clone M22: Mutations: R525G and F694S

[0291] Plate 2_Clone G9: Mutations: Q491H and T516S

[0292] Plate 2_Clone N19: Mutations: S515F and T666M

[0293] Plate 2_Clone N7: Mutations: E402V, V555A, and N582D

[0294] Plate 3_Clone A24: Mutations: A737T and A759T

[0295] Plate 3_Clone F21: Mutations: L521Q and T546A

[0296] Plate 3_Clone G10: Mutation: N668S

[0297] Plate 3_Clone I14: Mutation: A456T

[0298] Plate 4_Clone G23: Mutations: K507M, T571A and S652F

[0299] Plate 4_Clone K12: Mutations: S515F and A832V

[0300] Plate 5_Clone C6: Mutations: D498E, L524V, R598G, and M616I

[0301] Plate 5_Clone G20: Mutations: A444T, D498E, M660K, and Y673N

[0302] Plate 5_Clone G23: Mutations: E493D, T511S, M648I, and M749L

[0303] Plate 5_Clone H21: Mutation: Q635K

[0304] The mutations are spread throughout the polymerase domain. These mutations include the following "hot spots" or segments in the primary amino acid sequence: 515–516, 521–525, 633–636, 666–668, 693–694.

[0305] Nuclease activity: Figure 5A and 5B Results from two plates where no dNTPs were added to the extension reaction are shown. Most clones did not show any fluorescent signal. It should be noted that some wells showed fluorescent signals, but they were at background levels and no higher than 0.5 relative fluorescent units (RFU).

[0306] This example demonstrates that the mutant polymerases have increased or enhanced strand displacement activity compared to the G46E parent enzyme. The mutant polymerases can also function at high temperatures (i.e., they withstand a denaturation temperature of 95°C, an annealing temperature of 60°C, and an extension temperature of 65°C. In addition, the mutant polymerases exhibit very little exonuclease or endonuclease activity.

[0307] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in view of them will be suggested to those skilled in the art, and will be included within the spirit and limits of this application and within the scope of the appended claims of the present invention. All publications, serial accession numbers, patents and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

Claims

1. A DNA polymerase having increased 5'-3' strand displacement activity and significantly reduced 5'-3' exonuclease and endonuclease activities compared to a control DNA polymerase consisting of the amino acid sequence of SEQ ID NO:40, wherein the amino acid sequence of the DNA polymerase is identical to the amino acid sequence of SEQ ID NO:40 except for a combination of mutations at positions corresponding to positions 686 and 693 of SEQ ID NO:40; The mutations described are I686V and A693V.

2. The DNA polymerase of claim 1, wherein the increased 5'-3' strand displacement activity and significantly reduced 5'-3' exonuclease and endonuclease activities occur at high temperatures.

3. A recombinant nucleic acid encoding the DNA polymerase according to any one of claims 1 to 2.

4. A method for performing primer extension, comprising: The DNA polymerase according to any one of claims 1 to 2 is contacted with the primer, the polynucleotide template and nucleoside triphosphates under conditions suitable for primer extension, thereby generating an extended primer.

5. The method according to claim 4, wherein: The primer extension method comprises a strand displacement reaction, a polymerase chain reaction, an isothermal amplification, or an amplification reaction selected from loop-mediated amplification, cross-primer amplification and a polymerase chain displacement reaction.

6. The method according to any one of claims 4 and 5, wherein the conditions include an elevated temperature.

7. A kit for producing extended primers, comprising: At least one container comprising the DNA polymerase according to any one of claims 1 to 2.

8. The kit according to claim 7, further comprising one or more other containers selected from the group consisting of: (a) providing a container for a primer that can hybridize to a predetermined polynucleotide template under primer extension conditions; (b) providing a container for nucleoside triphosphates; and (c) providing a container for a buffer suitable for primer extension.

9. A reaction mixture comprising the DNA polymerase according to any one of claims 1 to 2, at least one primer, a polynucleotide template and nucleoside triphosphates.

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