DNA polymerases with improved activity

Abstract

Disclosed are novel modified DNA polymerases having improved reverse transcriptase efficiency, improved specificity, increased mismatch tolerance, increased extension rate, and increased tolerance of inhibitors of reverse transcription (RT) and polymerase, relative to a corresponding, unmodified polymerase. These novel modified polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the novel modified DNA polymerases.

Description

[0001] DNA POLYMERASES WITH IMPROVED ACTIVITY

[0002] SEQUENCE LISTING

[0003] The instant patent application contains a Sequence Listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created December 13, 2024, is named “P39291-US_SL” and is 86,282 bytes in size.

[0004] FIELD OF THE INVENTION

[0005] The present invention provides DNA polymerases with improved activities, including increased reverse transcriptase efficiency, mismatch tolerance, extension rate and / or tolerance of reverse transcriptase (RT) and polymerase inhibitors, as well as use of such polymerases in various applications, including nucleic acid polynucleotide extension and amplification.

[0006] BACKGROUND OF THE INVENTION DNA polymerases are responsible for the replication and maintenance of the genome, a role that is central to accurately transmitting genetic information from generation to generation. DNA polymerases function in cells as the enzymes responsible for the synthesis of DNA. They polymerize deoxyribonucleoside triphosphates in the presence of a metal activator, such as Mg2+or Mn2+, in an order dictated by the DNA template or polynucleotide template that is copied. In vivo, DNA polymerases participate in a spectrum of DNA synthetic processes including DNA replication, DNA repair, recombination, and gene amplification. During each DNA synthetic process, the DNA template is copied once or at most a few times to produce identical replicas. In contrast, in vitro, DNA replication can be repeated many times such as, for example, during polymerase chain reaction (see, e.g., U.S. Patent No. 4,683,202).

[0007] In the initial studies with polymerase chain reaction (PCR), the DNA polymerase was added at the start of each round of DNA replication (see U.S. Patent No. 4,683,202, supra). PCR is a method for making billions of copies of a specific nucleic acid or DNA / RNA sample rapidly, allowing for amplification of a very small sample of nucleic acid or DNA / RNA. Subsequently, it was determined that thermostable DNA polymerases could be obtained from bacteria that grow at elevated temperatures, and that these enzymes need to be added only once (see U.S. Patent No. 4,889,818 and U.S. Patent No.

[0008] 4,965,188). At the elevated temperatures used during PCR, these enzymes are not irreversibly inactivated. As a result, one can carry out repetitive cycles of polymerase chain reactions without adding fresh enzymes at the start of each synthetic addition process. DNA polymerases, particularly thermostable polymerases, are the key to a large number of techniques in recombinant DNA studies and in medical diagnosis of disease. For diagnostic applications in particular, a target nucleic acid sequence may be only a small portion of the DNA or RNA in question, so it may be difficult to detect the presence of a target nucleic acid sequence without amplification.The overall folding patern of DNA polymerases resembles the human right hand and contains three distinct subdomains of palm, fingers, and thumb (see Beese et al., Science 260:352-355, 1993); Patel et al., Biochemistry 34:5351-5363, 1995). While the structure of the fingers and thumb subdomains vary greatly between polymerases that differ in size and in cellular functions, the catalytic palm subdomains are all superimposable. For example, motif A, which interacts with the incoming dNTP and stabilizes the transition state during chemical catalysis, is superimposable with a mean deviation of about one A amongst mammalian pol a and prokaryotic pol I family DNA polymerases (Wang et al., Cell 89:1087-1099, 1997). Motif A begins structurally at an antiparallel 0-strand containing predominantly hydrophobic residues and continues to an a-helix. The primary amino acid sequence of DNA polymerase active sites is exceptionally conserved. In the case of motif A, for example, the sequence DYSQIELR (SEQ ID NO:22) is retained in polymerases from organisms separated by many millions years of evolution, including, e.g., Thermits aquaticus, Chlamydia trachomatis, and Escherichia coli.

[0009] In addition to being well-conserved, the active site of DNA polymerases has also been shown to be relatively mutable, capable of accommodating certain amino acid substitutions without reducing DNA polymerase activity significantly (see, e.g., U.S. Patent No. 6,602,695). Such mutant DNA polymerases can offer various selective advantages in, e.g., diagnostic and research applications comprising nucleic acid synthesis reactions.

[0010] There are at least two steps in the enzymatic process of DNA polymerization; 1) the incorporation of the incoming nucleotide and 2) the extension of the newly incorporated nucleotide. The overall faithfulness or “fidelity” of the DNA polymerase is generally thought of as a conglomerate of these two enzymatic activities, but the steps are distinct. A DNA polymerase may misincorporate the incoming nucleotide, but if it is not efficiently extended the extension rate will be severely decreased and overall product formation would be minimal. Alternatively, it is possible to have a DNA polymerase misincorporate the incoming nucleotide and readily misextend the newly formed mismatch. More particularly, the DNA polymerase may incorporate an incorrect incoming nucleotide with a relatively high rate of occurrence, thereby forming a 3’ terminal mismatch, and once this error has occurred, the mismatched nucleotide may be readily extended. In this case, the overall extension rate would be high, but the overall fidelity would be low (i.e., poor fidelity). An example of this type of enzyme would be ESI 12 DNA polymerase (E683R Z05 DNA polymerase; see U.S. Patent No.

[0011] 7,179,590) when using Mn2+as the divalent metal ion activator. The enzyme has a very high efficiency because unlike typical DNA polymerases that tend to hesitate / stall when a mismatch is encountered, the ESI 12 DNA polymerase readily extends the mismatch. The phenotype displayed in ESI 12 is more pronounced during the RT step, presumably because of structural effects of the RNA / DNA heteroduplex vs. the DNA / DNA homoduplex.In another aspect, it may be desirable to provide a DNA polymerase that does not readily misincorporate (i.e., may be even less likely to misincorporate) an incoming nucleotide but does have increased or otherwise improved capacity to extend a mismatch. In this case, the fidelity is not significantly altered for the overall product as the rate of misincorporation events resulting in mismatches is low, while the rate of extension of such mismatched nucleotides remains high. Fidelity of DNA replication refers to the accuracy with which the DNA sequence is copied, which is maintained by the action of DNA polymerases. In general, this type of enzyme is more favorable for extension reactions than an enzyme having the characteristics of ESI 12 in Mn2+because the fidelity of the product is improved. However, this attribute can be utilized to allow the extension of a mismatched oligonucleotide primer such as when an oligonucleotide primer of a single sequence is hybridized to a target that has sequence heterogeneity (e.g., viral targets), but the normal or lower misincorporation rate allows for completion of DNA synthesis beyond the original oligonucleotide primer. An example of this type of DNA polymerase is Z05 D580G DNA polymerase (see U.S. Patent Publication No.

[0012] 2009 / 0148891). This type of activity is referred to as “mismatch tolerant” because it is more tolerant to mismatches in the oligonucleotide primer. While the examples above have discussed primer extension type reactions, the activity can be more significant in reactions such as RT-PCR and PCR where primer extension is reoccurring frequently. Data suggests that while enzymes such as Z05 D580G are more “tolerant” to mismatches, they also have enhanced ability to extend oligonucleotide primers containing modified bases (e.g., t-butyl benzyl modified bases) or in the presence of DNA binding dyes such as SYBR Green I (see U.S. Patent Publication No. 2009 / 028053).

[0013] Reverse transcription polymerase chain reaction (RT-PCR) is a technique used in many applications to detect and / or quantify RNA targets by amplification. RT-PCR is a method / process which involves: (i) transcription of RNA into DNA, and (ii) amplification of specific DNA targets using PCR. In order to amplify RNA targets by PCR, it is necessary to first reverse transcribe the RNA template into cDNA. Typically, RT-PCR assays rely on a non-thermostable reverse transcriptase (RNA dependent DNA polymerase), derived from a mesophilic organism, for the initial cDNA synthesis step (RT). An additional thermostable DNA polymerase is required for amplification of cDNA to tolerate elevated temperatures required for nucleic acid denaturation in PCR. There are several potential benefits of using thermoactive or thermostable DNA polymerases engineered to perform more efficient reverse transcription for RT-PCR assays. Increased reverse transcriptase activity coupled with the ability to use higher reverse transcription incubation temperatures, which allow for relaxing of RNA template secondary structure, can result in overall higher cDNA synthesis efficiency and assay sensitivity. Reverse transcriptase activity refers to the process of converting RNA into DNA using the enzyme reverse transcriptase. In particular, reverse transcription refers to when a reverse transcriptase catalyzes the conversion of RNA template molecules into a DNA double helix. Higher temperature incubationcould also increase specificity of the amplification reaction by reducing false priming in the reverse transcription step. False priming is a phenomenon that can occur in polymerase chain reaction (PCR) and reverse transcriptase polymerase chain reaction (RT-PCR) and occurs when a primer binds to a non-target sequence on a template, resulting in the amplification of unwanted products. Specify, as it relates to a polymerase, is the ability of a polymerase to select the correct nucleoside triphosphate (dNTP) from a pool of similar molecules. Enzymes with improved reverse transcription activity can simplify assay design by allowing for reduced RT incubation times and / or enzyme concentration. When using dUTP and uracil N-glycosylase (UNG), non-specific extension products containing dUMP that are formed during nonstringent set-up conditions are degraded by UNG and cannot be utilized either as primers or as templates. When using a non-thermostable reverse transcriptase (RNA dependent DNA polymerase) derived from a mesophilic organism, it is not possible to utilize the dUTP and UNG methodologies. (Myers, T.W. et al., Amplification of RNA: High Temperature Reverse Transcription and DNA Amplification with Thermus thermophilus DNA Polymerase, in PCR Strategies, Innis, M.A., Gelfand, D.H., and Sninsky, J. J., Eds., Academic Press, San Diego, CA, 58-68, (1995)). However, the use of a thermoactive or thermostable DNA polymerase of the invention for the reverse transcription step enables the reaction to be completely compatible with the utilization of the dUTP / UNG carry-over prevention system (Longo et al., Use of Uracil DNA Glycosylase to Control Carry-over Contamination in Polymerase Chain Reactions. Gene 93:125-128, (1990). In addition to providing carry-over contamination control, the use of dUTP and UNG provides a “hot-start” to reduce non-specific amplification (Innis and Gelfand 1999).

[0014] As already discussed above and in various situations, it is useful to provide a DNA polymerase that exhibits RT activity. Previously, it has been demonstrated that Thermus sp. DNA polymerases, such as Z05 (SEQ ID NO:1), Z05D, and Z05D I616M I709K, exhibit RT activity in the presence of manganese (Mn2+); however, it has been observed that under certain conditions, there is a loss of fidelity associated with the use of Mn2+, as well as an increase in non-specific amplification, including as the result of primer-primer interactions. The Z05 polymerase is disclosed in U.S. Patent No.

[0015] 8,735,120. While loss of fidelity and non-specific amplification can be mitigated with techniques such as the use of alkylated primers, such non-specific amplification is exacerbated by the need for increased multiplexing. Accordingly, there is a need for improved DNA polymerases exhibiting RT activity that are further characterized by increased specificity and a corresponding reduction in non-specific amplification.

[0016] BRIEF SUMMARY OF THE INVENTION

[0017] The present disclosure overcomes the aforementioned challenges by providing improved DNA polymerases exhibiting RT activity that are further characterized by increased specificity and a corresponding reduction in non-specific amplification.In one aspect, the present disclosure provides DNA polymerases having improved activities, including increased specificity, reverse transcriptase activity, mismatch tolerance, extension rate and / or tolerance of RT and polymerase inhibitors, relative to a corresponding, unmodified control polymerase, and methods of making and using such DNA polymerases. In some embodiments, the improved DNA polymerase has increased specificity and / or reverse transcriptase activity as compared with a control DNA polymerase. In some embodiments, the improved DNA polymerase has the same or substantially similar DNA-dependent polymerase activity as compared with a control DNA polymerase.

[0018] Thus, in some embodiments, the improved DNA polymerase comprises an amino acid sequence that is substantially identical (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% identical) to SEQ ID NO: 1 (i.e., the Z05 polymerase), wherein the amino acid of the DNA polymerase corresponding to position 542 of SEQ ID NO: 1 is any amino acid other than K. In certain embodiments, the DNA polymerase comprises an amino acid sequence at least 90% identical to SEQ ID NO:1. In certain embodiments, the DNA polymerase comprises an amino acid sequence at least 95% identical to SEQ ID NO:1. In some embodiments, the amino acid of the improved DNA polymerase corresponding to position 542 of SEQ ID NO:1 is selected from the group consisting of Y or F. In some embodiments, the amino acid of the improved DNA polymerase corresponding to position 542 of SEQ ID NO:1 is Y. In some embodiments, the amino acid of the improved DNA polymerase corresponding to position 542 of SEQ ID NO:1 is F. In some embodiments, the control DNA polymerase has the same amino acid sequence as the improved DNA polymerase except that the amino acid of the control DNA polymerase corresponding to position 542 of SEQ ID NO: 1 is K. For example, in some embodiments, the amino acid at the position corresponding to position 542 of SEQ ID NO:1 of the improved DNA polymerase 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 the position corresponding to position 542 of SEQ ID NO:1 of the improved DNA polymerase is Y or F. In some embodiments, in addition the amino acid of the improved DNA polymerase corresponding to position 616 of SEQ ID NO: 1 is any amino acid other than I and / or the amino acid sequence of the improved DNA polymerase corresponding to position 709 of SEQ ID NO: 1 is any amino acid other than I. In certain embodiments, the amino acid of the improved DNA polymerase corresponding to position 616 of SEQ ID NO:1 is M and / or the amino acid of the improved DNA polymerase corresponding to position 709 of SEQ ID NO: 1 is K. In some embodiments, the improved DNA polymerase comprises an amino acid sequence that is substantially identical (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% identical) to SEQ ID NO: 1, wherein the amino acid of the DNA polymerase corresponding to position 580 of SEQ ID NO: 1 further is any amino acid other than D or E. In some embodiments, the amino acid of the DNA polymerase corresponding to position 580 of SEQ ID NO: 1 further is any amino acid other than D. In certain embodiments, the amino acid of the DNA polymerase corresponding toposition 580 of SEQ ID NO:1 is selected from the group consisting of L, G, T, Q, A, S, N, R, and K. In certain embodiments, the amino acid of the DNA polymerase corresponding to position 580 of SEQ ID NO:1 is G.

[0019] In some embodiments, the improved DNA polymerase comprises an amino acid sequence that is substantially identical (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% identical) to SEQ ID NO: 1 (i.e., the Z05 polymerase), wherein the amino acid of the DNA polymerase corresponding to position 616 of SEQ ID NO: 1 is any amino acid other than I. In certain embodiments, the DNA polymerase comprises an amino acid sequence at least 90% identical to SEQ ID NO:1. In certain embodiments, the DNA polymerase comprises an amino acid sequence at least 95% identical to SEQ ID NO:1. In some embodiments, the control DNA polymerase has the same amino acid sequence as the improved DNA polymerase except that the amino acid of the control DNA polymerase corresponding to position 616 of SEQ ID NO:1 is I. In some embodiments, the amino acid of the improved DNA polymerase corresponding to position 616 of SEQ ID NO:1 is M. In some embodiments, the amino acid of the improved DNA polymerase corresponding to position 542 of SEQ ID NO:1 is any amino acid other than K. In some embodiments, the amino acid at the position corresponding to position 542 of SEQ ID NO: 1 of the improved DNA polymerase is Y or F.

[0020] In some embodiments, the improved DNA polymerase comprises an amino acid sequence that is substantially identical (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% identical) to SEQ ID NO: 1 (i.e., the Z05 polymerase), wherein the amino acid of the DNA polymerase corresponding to position 709 of SEQ ID NO: 1 is any amino acid other than I. In certain embodiments, the DNA polymerase comprises an amino acid sequence at least 90% identical to SEQ ID NO:1. In certain embodiments, the DNA polymerase comprises an amino acid sequence at least 95% identical to SEQ ID NO:1. In some embodiments, the control DNA polymerase has the same amino acid sequence as the improved DNA polymerase except that the amino acid of the control DNA polymerase corresponding to position 709 of SEQ ID NO:1 is I. In some embodiments, the amino acid of the improved DNA polymerase corresponding to position 709 of SEQ ID NO:1 is K. In some embodiments, the amino acid of the improved DNA polymerase corresponding to position 542 of SEQ ID NO:1 is any amino acid other than K. In some embodiments, the amino acid at the position corresponding to position 542 of SEQ ID NO: 1 of the improved DNA polymerase is Y or F

[0021] In some embodiments, the improved DNA polymerase comprises an amino acid sequence that is substantially identical (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% identical) to SEQ ID NO: 42, wherein the amino acid of the DNA polymerase corresponding to position 542 of SEQ ID NO: 42 is any amino acid other than K. In certain embodiments, the DNA polymerase comprises an amino acid sequence at least 90% identical to SEQ ID NO:42. In certain embodiments, the DNA polymerase comprises an amino acid sequence at least 95% identical to SEQ ID NO: 42. Insome embodiments, the amino acid of the improved DNA polymerase corresponding to position 542 of SEQ ID NO:42 is selected from the group consisting of Y or F. In some embodiments, the amino acid of the improved DNA polymerase corresponding to position 542 of SEQ ID NO:42 is Y. In some embodiments, the amino acid of the DNA polymerase corresponding to position 542 of SEQ ID NO:42 is F. In some embodiments, the control DNA polymerase has the same amino acid sequence as the improved DNA polymerase except that the amino acid of the control DNA polymerase corresponding to position 542 of SEQ ID NO: 42 is K. For example, in some embodiments, the amino acid at the position corresponding to position 542 of SEQ ID NO: 42 of the improved DNA polymerase 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 improved DNA polymerase comprises an amino acid sequence that is substantially identical (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% identical) to SEQ ID NO:42, wherein the amino acid of the DNA polymerase corresponding to position 580 of SEQ ID NO:42 further is any amino acid other than D or E. In some embodiments, the amino acid of the DNA polymerase corresponding to position 580 of SEQ ID NO:42 further is any amino acid other than D. In certain embodiments, the amino acid of the DNA polymerase corresponding to position 580 of SEQ ID NO: 42 is selected from the group consisting of L, G, T, Q, A, S, N, R, and K. In certain embodiments, the amino acid of the DNA polymerase corresponding to position 580 of SEQ ID NO:42 is G.

[0022] In some embodiments, the improved DNA polymerase comprises an amino acid sequence that is substantially identical (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% identical) to SEQ ID NO:1, wherein the amino acid of the improved DNA polymerase corresponding to position 616 of SEQ ID NO:1 is any amino acid other than I, the amino acid corresponding to position 709 of SEQ ID NO: 1 is any amino acid other than I, and the amino acid corresponding to position 542 of SEQ ID NO: 1 is any amino acid other than K. In some embodiments, the improved DNA polymerase comprises an amino acid sequence that is substantially identical (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% identical) to SEQ ID NO:1, wherein the amino acid of the DNA polymerase corresponding to position 616 of SEQ ID NO: 1 is any amino acid other than I, wherein the amino acid corresponding to position 709 of SEQ ID NO:1 is K; and wherein the amino acid corresponding to position 542 of SEQ ID NO: 1 is selected from the group consisting of Y and F. In some embodiments, the improved DNA polymerase comprises an amino acid sequence that is substantially identical (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% identical) to SEQ ID NO: 1, wherein the amino acid of the DNA polymerase corresponding to position 616 of SEQ ID NO: 1 is M, wherein the amino acid corresponding to position 709 of SEQ ID NO: 1 is any amino acid other than I; and wherein the amino acid corresponding to position 542 of SEQ ID NO: 1 is selected from the group consisting of Y and F. In some embodiments, the improved DNA polymerase comprises an amino acid sequence that is substantially identical (e.g., at least about 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% identical) to SEQ ID NO: 1, wherein the amino acid of the DNA polymerase corresponding to position 616 of SEQ ID NO:1 is M, wherein the amino acid corresponding to position 709 of SEQ ID NO:1 is K; and wherein the amino acid corresponding to position 542 of SEQ ID NO: 1 is any amino acid other than K. In some embodiments, the improved DNA polymerase comprises an amino acid sequence that is substantially identical (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% identical) to SEQ ID NO:1, wherein the amino acid of the DNA polymerase corresponding to position 616 of SEQ ID NO: 1 is M, wherein the amino acid corresponding to position 709 of SEQ ID NO:1 K; and wherein the amino acid corresponding to position 542 of SEQ ID NO: 1 is selected from the group consisting of Y and F. In some embodiments, the improved DNA polymerase comprises an amino acid sequence that is substantially identical (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% identical) to SEQ ID NO: 1, wherein the amino acid of the DNA polymerase corresponding to position 616 of SEQ ID NO: 1 is M, wherein the amino acid corresponding to position 709 of SEQ ID NO:1 K; and wherein the amino acid corresponding to position 542 of SEQ ID NO:1 is Y. In some embodiments, the improved DNA polymerase comprises an amino acid sequence that is substantially identical (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% identical) to SEQ ID NO: 1, wherein the amino acid of the DNA polymerase corresponding to position 616 of SEQ ID NO:1 is M, wherein the amino acid corresponding to position 709 of SEQ ID NO:1 K; and wherein the amino acid corresponding to position 542 of SEQ ID NO:1 is F. In some embodiments the DNA polymerase has the same or substantially similar or substantially improved or better DNA-dependent or RNA-dependent polymerase activity, as compared with the control DNA polymerase. In some embodiments the DNA polymerase has the improved or better RNA-dependent polymerase activity, as compared with the control DNA polymerase. In some embodiments the DNA polymerase has improved or better reverse transcriptase efficiency, as compared with the control DNA polymerase. In some embodiments, the DNA polymerase has improved or better specificity, as compared with the control DNA polymerase. In another aspect, a method of conducting primer extension is provided, wherein the method comprises contacting a modified DNA polymerase as disclosed herein and above with a primer, a polynucleotide template, and a nucleoside triphosphate under conditions suitable for extension of the primer, thereby producing an extended primer. In some embodiments, the modified DNA polymerase is modified relative to a reference DNA polymerase, wherein the reference DNA polymerase has an amino acid sequence of SEQ ID NO:1 or has an amino acid sequence of SEQ ID NO:42, and wherein the amino acid corresponding to position 542 is an amino acid other than K. In some embodiments, modified DNA polymerase comprises an amino acid sequence at least 90% identical to SEQ ID NO:1 or SEQ ID NO:42. In some embodiments, the amino acid corresponding to position 542 is an amino acid selected from a group of amino acids, wherein the group of amino acids consists of F and Y. In certainembodiments, the amino acid corresponding to position 542 is F. In certain embodiments, the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:41. In other embodiments, the amino acid corresponding to position 542 is Y. Herein, in the certain embodiments the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:40. In some embodiments, the modified DNA polymerase has increased reverse transcriptase efficiency as compared to the reference polymerase. In some embodiments, the modified DNA polymerase has improved specificity as compared to the reference DNA polymerase. In some embodiments, the polynucleotide template is RNA. In other embodiments, the polynucleotide template is DNA. In some embodiments, the method for conducting primer extension comprises a polymerase chain reaction (PCR).

[0023] In another aspect, a method of conducting primer extension is provided, wherein the method comprises contacting a modified DNA polymerase as disclosed herein and above with a primer, a polynucleotide template, and a nucleoside triphosphate under conditions suitable for extension of the primer, thereby producing an extended primer, wherein: (i) the modified polymerase is modified relative to a reference DNA, wherein the reference DNA polymerase has an amino acid sequence of SEQ ID NO:1; and (ii) the amino acid corresponding to position 616 is an amino acid other than I, the amino acid corresponding to position 709 is an amino acid other than I, and the amino acid corresponding to position 542 is an amino acid other than K. In some embodiments, the modified DNA polymerase comprises an amino acid sequence at least 90% identical to SEQ ID NO:1. In certain embodiments, the modified DNA polymerase comprises an amino acid sequence at least 95% identical to SEQ ID NO:1. In some embodiments, the amino acid corresponding to position 616 is M. In some embodiments, the amino acid corresponding to position 709 is K. In some embodiments, the amino acid corresponding to position 542 is selected from a group of amino acids, wherein the group of amino acids consists of F and Y. In certain embodiments, the amino acid corresponding to position 542 is F. Herein, in certain embodiments the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:41. In certain embodiments, the amino acid corresponding to position 542 is Y. Herein, in certain embodiments the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the amino acid corresponding to position 616 is M, the amino acid corresponding to position 709 is K, and the amino acid corresponding to position 542 is F. In certain embodiments, the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the amino acid corresponding to position 616 is M, the amino acid corresponding to position 709 is K, and the amino acid corresponding to position 542 is Y. In certain embodiments, the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:40. In some embodiments, the modified DNA polymerase has increased reverse transcriptase efficiency as compared to the reference polymerase. In some embodiments, the modified DNA polymerase has improved specificity as compared to the reference DNA polymerase. In some embodiments, thepolynucleotide template is RNA. In other embodiments, the polynucleotide template is DNA. In some embodiments, the method for conducting primer extension comprises a polymerase chain reaction (PCR).

[0024] In another aspect, a modified DNA polymerase is provided, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:41.

[0025] In another aspect, a modified DNA polymerase, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:40.

[0026] In another aspect, a reaction mixture for performing a polynucleotide extension method is provided comprising a DNA polymerase as disclosed herein and above. In some embodiments, the reaction mixture comprises one or more additional reagents selected from the group of nucleoside triphosphates, a buffer suitable for polynucleotide extension, and / or one or more primer or probe polynucleotides suitable for hybridizing to a predetermined polynucleotide template under polynucleotide extension conditions.

[0027] In another aspect, a kit for performing a polynucleotide extension method is provided comprising a DNA polymerase as disclosed herein and above. In some embodiments, the DNA polymerase may be provided in a container. In some embodiments, the kit further comprises one or more additional containers providing one or more additional reagents selected from the group of nucleoside triphosphates, a buffer suitable for polynucleotide extension, and / or one or more primer or probe polynucleotides suitable for hybridizing to a predetermined polynucleotide template under polynucleotide extension conditions.

[0028] Various DNA polymerases are amenable to mutation according to the present invention. Particularly suitable are thermostable polymerases, including wild-type or naturally occurring thermostable polymerases from various species of thermophilic bacteria, as well as synthetic thermostable polymerases derived from such wild-type or naturally occurring enzymes by amino acid substitution, insertion or deletion, or other modification. Exemplary unmodified forms of polymerase include, e.g., CS5, CS6 or Z05 DNA polymerase, 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 thereto. The CS5 polymerase is a chimeric thermostable DNA polymerase. The Z05 polymerase (SEQ ID NO: 1) is a modified DNA polymerase having increased 3’-mismatch discrimination. In certain embodiments the amino acid sequence identity is at least 80%, preferably at least 90% and more preferably at least 95%. Other unmodified polymerases include, e.g., DNA polymerases from any of the following species of thermophilic bacteria (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,' Thermits aquaticus,' Thermits thermophilus,' Thermus flavus,' Thermusfiliformis,' Thermits sp. spsl7; Thermits sp. Z05; Thermotoga neopoHlancr. Thermosipho africanus,' Thermus caldophilus, Deinococcus radiodurans, Bacillus stearothermophilus or Bacillus caldotenax. In certain embodiments the amino acid sequence identity is at least 80%, preferably at least 90% and more preferably at least 95%. Suitable polymerases also include those having reverse transcriptase (RT) activity and / or the ability to incorporate unconventional nucleotides, such as ribonucleotides or other 2'-modified nucleotides.

[0029] While thermostable DNA polymerases possessing efficient reverse transcription activity are particularly suited for performing RT-PCR, especially single enzyme RT-PCR, thermoactive, but not thermostable DNA polymerases possessing efficient reverse transcription activity also are amenable to mutation according to the present invention. For example, the attributes of increased reverse transcriptase efficiency, mismatch tolerance, extension rate, and / or tolerance of RT inhibitors are useful for the RT step in an RT-PCR, and this step does not need to be performed at temperatures that would inactivate a thermoactive but not thermostable DNA polymerase. Following the RT step, a thermostable DNA polymerase could either be added or it could already be included in the reaction mixture to perform the PCR amplification step. For example, the improved DNA polymerase described herein can be combined with a second thermostable DNA polymerase prior to the RT step in a buffer suitable for extension and amplification of RNA and DNA templates, as described in the Examples. Examples of suitable thermostable DNA polymerases are described in US Patent No. 4,889,818, and US Patent Nos. 5,773, 258 and 5,677,152. In some embodiments, the second thermostable DNA polymerase is AmpliTaq® DNA polymerase (Deoxy-nucleoside triphosphate: DNA Deoxynucleotidyltransferase, E.C.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 Gold® DNA polymerase (Roche Applied Science, Indianapolis, IN, USA). This second methodology would especially benefit by using a chemically modified thermostable DNA polymerase (or other HotStart technology to inactivate the thermostable DNA polymerase) so that it would not be fully active during the RT step. An example of a thermoactive but not thermostable DNA polymerase possessing efficient reverse transcription activity is the DNA polymerase from Carboxydothermus hydrogenoformans (Chy; SEQ ID NO:39); see, e.g., US Patent Nos. 6,468,775 and 6,399,320.

[0030] 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:

[0031] (a) a Thermus sp. Z05 DNA polymerase (Z05) (SEQ ID NO:1);

[0032] (b) a Thermus aquaticus DNA polymerase (Taq) (SEQ ID NO:2);

[0033] (c) a Thermus filiformis DNA polymerase (Tfi) (SEQ ID NO: 3);(d) a Thermits flavus DNA polymerase (TH) (SEQ ID NO:4);

[0034] (e) a Thermus sp. spsl7 DNA polymerase (Spsl7) (SEQ ID NO: 5);

[0035] (f) a Thermus thermophilus DNA polymerase (Tth) (SEQ ID NO:6);

[0036] (g) a Thermus caldophilus DNA polymerase (Tea) (SEQ ID NO:7); and

[0037] (h) Carboxydothermus hydrogenoformans DNA polymerase (Chy) (SEQ ID NO:39).

[0038] In certain embodiments the amino acid sequence identity is at least 80%, preferably at least 90% and more preferably at least 95%.

[0039] 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 to a polymerase selected from the group consisting of:

[0040] (a) a Thermotoga maritima DNA polymerase (Tma) (SEQ ID NO: 34); and

[0041] (b) a Thermotoga neopolitana DNA polymerase (Tne) (SEQ ID NO:35).

[0042] In certain embodiments the amino acid sequence identity is at least 80%, preferably at least 90% and more preferably at least 95%.

[0043] In certain 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% ammo acid sequence identity to SEQ ID NO:1. In certain embodiments the amino acid sequence identity is at least 80%, preferably at least 90% and more preferably at least 95%. In some embodiments, the DNA polymerase is a Thermus sp. Z05 DNA polymerase (Z05) DNA polymerase (i.e., SEQ ID NO:1), and the amino acid at position 616 is any amino acid other than I. For example, in some embodiments, the amino acid at position 616 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 DNA polymerase is a Z05 DNA polymerase, and the amino acid at position 616 is M. 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 other than D or E. In some embodiments, the DNA polymerase is a Z05 DNA polymerase, and the amino acid at position 580 is any amino acid other than 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 other than 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.

[0044] In some embodiments, the control or reference DNA polymerase is a Z05 polymerase (SEQ ID NO: 1),Z05 D580G polymerase, Z05 D580GI709K polymerase, C21 polymerase (SEQ ID NO:43), parental type polymerase (SEQ ID NO:42, which is Z05 I616M I709K).

[0045] In some embodiments, this disclosure is directed to a modified DNA polymerase, wherein the modified DNA polymerase is modified relative to a reference DNA polymerase, wherein the reference DNA polymerase has an amino acid sequence of SEQ ID NO: 42, and wherein the amino acid corresponding to position 542 is an amino acid other than K. In some embodiments, the modified DNA polymerase comprises an amino acid sequence at least 90% identical to SEQ ID NO: 42. In some embodiments, the amino acid corresponding to position 542 is an amino acid selected from a group of amino acids, wherein the group of amino acids consists of F and Y. In some embodiments, the amino acid corresponding to position 542 is F. In some embodiments, the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the modified DNA polymerase of claim 3, wherein the amino acid corresponding to position 542 is Y. In some embodiments, the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the modified DNA polymerase has increased reverse transcriptase efficiency as compared to the reference polymerase. In some embodiments, the modified DNA polymerase has improved specificity compared to the reference DNA polymerase.

[0046] In some embodiments, the disclosure is directed to a modified DNA polymerase, wherein: (i) the modified DNA polymerase is modified relative to a reference DNA polymerase, wherein the reference DNA polymerase has an amino acid sequence of SEQ ID NO: 1; and (ii) the amino acid corresponding to position 616 is an amino acid other than I, the amino acid corresponding to position 709 is an amino acid other than I, and the amino acid corresponding to position 542 is an amino acid other than K. In some embodiments, the modified DNA polymerase of claim 10, wherein the modified DNA polymerase comprises an amino acid sequence at least 90% identical to SEQ ID NO:1. In some embodiments, the amino acid corresponding to position 616 is M. In some embodiments, the amino acid corresponding to position 709 is K. In some embodiments, the amino acid corresponding to position 542 is an amino acid selected from a group of amino acids, wherein the group of amino acids consists of F and Y. In some embodiments, the amino acid corresponding to position 542 is F. In some embodiments, the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the amino acid corresponding to position 542 is Y. In some embodiments, the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the amino acid corresponding to position 616 is M, the amino acid corresponding to position 709 is K, and the amino acid corresponding to position 542 is F. In some embodiments, the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the amino acid corresponding to position 616 is M, the amino acid corresponding to position 709 is K, and the amino acid corresponding to position 542 is Y. In some embodiments, themodified DNA polymerase comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the modified DNA polymerase has increased reverse transcriptase efficiency as compared to the reference polymerase. In some embodiments, the modified DNA polymerase has improved specificity as compared to the reference DNA polymerase.

[0047] In some embodiments, the disclosure is directed to a modified DNA polymerase, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:41.

[0048] In some embodiments, the disclosure is directed to a modified DNA polymerase, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO: 40.

[0049] In some embodiments, the disclosure is directed to a method of conducting primer extension, wherein the method comprising contacting a modified DNA polymerase with a primer, a polynucleotide template, and a nucleoside triphosphate under conditions suitable for extension of the primer, thereby producing an extended primer, and wherein the modified DNA polymerase is modified relative to a reference DNA polymerase, wherein the reference DNA polymerase has an amino acid sequence of SEQ ID NO:42, and wherein the amino acid corresponding to position 542 is an amino acid other than K. In some embodiments, the modified DNA polymerase comprises an amino acid sequence at least 90% identical to SEQ ID NO:42. In some embodiments, the amino acid corresponding to position 542 is an amino acid selected from a group of amino acids, wherein the group of amino acids consists of F and Y. In some embodiments, the amino acid corresponding to position 542 is F. In some embodiments, the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the amino acid corresponding to position 542 is Y. In some embodiments, the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:40. In some embodiments, the modified DNA polymerase has increased reverse transcriptase efficiency as compared to the reference polymerase. In some embodiments, the modified DNA polymerase has improved specificity as compared to the reference DNA polymerase. In some embodiments, the polynucleotide template is RNA. In some embodiments, the polynucleotide template is DNA. In some embodiments, the method for conducting primer extension comprises a polymerase chain reaction (PCR).

[0050] In some embodiments, the disclosure is directed to a method of conducting primer extension, the method comprising contacting a modified DNA polymerase with a primer, a polynucleotide template, and a nucleoside triphosphate under conditions suitable for extension of the primer, thereby producing an extended primer, wherein: (i) the modified polymerase is modified relative to a reference DNA, wherein the reference DNA polymerase has an amino acid sequence of SEQ ID NO:1; and (ii) the amino acid corresponding to position 616 is an amino acid other than I, the amino acid corresponding to position 709 is an amino acid other than I, and the amino acid corresponding to position 542 is an amino acid other than K. In some embodiments, the modified DNA polymerase comprises an amino acid sequence at least 90% identical to SEQ ID NO:1. In some embodiments, the amino acidcorresponding to position 616 is M. In some embodiments, the amino acid corresponding to position 709 is K. In some embodiments, the amino acid corresponding to position 542 is selected from a group of amino acids, wherein the group of amino acids consists of F and Y. In some embodiments, the amino acid corresponding to position 542 is F. In some embodiments, the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the amino acid corresponding to position 542 is Y. In some embodiments, the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:40. In some embodiments, the amino acid corresponding to position 616 is M, the amino acid corresponding to position 709 is K, and the amino acid corresponding to position 542 is F. In some embodiments, the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the amino acid corresponding to position 616 is M, the amino acid corresponding to position 709 is K, and the amino acid corresponding to position 542 is Y. In some embodiments, the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:40. In some embodiments, the modified DNA polymerase has increased reverse transcriptase efficiency as compared to the reference polymerase. In some embodiments, the modified DNA polymerase has improved specificity as compared to the reference DNA polymerase. In some embodiments, the polynucleotide template is RNA. In some embodiments, the polynucleotide template is DNA. In some embodiments, the method for conducting primer extension comprises a polymerase chain reaction (PCR).

[0051] The mutant or improved polymerases can include other, non-substitutional modifications. One such modification is a thermally reversible covalent modification that inactivates the enzyme, but which is reversed to activate the enzyme upon incubation at an elevated temperature, such as a temperature typically used for polynucleotide extension. Exemplary reagents for such thermally reversible modifications are described in U.S. Patent Nos. 5,773, 258 and 5,677,152.

[0052] In some embodiments, the reverse transcriptase activity is determined by performing real-time RT-PCR amplification and detection of a Hepatitis C Virus (HCV) transcript generated from the first 800 bases of HCV genotype lb 5’NTR in pSP64 poly(A) (Promega). Two or more reaction mixtures can have titrated numbers of copies of the Hepatitis C Virus (HCV) transcript (e.g., 1:5 titrations, 1:10 titrations, e.g., 10,000 copies, 1000 copies, 100 copies, 10 copies, 1 copy, 0 copies in several reaction mixtures). The reverse transcriptase ability of a polymerase of the invention can be compared to the reverse transcriptase ability of a reference polymerase (e.g., a naturally occurring, unmodified, or control polymerase), over a preselected unit of time, as described herein. Polymerases with improved reverse transcriptase ability will amplify the transcript with greater efficiency or will require a lower number of PCR cycles to amplify the transcript (i.e., exhibit a lower Cp value, as calculated herein), in comparison to a naturally occurring or unmodified polymerase. Moreover, in some embodiments, polymerases with improved RT function also have improved replication of long RNA (e.g., at least500 or 1000 or 2000 or 5000 or more nucleotides long) templates. In some embodiments, the improved reverse transcriptase efficiency includes a shorter reverse transcription time in comparison to a control polymerase. Thus, in some embodiments, polymerases with increased reverse transcriptase efficiency will reverse transcribe an RNA template faster than a control or reference polymerase.

[0053] In various other aspects, the present invention provides a recombinant nucleic acid encoding a mutant or improved DNA polymerase as described herein, a vector comprising the recombinant nucleic acid, and a host cell transformed with the vector. In certain embodiments, the vector is an expression vector. Host cells comprising such expression vectors are useful in methods of the invention for producing the mutant or improved polymerase by culturing the host cells under conditions suitable for expression of the recombinant nucleic acid. The polymerases of the invention may be contained in reaction mixtures and / or kits. The embodiments of the recombinant nucleic acids, host cells, vectors, expression vectors, reaction mixtures and kits are as described above and herein.

[0054] In yet another aspect, a method for conducting polynucleotide extension is provided. The method generally includes contacting a DNA polymerase having increased / improved specificity, reverse transcriptase efficiency, mismatch tolerance, extension rate and / or tolerance of RT and polymerase inhibitors as described herein with a primer, a polynucleotide template, and nucleoside triphosphates under conditions suitable for extension of the primer, thereby producing an extended primer. The polynucleotide template can be, for example, an RNA or DNA template. In certain embodiments the primer extension comprises a reverse transcription step of less than about five minutes. In some embodiments the conditions suitable for extension comprise Mg2+. The nucleotide triphosphates can include unconventional nucleotides such as, e.g., ribonucleotides and / or labeled nucleotides. Further, the primer and / or template can include one or more nucleotide analogs. In some variations, the polynucleotide extension method is a method for polynucleotide amplification that includes contacting the mutant or improved DNA polymerase with a primer pair, the polynucleotide template, and the nucleoside triphosphates under conditions suitable for amplification of the polynucleotide. The polynucleotide extension reaction can be, e.g., PCR, isothermal extension, or sequencing (e.g., 454 sequencing reaction). In certain embodiments the primer extension method comprises a polymerase chain reaction (PCR). The polynucleotide template can be from any type of biological sample.

[0055] Optionally, the primer extension reaction comprises an actual or potential inhibitor of a reference or unmodified polymerase. The inhibitor can inhibit the nucleic acid extension rate and / or the reverse transcription efficiency 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 breakdown product, such as hemin, hematoporphyrin, or bilirubin. In some embodiments, the inhibitor is an iron-chelator or a purple pigment. In other embodiments, the inhibitor is heparin or melanin. In certain embodiments, the inhibitor is anintercalating dye. In some embodiments, the intercalating dye is [2-[N-bis-(3-dimethylaminopropyl)-amino]-4-[2,3-dihydro-3-methyl-(benzo-l,3-thiazol-2-yl)-methylidene]-l-phenyl-quinolinium]+. In some embodiments, the intercalating dye is [2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-l,3-thiazol-2-yl)-methylidene]-l-phenyl-quinolinium]+. In some embodiments, the intercalating dye is not [2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-l,3-thiazol-2-yl)-methylidene]-l-phenyl-quinolinium]+. In some embodiments, the conditions suitable for extension comprise Mg2+. In some embodiments, the conditions suitable for extension comprise Mn2+.

[0056] The present invention also provides a kit useful in such a polynucleotide extension method. Generally, the kit includes at least one container providing a mutant or improved DNA polymerase as described herein. In certain embodiments, the kit further includes one or more additional containers providing one or more additional reagents. For example, in specific variations, the one or more additional containers provide nucleoside triphosphates; a buffer suitable for polynucleotide extension; and / or one or more primer or probe polynucleotides, hybridizable, under polynucleotide extension conditions, to a predetermined polynucleotide template. The polynucleotide template can be from any type of biological sample.

[0057] Further provided are reaction mixtures comprising the polymerases of the invention. The reaction mixtures can also contain a template nucleic acid (DNA and / or RNA), one or more primer or probe polynucleotides, nucleoside triphosphates (including, e.g., deoxyribonucleoside triphosphates, ribonucleoside triphosphates, labeled nucleoside triphosphates, unconventional nucleoside triphosphates), buffers, salts, labels (e.g., fluorophores). In some embodiments, the reaction mixtures comprise an iron chelator or a purple dye. In certain embodiments, the reaction mixtures comprise hemoglobin, or a degradation product of hemoglobin. For example, in certain embodiments, the degradation products of hemoglobin include heme breakdown products such as hemin, hematin, hematophoryn, and bilirubin. In other embodiments, the reaction mixtures comprise heparin or a salt thereof. Optionally, the reaction mixture comprises an intercalating dye (including but not limited to those described above or elsewhere herein). In certain embodiments, the reaction mixture contains a template nucleic acid that is isolated from blood. In other embodiments, the template nucleic acid is RNA and the reaction mixture comprises heparin or a salt thereof. In some embodiments the reaction mixture further comprising Mg2+.

[0058] In certain embodiments the reaction mixture further comprising a second thermostable DNA polymerase. In some embodiments, the reaction mixture comprises two or more polymerases. For example, in some embodiments, the reaction mixture comprises an improved DNA polymerase having increased reverse transcription efficiency (e.g., increased activity extending an RNA-template) as described herein, and another polymerase having DNA-dependent polymerase activity. In oneembodiment, the reaction mixture comprises a blend of an improved DNA polymerase having increased reverse transcription efficiency 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 such that the enzyme is inactive at temperatures suitable for the reverse transcription step, but is activated under suitable conditions, for example, at elevated temperatures of about 90° C to 100° C for a period of time up to about 12 minutes. Suitable conditions for activation of a reversibly inactivated thermostable polymerase are provided, for example, in a Hot Start PCR reaction, as described in the Examples. Examples of suitable second thermostable DNA-dependent polymerases are described in U.S. Patent Nos. 5,773, 258 and 5,677,152, supra.

[0059] Further embodiments of the invention are described herein.

[0060] DEFINITIONS

[0061] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although essentially any methods and materials similar to those 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 below.

[0062] The terms “a,” “an,” and “the” include plural referents, unless the context clearly indicates otherwise. An “amino acid” refers to any monomer 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 alpha-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gin or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (He or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Vai or V). In cases where “X” residues are undefined, these should be defined as “any amino acid.” The structures of these twenty natural amino acids are shown in, e.g., Stryer et al., Biochemistry, 5thed., Freeman and Company (2002). Additional amino acids, such as selenocysteine and pyrrolysine, can also be genetically coded for (Stadtman (1996) “Selenocysteine,” Annu Rev Biochem, 65:83-100 and Ibba et al. (2002) “Genetic code: introducing pyrrolysine,” Curr Biol, 12(13):R464-R466). The term “amino acid” also includes unnatural amino acids, modified amino acids (e.g., having 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. U.S.A. 101(24):8882-8887, Anderson et al. (2004) “An expanded genetic code with a functional quadruplet codon” Proc. Natl. Acad. Sci. U.S.A.

[0063] 101(20):7566-7571, Ikeda et al. (2003) “Synthesis of a novel histidine analogue and its efficient incorporation into a protein in vivo,” Protein Eng, Des. Sei. 16(9):699-706, Chin et al. (2003) “AnExpanded Eukaryotic Genetic Code,” Science 301(5635):964-967, James et al. (2001) “Kinetic characterization of ribonuclease S mutants containing photoisomerizable phenylazophenylalanine residues,” Protein Eng, Des. Sei. 14(12):983-991, Kohrer et al. (2001) “Import of amber and ochre suppressor tRNAs into mammalian cells: A general approach to site-specific insertion of amino acid analogues into proteins,” Proc. Natl. Acad. Sci. U.S.A. 98(25): 14310-14315, Bacher et al. (2001) “Selection and Characterization of Escherichia coli Variants Capable of Growth on an Otherwise Toxic Tryptophan Analogue,” J. Bacteriol, 183(18): 5414-5425, Hamano-Takaku et al. (2000) “A Mutant Escherichia coli Tyrosyl-tRNA Synthetase Utilizes the Unnatural Amino Acid Azatyrosine More Efficiently than 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).

[0064] To further illustrate, an amino acid is typically an organic acid that includes a substituted or unsubstituted amino group, a substituted or unsubstituted carboxy group, and one or more side chains or groups, or analogs of any of these groups. Exemplary side chains include, e.g., thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynl, ether, borate, boronate, phospho, phosphono, phosphine, heterocyclic, 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 photoactivatable cross-linkers, metal binding amino acids, spin-labeled amino acids, fluorescent amino acids, metal-containing amino acids, amino acids with novel functional groups, amino acids that covalently or noncovalently interact with other molecules, photocaged and / or photoisomerizable amino acids, radioactive amino acids, amino acids comprising biotin or a biotin analog, glycosylated amino acids, other carbohydrate modified amino acids, amino acids comprising polyethylene glycol or polyether, heavy atom substituted amino acids, chemically cleavable and / or photocleavable amino acids, carbon-linked sugar-containing amino acids, redox-active amino acids, amino thioacid containing amino acids, and amino acids comprising one or more toxic moieties.

[0065] The term "biological sample" encompasses a variety of sample types obtained from an organism and can be used in a diagnostic or monitoring assay. The term encompasses urine, urine sediment, blood, saliva, and other liquid samples of biological origin, solid tissue samples, such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The term encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, sedimentation, or enrichment for certain components. The term encompasses a clinical sample, and also includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.

[0066] The term “mutant,” in the context of DNA polymerases of the present invention, means a polypeptide,typically recombinant, that comprises one or more amino acid substitutions relative to a corresponding, functional DNA polymerase.

[0067] The term “unmodified form,” in the context of a mutant polymerase, is a term used herein for purposes of defining a mutant DNA polymerase of the present invention: the term “unmodified form” refers to a functional DNA polymerase that has the amino acid sequence of the mutant polymerase except at one or more amino acid position(s) specified as characterizing the mutant polymerase. Thus, reference to a mutant DNA polymerase in terms of (a) its unmodified form and (b) one or more specified amino acid substitutions means that, with the exception of the specified amino acid substitution(s), the mutant polymerase otherwise has an amino acid sequence identical to the unmodified form in the specified motif. The “unmodified polymerase” (and therefore also the modified form having increased reverse transcriptase efficiency, mismatch tolerance, extension rate and / or tolerance of RT and polymerase inhibitors) may contain additional mutations to provide desired functionality, e.g., improved incorporation of dideoxyribonucleotides, ribonucleotides, ribonucleotide analogs, dye-labeled nucleotides, modulating 5 ’-nuclease activity, modulating 3 ’-nuclease (or proofreading) activity, or the like. Accordingly, in carrying out the present invention as described herein, the unmodified form of a DNA polymerase is predetermined. The unmodified form of a DNA polymerase can be, for example, a wild-type and / or a naturally occurring DNA polymerase, or a DNA polymerase that has already been intentionally modified. An unmodified form of the polymerase is preferably a thermostable DNA polymerase, such as DNA polymerases from various thermophilic bacteria, as well as functional variants thereof having substantial sequence identity to a wild-type or naturally occurring thermostable polymerase. Such variants can include, for example, chimeric DNA polymerases such as, for example, the chimeric DNA polymerases described in U.S. Patent Nos. 6,228,628 and 7,148,049. In certain embodiments, the unmodified form of a polymerase has reverse transcriptase (RT) activity.

[0068] The term “reverse transcription efficiency” refers to the fraction of RNA molecules that are reverse transcribed as cDNA in a given reverse transcription reaction. In certain embodiments, the mutant DNA polymerases of the invention have improved reverse transcription efficiencies relative to unmodified forms of these DNA polymerases. That is, these mutant DNA polymerases reverse transcribe a higher fraction of RNA templates than their unmodified forms under a particular set of reaction conditions. Without being limited by theory, the ability of a mutant DNA polymerase described herein to reverse transcribe a higher fraction of RNA templates can be due to an increased reverse transcription activity, for example, an increased nucleotide incorporation rate and / or increased processivity of the enzyme. “Processivity” is a term that refers to the number of nucleotides incorporated by a polymerase in a single binding event (before dissociation). Reverse transcription efficiency can be measured, for example, by measuring the crossing point (Cp) of a PCR reaction using a RNA template, and comparing the Cp value to a Cp value of a control reaction in which a DNAtemplate of the same sequence (except U's are replaced with T's) is amplified, wherein the RNA and DNA amplifications use a common primer set and the same polymerase, e.g., as described in the examples. A test polymerase has improved RT efficiency when the test polymerase has a decreased Cp value compared to a control polymerase when RNA is used as a template but has a substantially unchanged Cp value relative to the control polymerase when DNA is used as a template. In some embodiments, a 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. Improved RT efficiency of a test polymerase can be measured as described in the Examples.

[0069] The term “thermostable polymerase,” refers to an enzyme that is stable to heat, is heat resistant, and retains sufficient activity to effect subsequent polynucleotide extension reactions and does not become irreversibly denatured (inactivated) when subjected to the elevated temperatures for the time necessary to effect denaturation of double-stranded nucleic acids. The heating conditions necessary for nucleic acid denaturation are well known in the art and are exemplified in, e.g., U.S. Patent Nos. 4,683,202, 4,683,195, and 4,965,188. As used herein, a thermostable polymerase is suitable for use in a temperature cycling reaction such as the polymerase chain reaction ("PCR"). Irreversible denaturation for purposes herein refers to permanent and complete loss of enzymatic activity. For a thermostable polymerase, enzymatic activity refers to the catalysis of the combination of the nucleotides in the proper manner to form polynucleotide extension products that are complementary to a template nucleic acid strand. Thermostable DNA polymerases from thermophilic bacteria include, e.g., DNA polymerases from Thermotoga maritima, Thermus aquaticus, Thermus thermophilus, Thermus flavus, Thermus filiformis, Thermus species spsl7, Thermus species Z05, Thermus caldophilus, Bacillus caldotenax, Thermotoga neopolitana, and Thermosipho africanus.

[0070] The term “thermoactive” refers to an enzyme that maintains catalytic properties at temperatures commonly used for reverse transcription or anneal / extension steps in RT-PCR and / or PCR reactions (i.e., 45-80°C). Thermostable enzymes are those which are not irreversibly inactivated or denatured when subjected to elevated temperatures necessary for nucleic acid denaturation. Thermoactive enzymes may or may not be thermostable. Thermoactive DNA polymerases can be DNA or RNA dependent from thermophilic species or from mesophilic species including, but not limited to, Escherichia coli, Moloney murine leukemia viruses, and Avian myoblastosis virus.

[0071] As used herein, a "chimeric" protein refers to a protein whose amino acid sequence represents a fusion product of subsequences of the amino acid sequences from at least two distinct proteins. A chimeric protein typically is not produced by direct manipulation of amino acid sequences, but, rather, is expressed from a "chimeric" gene that encodes the chimeric amino acid sequence. In certain embodiments, for example, an unmodified form of a mutant DNA polymerase of the present inventionis a chimeric protein that consists of an amino-terminal (N-terminal) region derived from a Thermus species DNA polymerase and a carboxy-terminal (C-terminal) region derived from 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.

[0072] The term "aptamer" refers to a single-stranded DNA that recognizes and binds to DNA polymerase and efficiently inhibits the polymerase activity as described in U.S. Patent No. 5,693,502. Use of aptamer and dUTP / UNGin RT-PCR is also discussed, for example, in Smith, E.S. 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, C.W. andDveksler, G.S., Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 211-219, (2003)).

[0073] In the context of mutant DNA polymerases, "correspondence" to another sequence (e.g., regions, fragments, nucleotide or amino acid positions, or the like) is based on the convention of numbering according to nucleotide or amino acid position number and then aligning the sequences in a manner that maximizes the percentage of sequence identity. An amino acid "corresponding to position [X] of [specific sequence]" refers to an amino acid in a polypeptide of interest that aligns with the equivalent amino acid of a specified sequence. Generally, as described herein, the amino acid corresponding to a position of a polymerase can be determined using an alignment algorithm such as BLAST as described below. Because not all positions within a given "corresponding region" need be identical, nonmatching positions within a corresponding region may be regarded as "corresponding positions." Accordingly, as used herein, referral to an "amino acid position corresponding to amino acid position [X]" of a specified DNA polymerase refers to equivalent positions, based on alignment, in other DNA polymerases and structural homologues and families. In some embodiments of the present invention, "correspondence" of amino acid positions are determined with respect to a region of the polymerase comprising one or more motifs of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 32, 33, 34, 35, 36, 37, or 39. When a polymerase polypeptide sequence differs from SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 32, 33, 34, 35, 36, 37, or 39 (e.g. , by changes in amino acids or addition or deletion of amino acids), it may be that a particular mutation associated with improved activity as discussed herein will not be in the same position number as it is in SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 32, 33, 34, 35, 36, 37, or 39. This is illustrated, for example, in Table 1.

[0074] “Recombinant,” as used herein, refers to an amino acid sequence or a nucleotide sequence that has been intentionally modified by recombinant methods. By the term "recombinant nucleic acid" herein is meant a nucleic acid, originally formed in vitro, in general, by the manipulation of a nucleic acid by restriction endonucleases, in a form not normally found in nature. Thus, an isolated, mutant DNApolymerase nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this invention. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell, it will replicate non-recombinantly, i. e. , using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention. A "recombinant protein" is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid as depicted above.

[0075] 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 sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.

[0076] The term "host cell" refers to both single-cellular prokaryote and eukaryote organisms (e.g., bacteria, yeast, and actinomycetes) and single cells from higher order plants or animals when being grown in cell culture.

[0077] The term “vector” refers to a piece of DNA, typically double-stranded, which may have inserted into it a piece of foreign DNA. The vector or may be, for example, of plasmid origin. Vectors contain “replicon” polynucleotide sequences that facilitate the autonomous replication of the vector in a host cell. Foreign DNA is defined as heterologous DNA, which is DNA not naturally found in the host cell, which, for example, replicates the vector molecule, encodes a selectable or screenable marker, or encodes a transgene. The vector is used to transport the foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector can replicate independently of or coincidental with the host chromosomal DNA, and several copies of the vector and its inserted DNA can be generated. In addition, the vector can also contain the necessary elements that permit transcription of the inserted DNA into an mRNA molecule or otherwise cause replication of the inserted DNA into multiple copies of RNA. Some expression vectors additionally contain sequence elements adjacent to the inserted DNA that increase the half-life of the expressed mRNA and / or allow translation of the mRNA into a protein molecule. Many molecules of mRNA and polypeptide encoded by the inserted DNA can thus be rapidly synthesized.

[0078] The term “nucleotide” in addition to referring to the naturally occurring ribonucleotide or deoxyribonucleotide monomers, shall herein be understood to refer to related structural variants thereof, including derivatives and analogs, that are functionally equivalent with respect to the particular context in which the nucleotide is being used (e.g., hybridization to a complementary base), unless the context clearly indicates otherwise.

[0079] The term “nucleic acid” or “polynucleotide” refers to a polymer that can be corresponded to a ribosenucleic acid (RNA) or deoxyribose nucleic 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 an analog, intemucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, and the like), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, and the like), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids and the like). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Typically, nucleotide monomers are linked via phosphodiester bonds, although synthetic forms of nucleic acids can comprise other linkages (e.g., peptide nucleic acids as described in Nielsen et al. (Science 254:1497-1500, 1991). A nucleic acid can be or can include, e.g., a chromosome or chromosomal segment, a vector (e.g., an expression vector), an expression cassette, a naked DNA or RNA polymer, the product of a polymerase chain reaction (PCR), an oligonucleotide, a probe, and a primer. A nucleic acid can be, e.g., single-stranded, doublestranded, or triple-stranded and is not limited to any particular length. Unless otherwise indicated, a particular nucleic acid sequence optionally comprises or encodes complementary sequences, in addition to any sequence explicitly indicated.

[0080] The term "oligonucleotide" refers to a nucleic acid that includes at least two nucleic acid monomer units (e.g., nucleotides). An oligonucleotide typically includes from about six to about 175 nucleic acid monomer units, more typically from about eight to about 100 nucleic acid monomer units, and still more typically from 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 will depend on many factors, including the ultimate function or use of the oligonucleotide. Oligonucleotides are optionally prepared by any suitable method, including, but not limited to, isolation of an existing or natural sequence, DNA replication or amplification, reverse transcription, cloning and restriction digestion of appropriate sequences, or direct chemical synthesis by a method such as the phosphotriester method of Narang etal. (Meth. Enzymol. 68:90-99, 1979); the phosphodiester method of Brown et al. (Meth. Enzymol. 68:109-151, 1979); the di ethylphosphorami dite 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 methods; or the solid support method of U.S. Patent No. 4,458,066 or other methods known to those skilled in the art.

[0081] The term "primer" as used herein refers to a polynucleotide capable of acting as a point of initiation of template-directed nucleic acid synthesis when placed under conditions in which polynucleotide extension is initiated (e.g., under conditions comprising the presence of requisite nucleosidetriphosphates (as dictated by the template that is copied) and a polymerase in an appropriate buffer and at a suitable temperature or cycle(s) of temperatures (e.g., as in a polymerase chain reaction)). To further illustrate, primers can also be used in a variety of other oligonucleotide-mediated synthesis processes, including as initiators of de novo RNA synthesis and in vitro transcription-related processes (e.g., nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), etc.). A primer is typically a single-stranded oligonucleotide (e.g., oligodeoxyribonucleotide). The appropriate length of a primer depends on the intended use of the primer but typically ranges from 6 to 40 nucleotides, more typically from 15 to 35 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template for primer elongation to occur. In certain embodiments, the term "primer pair" means a set of primers including a 5' sense primer (sometimes called “forward”) that hybridizes with the complement of the 5' end of the nucleic acid sequence to be amplified and a 3' antisense primer (sometimes called “reverse”) that hybridizes with the 3' end of the sequence to be amplified (e.g., if the target sequence is expressed as RNA or is an RNA). A primer can be labeled, if desired, by incorporating a label detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include32P, fluorescent dyes, electron-dense reagents, enzymes (as commonly used in ELISA assays), biotin, or haptens and proteins for which antisera or monoclonal antibodies are available.

[0082] The term "conventional" or "natural" when referring to nucleic acid bases, nucleoside triphosphates, or nucleotides refers to those which occur naturally in the polynucleotide being described (i.e., for DNA these are dATP, dGTP, dCTP and dTTP). Additionally, diTP, and 7-deaza-dGTP are frequently utilized in place of dGTP and 7-deaza-dATP can be utilized in place of dATP in in vitro DNA synthesis reactions, such as sequencing. Collectively, these may be referred to as dNTPs.

[0083] The term "unconventional" or "modified" when referring to a nucleic acid base, nucleoside, or nucleotide includes modification, derivations, or analogues of conventional bases, nucleosides, or nucleotides that naturally occur in a particular polynucleotide. Certain unconventional nucleotides are modified at the 2' position of the ribose sugar in comparison to conventional dNTPs. Thus, although for RNA the naturally occurring nucleotides are ribonucleotides (i.e., ATP, GTP, CTP, UTP, collectively rNTPs), because these nucleotides have a hydroxyl group at the 2' position of the sugar, which, by comparison is absent in dNTPs, as used herein, ribonucleotides are unconventional nucleotides as substrates for DNA polymerases. 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 that have a 2', 3' di deoxy structure and are referred to as dideoxynucleoside triphosphates. The dideoxynucleoside triphosphatesddATP, ddTTP, ddCTP and ddGTP are referred to collectively as ddNTPs. Additional examples of terminator compounds include 2'-PO4 analogs of ribonucleotides (see, e.g., U.S. Application Publication Nos. 2005 / 0037991 and 2005 / 0037398). Other unconventional nucleotides include phosphorothioate dNTPs ([a-S]dNTPs), 5'-[a-borano]-dNTPs, [a]-methyl-phosphonate dNTPs, and ribonucleoside triphosphates (rNTPs). Unconventional bases may be labeled with radioactive isotopes such as32P,33P, or35S; fluorescent labels; chemiluminescent labels; bioluminescent labels; hapten labels such as biotin; or enzyme labels such as streptavidin or avidin. Fluorescent labels may include dyes that are negatively charged, such as dyes of the fluorescein family, or dyes that are neutral in charge, such as dyes of the rhodamine family, or dyes that are positively charged, such as dyes of the cyanine family. Dyes of the fluorescein family include, e.g., 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 marketed by Perkin-Elmer (Boston, MA), Applied Biosystems (Foster City, CA), or Invitrogen / Molecular Probes (Eugene, OR). Dyes of the cyanine family include Cy2, Cy3, Cy5, and Cy7 and are marketed by GE Healthcare UK Limited (Amersham Place, Little Chalfont, Buckinghamshire, England).

[0084] As used herein, "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence in the comparison window can comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0085] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same. Sequences are "substantially identical" to each other if they have a specified percentage of nucleotides or amino acid residues that are the same (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 over a specified region)), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Sequences are "substantially identical" to each other if they are 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% identical. These definitions also refer to the complement of a test sequence. Optionally, the identity exists over a regionthat is at least about 50 nucleotides in length, or more typically over a region that is 100 to 500 or 1000 or more nucleotides in length.

[0086] The terms "similarity" or "percent similarity," in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of amino acid residues that are either the same or similar as defined by a conservative amino acid substitutions (e.g., 60% similarity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% similar over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Sequences are "substantially similar" to each other if they are 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% similar to each other. Optionally, this similarly exists over a region that is at least about 50 amino acids in length, or more typically over a region that is at least about 100 to 500 or 1000 or more amino acids in length. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters are commonly used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities or similarities for the test sequences relative to the reference sequence, based on the program parameters.

[0087] A "comparison window," as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, 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 etal., Current Protocols in Molecular Biology (1995 supplement)).

[0088] Examples of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the 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), respectively. Software for performing BLAST analyses is publicly available through the National Center forBiotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0089] 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 measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, typically less than about 0.01, and more typically less than about 0.001.

[0090] The term “reaction efficiency” refers to the fraction of substrate molecules that are converted into a product in a given reaction. One specific example of reaction efficiency is reverse transcription reaction efficiency. The terms “reverse transcription reaction efficiency”, “reverse transcription efficiency” and “RT efficiency” each refer to the fraction of target RNA molecules that are reverse transcribed as cDNA in a given reverse transcription reaction. In certain embodiments, the mutant DNA polymerases of the invention have improved reverse transcription reaction efficiencies relative to unmodified forms of these DNA polymerases. That is, these mutant DNA polymerases reverse transcribe a higher fraction of RNA templates than their unmodified forms under a particular set of reaction conditions. Without being limited by theory, the ability of a mutant DNA polymerase described herein to reverse transcribea higher fraction of RNA templates can be due to an increased reverse transcription activity, for example, an increased nucleotide incorporation rate and / or increased processivity of the enzyme. Reverse transcription efficiency can be measured, for example, by measuring the crossing point (Cp) of a PCR reaction using a RNA template, and comparing the Cp value to a Cp value of a control reaction in which a DNA template of the same sequence (except U’s are replaced with T’s) is amplified, wherein the RNA and DNA amplifications use a common primer set and the same polymerase, e.g., as described in the examples. A test polymerase has improved RT efficiency when the test polymerase has a decreased Cp value compared to a control polymerase when RNA is used as a template but has a substantially unchanged Cp value relative to the control polymerase when DNA is used as a template. In some embodiments, a 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. Improved RT efficiency of a test polymerase can be measured as described in the Examples.

[0091] The term “mismatch tolerance” refers to the ability of a polymerase to tolerate a mismatch-containing sequence when extending a nucleic acid (e.g., a primer or other oligonucleotide) in a templatedependent manner by attaching (e.g., covalently) one or more nucleotides to the nucleic acid. The term “3 ’ mismatch tolerance” refers to the ability of a polymerase to tolerate a mismatch-containing (nearly complementary) sequence 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. Mismatches to the template may also be located at the 3 ’ penultimate nucleotide of the primer, or at another position within the sequence of the primer.

[0092] The term “mismatch discrimination” refers to the ability of a polymerase to distinguish a fully complementary sequence from a mismatch-containing sequence when extending a nucleic acid (e.g., a primer or other oligonucleotide) in a template-dependent manner by attaching (e.g., covalently) one or more nucleotides to the nucleic acid. The term “3 ’-mismatch discrimination” refers to the ability of a polymerase to distinguish a fully complementary sequence from a mismatch-containing (nearly complementary) sequence where the nucleic acid to be extended (e.g., a primer or other oligonucleotide) has a mismatch at the nucleic acid’s 3’ terminus compared to the template to which the nucleic acid hybridizes. The term “mismatch” refers to the existence of one or more base mispairings (or “noncomplementary base oppositions”) within a stretch of otherwise complementary duplex-forming (or potentially duplex-forming) sequences.

[0093] The term “Cp value” or “crossing point” value refers to a value that allows quantification of input target nucleic acids. 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, February2005, pp. 287-293). In the second derivative method, a Cp corresponds to the first peak of a second derivative curve. This peak corresponds to the beginning of a log-linear phase. The second derivative method calculates a second derivative value of the real-time fluorescence intensity curve, and only one value is obtained. The original Cp method is based on a locally defined, differentiable approximation of the intensity values, e.g., by a polynomial function. Then the third derivative is computed. The Cp value is the smallest root of the third derivative. The Cp can also be determined using the fit point method, in which the Cp is determined by the intersection of a parallel to the threshold line in the log-linear region (Van Luu-The, et al., BioTechniques, Vol. 38, No. 2, February 2005, pp. 287-293). The Cp value provided by the LightCycler® instrument offered by Roche by calculation according to the second-derivative maximum method.

[0094] The term “PCR efficiency” refers to an indication of cycle to cycle amplification efficiency. PCR efficiency is calculated for each condition using the equation: % PCR efficiency = (10('slope)-l) x 100, wherein the slope was calculated by linear regression with the log copy number plotted on the y-axis and Cp plotted on the x-axis. PCR efficiency can be measured using a perfectly matched or mismatched primer template.

[0095] The term “nucleic acid extension rate” refers the rate at which a biocatalyst (e.g., an enzyme, such as a polymerase, ligase, or the like) extends a nucleic acid (e.g., a primer or other oligonucleotide) in a template-dependent or template-independent manner by attaching (e.g., covalently) one or more nucleotides to the nucleic acid. To illustrate, certain mutant DNA polymerases described herein have improved nucleic acid extension rates relative to unmodified forms of these DNA polymerases, such that they can extend primers at higher rates than these unmodified forms under a given set of reaction conditions.

[0096] The term “tolerance of 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 would inhibit the polymerase activity or reverse transcription activity of a control polymerase. In some embodiments, the improved polymerase is capable of polymerase or reverse transcription activity in the presence of an amount of the inhibitor that would essentially eliminate the control polymerase activity.

[0097] The term “5 ’-nuclease probe” refers to an oligonucleotide that comprises at least one light emitting labeling moiety and that is used in a 5 ’-nuclease reaction to effect target nucleic acid detection. In some embodiments, for example, a 5’-nuclease probe includes only a single light emitting moiety (e.g., a fluorescent dye, etc.). In certain embodiments, 5’-nuclease probes include regions of selfcomplementarity such that the probes are capable of forming hairpin structures under selected conditions. To further illustrate, in some embodiments a 5 ’-nuclease probe comprises at least two labeling moieties and emits radiation of increased intensity after one of the two labels is cleaved orotherwise separated from the oligonucleotide. In certain embodiments, a 5 ’-nuclease probe is labeled with two different fluorescent dyes, e.g., a 5’ terminus reporter dye and the 3’ terminus quencher dye or moiety. In some embodiments, 5 ’-nuclease probes are labeled at one or more positions other than, or in addition to, terminal positions. When the probe is intact, energy transfer typically occurs between the two fluorophores such that fluorescent emission from the reporter dye is quenched at least in part. During an extension step of a 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, e.g., a Taq polymerase or another polymerase having this activity such that the fluorescent emission of the reporter dye is no longer quenched. Exemplary 5’-nuclease probes are also described in, e.g., U.S. Patent No. 5,210,015, U.S. Patent No. 5,994,056, and U.S. Patent No. 6,171,785. In other embodiments, a 5’ nuclease probe may be labeled with two or more different reporter dyes and a 3’ terminus quencher dye or moiety.

[0098] The term “FRET” or “fluorescent resonance energy transfer” or “Foerster resonance energy transfer” refers to a transfer of energy between at least two chromophores, a donor chromophore and an acceptor chromophore (referred to as a quencher). The donor typically transfers energy to the acceptor when the donor is excited by light radiation with a suitable wavelength. The acceptor typically re-emits the transferred energy in the form of light radiation with 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 member 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 BlackHole Quenchers™ (BHQ), (Biosearch Technologies, Inc., Novato, Cal.), Iowa Black™ (Integrated DNA Tech., Inc., Coralville, Iowa), and BlackBerry™ Quencher 650 (BBQ-650) (Berry & Assoc., Dexter, Mich.).

[0099] The term “Polymerase Specificity” refers to the ability of a DNA polymerase enzyme to accurately select and incorporate the correct nucleotide during DNA replication or amplification. It involves the enzyme's ability to recognize the complementary base pairs and incorporate the corresponding nucleotides in the growing DNA strand. Polymerase specificity ensures that the correct nucleotide is added at the appropriate position in the DNA sequence. In one aspect, polymerase specificity can have an effect on the occurrence of non-specific amplification, a process that produces unintended nucleic acid copies during a reaction, which can cause false positives and make it difficult to detect specific targets.

[0100] The terms “non-specific amplification” and “off-target amplification” refer to the generation of unintended or undesirable DNA products during the amplification process. Non-specific amplification can occur, for example, when PCR primers bind to non-target DNA sequences, leading to the amplification of unintended fragments. Several common causes of non-specific amplification in PCR include i) contamination due the presence of extraneous DNA, ii) primer design, including primersequences that are not specific enough to the target region, iii) reaction conditions, such as annealing temperature and choice of metal ion (e.g., Mg2+vs. Mn2+), iv) template complexity, including the presence of repetitive sequences or regions with high sequence similarity, and v) polymerase specificity. In the case of a polymerase with poor specificity, such a polymerase may be more tolerant of mismatches, either due to incorporation of an incorrect nucleotide or extension of a mismatched nucleotide, thereby resulting in an increased potential for non-specific amplification.

[0101] BRIEF DESCRIPTION OF THE DRAWINGS

[0102] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0103] FIG. 1 depicts an amino acid sequence alignment of a region from the polymerase domain of exemplary DNA polymerases from various species of bacteria: Thermus species Z05 (Z05) (SEQ ID NO: 12), Thermus aquaticus (Taq) (SEQ ID NO: 13), Thermus filiformus (Tfi) (SEQ ID NO: 14), Thermus flavus (TH) (SEQ ID NO:15), Thermus species spsl7 (Spsl7) (SEQ ID NO:16), Thermus thermophilus (Tth) (SEQ ID NO: 17), Thermus caldophilus (Tea) (SEQ ID NO: 18), Thermotoga maritima (Tma) (SEQ ID NO: 19), Thermotoga neopolitana (Tne) (SEQ ID NO:20), Thermosipho africanus (Taf) (SEQ ID NO:21), Deinococcus radiodurans (Dra) (SEQ ID NO:23), Bacillus stearothermophilus (Bst) (SEQ ID NO:24), and Bacillus caldotenax (Bea) (SEQ ID NO:25). In addition, the polypeptide regions shown comprise the amino acid motif X1-X2-X3-X4-D-Y-S-Q-X5-E-L-R-X6-L-A-H-X7-X8-X9-D (SEQ ID NO:26), the variable positions of which are further defined herein. This motif is highlighted in bold type for each polymerase sequence. Amino acid positions amenable to mutation in accordance with the present invention are indicated with an asterisk (*). Gaps in the alignments are indicated with a dot (.).

[0104] FIG. 2 provides sequence identities among the following DNA Polymerase I enzymes: Thermus sp. Z05 DNA polymerase (Z05); Thermus aquaticus DNA polymerase (Taq); Thermus filiformis DNA polymerase (Tfi); Thermus flavus DNA polymerase (TH); Thermus sp. sps!7 DNA polymerase (Spsl7); Thermus thermophilus DNA polymerase (Tth); Thermus caldophilus DNA polymerase (Tea); Deinococcus radiodurans DNA polymerase (Dra); Thermotoga maritima DNA polymerase (Tma); Thermotoga neopolitana DNA polymerase (Tne); Thermosipho africanus DNA polymerase (Taf); Bacillus stearothermophilus DNA polymerase (Bst); and Bacillus caldotenax DNA polymerase (Bea).

[0105] (A) sequence identities over the entire polymerase I enzyme (corresponding to amino acids 1-834 of Z05); and (B) sequence identities over the polymerase sub domain corresponding to amino acids 420-834 of Z05.

[0106] FIG. 3 provides sequence identities among various Thermus sp DNA Polymerase I enzymes: Thermus sp. Z05 DNA polymerase (Z05); Thermus aquaticus DNA polymerase (Taq); Thermus filiformis DNApolymerase (Tfi); Thermits flavus DNA polymerase (TH); Thermits sp. sps!7 DNA polymerase (Spsl7); Thermus thermophilus DNA polymerase (Tth); and Thermus caldophilus DNA polymerase (Tea). (A) sequence identities over the entire polymerase I enzyme (corresponding to amino acids 1-834 of Z05); and (B) sequence identities over the polymerase sub domain corresponding to amino acids 420-834 of Z05.

[0107] As described in Example 5, FIG. 4 depicts a cycle of directed evolution, mimicking the process of biological evolution, with the goal to generate enzymes with improved or altered activity for specific applications. FIG. 4 is taken from Packer and Liu, “Method of the directed evolution of proteins,” Nature Reviews: Genetics 16:379-394 (2015). This method involves the following steps: (1) step 1 is a divarication step, which involves the generation of a diverse library of a single gene, (2) step 2 is a translation step, which involves translating the library into corresponding gene products, (3) step 3 is a screening step, which involves isolating members with the desired properties, and (4) step 4 is a replication steps, wherein the best variants undergo subsequent rounds of diversification and screening / selection.

[0108] As described in Example 5, FIG.5 depicts the Z05 library screening workflow.

[0109] As discussed in Examples 5 and 6, under primary screening, roughly 6,000 clones were expressed and screened by primary MS2 singleplex Mg-based RT-PCR with 1 minute RT incubation time. Hits or results with early Cps and high RFI were sequenced, re-arrayed, and screened by secondary assays for RT efficiency and specificity. Sequencing hits from the primary screen are depicted in FIG.6.

[0110] As described in Example 5, the substitution at the 542 position (i.e., K542F) (SEQ ID NO:41) emerged as one of the leading candidates with good RT efficiency and specificity (i.e., RFI). K542F (SEQ ID NO:41) was purified and characterized in Roche cobas® LIAT “RAB” multiplex system (CRAB-COVID19) with C21 and parental Z05 I616M I709K controls. FluB Wild Type and mutant RNA templates (mismatch under RT primer) were employed in four-plex with RSV, FluB, FluA, and internal control (IC) oligonucleotides. Mg-based RT-PCR was performed on Roche LC 480 with unmodified primers, with results depicted in FIG.7A and FIG.7B. In FIG.7A and FIG.7B, “purified DM” refers to the parental polymerase (SEQ ID NO:42), “C21” refers to the C21 polymerase (SEQ ID NO:43), and “DM K542F” refers to the K542F mutant polymerase (SEQ ID NO:41). The results show that that the K542F variant (SEQ ID NO:41) was the only polymerase to pick up the mutant FluB target, as shown in FIG.7A and FIG.7B. The results also show that the K542F variant (SEQ ID NO:41) shows a higher RFI than the C21 polymerase and the parental polymerase (see, FIG. 7B).

[0111] FIG. 8 depicts the assay conditions and thermal profile for the RT assay described in Example 6. To measure RT efficiency, the variants were screened by Hepatitis C Virus (HCV) RT-PCR. An HCV RT-PCR was established with 0.5X RAB oligos, 3,000 copies of genomic DNA, 105copies of HCV RNA, with 75 seconds of RT reaction (see, FIG.8).FIG. 9A depicts the HCV RT-PCR results for all samples, and FIG. 9B shows the results for the top samples, as described in Example 6.

[0112] FIG. 10 shows the assay conditions and thermal profile for the SYBR test (in the absence of template RNA), which measures specificity, as described in Example 6. SYBR binds double-stranded DNA non-specifically. When this PCR is run without a template, SYBR signal is generated by the amplification of non-specific products (e.g., primer dimers, etc.). In this screen, enzyme variants that generate more non-specific products will have earlier Cps in the PCR.

[0113] FIG. 11 depicts the results of the SYBR test, described in Example 6.

[0114] FIG. 12A shows a bar graph and FIG. 12B shows a JMP graph of the Sanger sequencing results, described in Example 6. The samples (38 in total) were sequenced via Sanger sequencing to uncover and ascertain which amino acid, at the 542 position, was most common in the top performing samples. Sanger sequencing results show that all six (6) top samples exhibit the K542Y substitution, as shown in the bar graph of FIG. 12A, and the IMP graph of FIG. 12B.

[0115] FIG. 13 shows an SDS-PAGE conducted to determine the lysate concentrate was assessed for the top samples and controls, as described in Example 6. These results show that the lysate concentrations of the top samples was higher than the controls (by roughly 1.5-2 times higher), as shown in FIG. 13, suggesting, perhaps, better and higher expression of the K542F and K542Y mutations.

[0116] FIG. 14 depicts a table showing all of the data from the HCV RT-PCR tests and SYBR tests results with 6 minutes and 75 seconds RT time, as described in Example 6. These results show that from the K542X library, the K542Y (SEQ ID NO:40) is shown to be an excellent mutation, in terms of RT efficiency and specificity.

[0117] FIG. 15 shows the primary screening workflow, as described in Example 7. A Single Site Variant Library consisting of Z05 I616M I709K (Z05DM) backbone (SEQ ID NO:42) with one additional amino acid in the polymerase domain was cloned and transformed into E. coli BL21 expression strain. Clonal mutant library glycerol culture stocks were arrayed in 384 well plates. Plate-based expression and partial purification was done to make corresponding clone lysates. Screening of diluted lysates was conducted in Mg-based MS2 RT-PCR.

[0118] For the primary screening workflow, described in Example 7, FIG. 16 shows that on plate 1910, the top clones were chosen for early Cps and high fluorescence signal, and were in wells Hl 9 and El 3.

[0119] FIG. 17 depicts the results of the HCV RT-PCR assay, as described in Example 7. Here, the K542F (SEQ ID NO:41) and K542Y (SEQ ID NO:40) mutants were purified and characterized further in an HCV RT-PCR system. In particular, an HCV Mg-based RT-PCR reaction was conducted, comparing the purified K542Y (SEQ ID NO:40) against the Z05DM parental (SEQ ID NO:42). In this reaction, 50nM enzyme was used, with a 2 minute RT incubation at 65°C, with RNA and DNA as templates. The results show that for the K542Y mutant (SEQ ID NO: 40), RNA Cps are 5 cycles earlier than theZ05DM parental (SEQ ID NO: 42), that the RT efficiency for the K542Y (SEQ ID NO: 40) is ~ 60 times higher than the Z05DM parental (SEQ ID NO:42), and that the PCR efficiencies are comparable between the two enzymes, but the Z05DM parental (SEQ ID NO: 42) DNA Cps come up 1 cycle earlier.

[0120] FIG. 18 depicts the result of the SYBR-NTC assay, as described in Example 7. Here, the RT and PCR efficiency of pure K542Y (SEQ ID NO:40) was assessed in a SYBR-NTC assay. In particular, the specificity was assessed in an HCV Mg2+-based RT-PCR multiplex (MMx) assay was conducted comparing the following three (3) enzymes: (i) K542Y mutant (SEQ ID NO:40), (ii) the Z05DM parental (SEQ ID NO:42), and (iii) the Z05 wild type polymerase (SEQ ID NO:1). The reaction conditions were 50nM enzyme, 2 minute RT incubation at 65°C, no template, 0.2X SYBR Green, with four replicates for each enzyme. Cps represent non-specific amplification product (NS A), and the earlier the Cp, the more NSA present. The results showthat the K542Y mutant (SEQ ID NO:40) SYBR Cps are ~3 cycles delayed compared to the Z05DM parental (SEQ ID NO:42), and the Z05 wild type polymerase (SEQ ID NO:1). Additionally, the K542Y mutant (SEQ ID NO:40) exhibits improved specificity compared to the Z05DM parental (SEQ ID NO:42), and the Z05 wild type polymerase (SEQ ID NO:1).

[0121] DETAILED DESCRIPTION

[0122] The present invention provides improved DNA polymerases in which one or more amino acids in the polymerase domain have been mutated relative to a functional DNA polymerase. The DNA polymerases of the invention are active enzymes having increased reverse transcriptase efficiency (e.g., in the presence of Mn2+and Mg2+divalent cations) relative to the unmodified form of the polymerase and / or increased mismatch tolerance, extension rate and tolerance of RT and polymerase inhibitors. In certain embodiments, the mutant DNA polymerases may be used at lower concentrations for superior or equivalent performance as the parent enzymes. In some embodiments, the mutant DNA polymerases have increased reverse transcriptase efficiency while retaining substantially the same DNA-dependent polymerase activity relative to an unmodified or control polymerase.

[0123] DNA polymerases that more efficiently perform reverse transcription are helpful, for example, in a variety of applications involving assays that employ RT-PCR to detect and / or quantify RNA targets. The DNA polymerases are therefore useful in a variety of applications involving polynucleotide extension as well as reverse transcription or amplification of polynucleotide templates, including, for example, applications in recombinant DNA studies and medical diagnosis of disease. The mutant DNA polymerases are also particularly useful, because of their tolerance for mismatches, for detecting targets that possibly have variable sequences (e.g., viral targets, or cancer and other disease genetic markers).

[0124] In some embodiments, DNA polymerases of the invention can be characterized by having the following motif:Xi-X2-X3-X4-Asp-Tyr-Ser-Gln-X5-Glu-Leu-Arg-X6-Leu-Ala-His-X7-Xs-X9-Asp (also referred to herein in the one-letter code as X1-X2-X3-X4-D-Y-S-Q-X5-E-L-R-X6-L-A-H-X7-X8-X9-D) (SEQ ID NO: 8); wherein:

[0125] Xi is Leu (L) or He (I);

[0126] X2is Vai (V), Leu (L), He (I) or Phe (F);

[0127] X3is Ala (A), Vai (V), Ser (S) or Gly (G);

[0128] X4 is Leu (L) or Ala (A);

[0129] Xs is any amino acid other than He (I), Lys (K); Asn (N), Gin (Q) and Thr (T);

[0130] Xe is Vai (V), He (I) or Leu (L);

[0131] X7 is Leu (L), Vai (V) or He (I);

[0132] Xs is Ser (S) or Ala (A);

[0133] X9 is Gly (G), Lys (K), Asp (D) or Glu (E).

[0134] In some embodiments, Xs is selected from G, A, W, P, S, F, Y, C, D, E, V, R. L, M, or H.

[0135] In some embodiments, DNA polymerases of the invention can be characterized by having the following motif:

[0136] Leu-Val-Xs-Leu-Asp-Tyr-Ser-Gln-Xs-Glu-Leu-Arg-Val-Leu-Ala-His-Leu-Ser-Gly-Asp

[0137] (also referred to herein in the one-letter code as L-V-X3-L-D-Y-S-Q-X5-E-L-R-V-L-A-H- L-S-G-D) (SEQ ID NO:9); wherein

[0138] X3 is Ala (A) or Vai (V); and

[0139] Xs any amino acid other than He (I), Lys (K); Asn (N), Gin (Q) and Thr (T).

[0140] In some embodiments, DNA polymerases of the invention can be characterized by having the following motif:

[0141] Leu-Val-Ala-Leu-Asp-Tyr-Ser-Gln-Xs-Glu-Leu-Arg-Val-Leu-Ala-His-Leu-Ser-Gly-Asp

[0142] (also referred to herein in the one-letter code as L-V-A-L-D-Y-S-Q-Xs-E-L-R-V-L-A-H- L-S-G-D) (SEQ ID NOTO); wherein:

[0143] Xs is any amino acid other than He (I), Lys (K); Asn (N), Gin (Q) and Thr (T).

[0144] In some embodiments, DNA polymerases of the invention can be characterized by having the following motif:

[0145] Leu-Val-Ala-Leu-Asp-Tyr-Ser-Gln-Xs-Glu-Leu-Arg-Val-Leu-Ala-His-Leu-Ser-Gly-Asp

[0146] (also referred to herein in the one-letter code as L-V-A-L-D-Y-S-Q-Xs-E-L-R-V-L-A-H-L-S-G-D) (SEQ ID NO: 11); wherein: X5is Met (M).

[0147] In some embodiments, the amino acid at position X3 of SEQ ID NO: 8 or 9 is not Vai (V). In some embodiments, the amino acid at position X3 of SEQ ID NO: 8 or 9 is not Asp (D). In some embodiments, the amino acid at position X3 of SEQ ID NO: 8 or 9 is Ala (A).

[0148] In some embodiments, DNA polymerases of the invention can be characterized by having the abovemotifs (e.g., SEQ ID NOs:8, 9, 10, and 11), optionally in combination with additional motifs described below. For example, in some embodiments, the DNA polymerase further comprises the motif of SEQ ID NO: 29 and / or SEQ ID NO: 38.

[0149] This motif is present within the “fingers” domain (L alpha helix) of many Family A type DNA- dependent DNA polymerases, particularly thermostable DNA polymerases from thermophilic bacteria (Li et al., EMBO J. 17:7514-7525, 1998). For example, Figure 1 shows an amino acid sequence alignment of a region from the “fingers” domain of DNA polymerases from several species of bacteria: Bacillus caldotenax, Bacillus stearothermophilus , Deinococcus radiodurans, Thermosipho africanus, Thermotoga maritima, Thermotoga neopolitana, Thermus aquaticus, Thermus caldophilus, Thermus filiformus, Thermus flavus, Thermus sp. spsl7, Thermus sp. Z05, and Thermus thermophilus. As shown, the native sequence corresponding to the motif above is present in each of these polymerases, indicating a conserved function for this region of the polymerase. Figure 2 provides sequence identities among these DNA polymerases.

[0150] Accordingly, in some embodiments, the invention provides for a polymerase comprising SEQ ID NO: 8, 9, 10, or 11, having the improved activity and / or characteristics described herein, and wherein the DNA polymerase is otherwise a wild-type or a naturally occurring DNA polymerase, such as, for example, a polymerase from any of the species of thermophilic bacteria listed above, or is substantially identical to such a wild-type or a naturally occurring DNA polymerase. For example, in some embodiments, the polymerase of the invention comprises SEQ ID NO: 8, 9, 10, or 11 and is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 32, 33, 34, 35, 36, 37, or 39. In one variation, 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 Thermus, e.g., Thermotoga. The full nucleic acid and amino acid sequence for numerous thermostable DNA polymerases are available. The sequences each of Thermus aquaticus (Taq) (SEQ ID NO:2), Thermus thermophilus (Tth) (SEQ ID NO:6), Thermus species Z05 (SEQ ID NO:1), Thermus species spsl7 (SEQ ID NO:5), Thermotoga maritima (Tma) (SEQ ID NO:34), and Thermosipho africanus (Taf) (SEQ ID NO:33) polymerase have been published in PCT International Patent Publication No. WO 92 / 06200. The sequence for the DNA polymerase from Thermus flavus (SEQ ID NO:4) has been published in Akhmetzjanov and Vakhitov (Nucleic Acids Research 20:5839, 1992). 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 filiformis can be recovered from ATCC Deposit No. 42380 using, e.g., the methods provided in U.S. Patent No. 4,889,818, as well as the sequence information provided in Table 1. The sequence of the Thermotoga neapolitana DNA polymerase (SEQ ID NO:35) is from GeneSeq Patent Data Base Accession No. R98144 and PCT WO 97 / 09451. The sequence of the thermostable DNA polymerasefrom Bacillus caldotenax (SEQ ID NO:37 is described in, e.g., Uemori et al. (J Biochem (Tokyo) 113(3):401-410, 1993; see also, Swiss-Prot database Accession No. Q04957 and GenBank Accession Nos. DI 2982 and BAA02361). Examples of unmodified forms of DNA polymerases that can be modified as described herein are also described in, e.g., 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. Representative full length polymerase sequences are also provided in the sequence listing. Also amenable to the mutations described herein are functional DNA polymerases that have been previously modified (e.g., by amino acid substitution, addition, or deletion). In some embodiments, such functional modified polymerases retain the amino acid motif of SEQ ID NO: 8 (or a motif of SEQ ID NO:9, 10 or 11), and optionally the amino acid motif of SEQ ID NO:38. Thus, suitable unmodified DNA polymerases also include functional variants of wild-type or naturally occurring polymerases. Such variants typically will have substantial sequence identity or similarity to the wild-type or naturally occurring polymerase, typically at least 80% sequence identity and more typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0151] In some embodiments, the polymerase of the invention, as well as having a polymerase domain comprising SEQ ID NOS: 8, 9, 10, or 11, also comprises a nuclease domain (e.g., corresponding to positions 1 to 291 ofZ05).

[0152] In some embodiments, a polymerase of the invention is a chimeric polymerase, i.e., comprising polypeptide regions from two or more enzymes. Examples of such chimeric DNA polymerases are described in, e.g., U.S. Patent No. 6,228,628. Particularly suitable are chimeric CS-family DNA polymerases, which include the CS5 (SEQ ID NO:27) and CS6 (SEQ ID NO:28) polymerases and variants thereof having substantial amino acid sequence identity or similarity to SEQ ID NO: 27 or SEQ ID NO:28 (typically at least 80% amino acid sequence identity and more typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity) and can thus be modified to contain SEQ ID NO: 8. The CS5 and CS6 DNA polymerases are chimeric enzymes derived from Thermus sp. Z05 and Thermotoga maritima (Tma) DNA polymerases. They comprise the N-terminal 5'-nuclease domain of the Thermus enzyme and the C-terminal 3'-5' exonuclease and the polymerase domains of the Tma enzyme. These enzymes have efficient reverse transcriptase activity, can extend nucleotide analog-containing primers, and can incorporate alpha-phosphorothioate dNTPs, dUTP, diTP, and also fluorescein- and cyanine-dye family labeled dNTPs. The CS5 and CS6 polymerases are also efficient Mg2+-activated PCR enzymes. The CS5 and CS6 chimeric polymerases are further described in, e.g., U.S. Patent No. 7,148,049.

[0153] In some embodiments, the amino acid substitutions are single amino acid substitutions. The DNA polymerases provided herein can comprise one or more amino acid substitutions in the active site relative to the unmodified polymerase. In some embodiments, the amino acid substitution(s) compriseat least position Xs of the motif set forth in SEQ ID NO:8 (or a motif of SEQ ID NO:9, 10 or 11). Amino acid substitution at this position confers increased reverse transcriptase efficiency, mismatch tolerance, extension rate and / or tolerance of RT and polymerase inhibitors, yielding a mutant DNA polymerase with an increased reverse transcriptase efficiency, mismatch tolerance, extension rate and / or tolerance of RT and polymerase inhibitors relative to the unmodified polymerase. Typically, the amino acid at position Xs is substituted with an amino acid that does not correspond to the native sequence within the motif set forth in SEQ ID NO:8 (or a motif of SEQ ID NO:9, 10 or 11). Thus, typically, the amino acid at position Xs, if substituted, is not He (I), as I occurs at this position in naturally-occurring polymerases. See, e.g., Figure 1. In certain embodiments, amino acid substitutions include G, A, W, P, S, T, F, Y, C, N, Q, D, E, K, V, R, L, M, or H at position Xs. In certain embodiments, the amino acid substitutions at postion Xs do not include I, K, N, Q or T. In certain embodiments, amino acid substitutions include Methionine (M) at position Xs. Other suitable amino acid substitution(s) at one or more of the identified sites can be determined using, e.g., known methods of site-directed mutagenesis and determination of polynucleotide extension performance in assays described further herein or otherwise known to persons of skill in the art.

[0154] In some embodiments, the polymerase of the invention comprises SEQ ID NO: 8, 9, 10, or 11 and further comprises one or more additional amino acid changes (e.g., by amino acid substitution, addition, or deletion) compared to a native polymerase. In some embodiments, such polymerases retain the amino acid motif of SEQ ID NO:8 (or a motif of SEQ ID NO:9, 10 or 11), and further comprise the amino acid motif of SEQ ID NO:38 (corresponding to the D580X mutation of Z05 (SEQ ID NO: 1)) as follows:

[0155] Thr-Gly-Arg-Leu-Ser-Ser-X7-Xs-Pro-Asn-Leu-Gln-Asn

[0156] (also referred to herein in the one-letter code as T-G-R-L-S-S-X7-X8-P-N-L-Q-N)

[0157] (SEQ ID NO: 38); wherein

[0158] X7 is Ser (S) or Thr (T); and

[0159] Xs is any amino acid other than Asp (D) or Glu (E)

[0160] The mutation characterized by SEQ ID NO: 38 is discussed in more detail in, e.g., US Patent Publication No. 2009 / 0148891. Such functional variant polymerases typically will have substantial sequence identity or similarity to the wild-type or naturally occurring polymerase (e.g., SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 32, 33, 34, 35, 36, 37, or 39), typically at least 80% amino acid sequence identity and more typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity.

[0161] In some embodiments, the polymerase of the invention comprises SEQ ID NO: 8, 9, 10, or 11 and further comprises the amino acid motif of SEQ ID NO:29 (corresponding to the I709X mutation of Z05 (SEQ ID NO:1)) as follows:Xi-X2-X3-X4-X5-X6-X7-X8-X9-Xio-Xii-Xi2-Xi3-Gly-Tyr-Val-Xi4-Thr-Leu (also referred

[0162] to herein in the one-letter code as X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-G-Y-V-X14-T-L) (SEQ ID NO:29); wherein

[0163] Xi is Ala (A), Asp (D), Ser (S), Glu (E), Arg (R) or Gin (Q);

[0164] X2 is Trp (W) or Tyr (Y);

[0165] X3 is any amino acid other than He (I), Leu (L) or Met (M);

[0166] X4 is Glu (E), Ala (A), Gin (Q), Lys (K), Asn (N) or Asp (D);

[0167] Xs is Lys (K), Gly (G), Arg (R), Gin (Q), His (H) or Asn (N);

[0168] X6is Thr (T), Vai (V), Met (M) or He (I);

[0169] X7 is Leu (L), Vai (V) or Lys (K);

[0170] X8is Glu (E), Ser (S), Ala (A), Asp (D) or Gin (Q);

[0171] X9 is Glu (E) or Phe (F);

[0172] X10 is Gly (G) or Ala (A);

[0173] Xu is Arg (R) or Lys (K);

[0174] X12 is Lys (K), Arg (R), Glu (E), Thr (T) or Gin (Q);

[0175] X13 is Arg (R), Lys (K) or His (H); and

[0176] X14 is Glu (E), Arg (R) or Thr (T).

[0177] In some embodiments, such functional variant polymerases typically will have substantial sequence identity or similarity to the wild-type or naturally occurring polymerase (e.g., SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 32, 33, 34, 35, 36, 37, or 39), typically at least 80% amino acid sequence identity and more typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity.

[0178] In some embodiments, the DNA polymerase of the invention comprises an amino acid substitution at position Xs (e.g., as in a motif selected from SEQ ID NO:8, 9, 10 or 11) and comprises an amino acid substitution corresponding to SEQ ID NO: 38 and SEQ ID NO:29.

[0179] In some embodiments, the amino acid at position Xs is substituted with an amino acid as set forth in SEQ ID NO:8, 9, 10 or 11, and the amino acid at position X8(of SEQ ID NO:38) is substituted with an amino acid as set forth in SEQ ID NO:38. Thus, in some embodiments, the amino acid at position Xs is any amino acid other than He (I) and the amino acid at position X8is any amino acid other than Asp (D) or Glu (E). In some embodiments, 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 X8of SEQ ID NO:38. In certain embodiments, amino acid substitutions independently include Methionine (M) at position Xs of SEQ ID NO: 8, 9, 10 or 11, and Glycine (G) at position X8of SEQ IDNO:38.

[0180] In some embodiments, the amino acid at position Xs is substituted with an amino acid as set forth inSEQ ID NO: 8, 9, 10 or 11, and the amino acid at position X3 is substituted with an amino acid as set forth in SEQ ID NO:29. Thus, in some embodiments, the amino acid at position Xs is any amino acid other than He (I) and the amino acid at position X3 is any amino acid other than He (I), Leu (L) or Met (M). In some embodiments, 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, amino acid substitutions independently include Methionine (M) at position Xs of SEQ ID NO: 8, 9, 10 or 11, and Lysine (K) at position X3 of SEQ ID NO:29.

[0181] Other suitable amino acid substitution(s) at one or more of the identified sites can be determined using, e.g., known methods of site-directed mutagenesis and determination of polynucleotide extension performance in assays described further herein or otherwise known to persons of skill in the art, e.g., amino acid substitutions described in U.S. Patent Application Publication Nos. 2009 / 0148891 and 2009 / 0280539.

[0182] Because the precise length of DNA polymerases varies, the precise amino acid positions corresponding to each of Xs (SEQ ID NO:8), Xs (SEQ ID NO:38) and X3 (SEQ ID NO:29) can vary depending on the particular mutant polymerase used. Amino acid and nucleic acid sequence alignment programs are readily available (see, e.g., those referred to supra) and, given the particular motifs identified herein, serve to assist in the identification of the exact amino acids (and corresponding codons) for modification in accordance with the present invention. The positions corresponding to each of Xs, Xs and X3 are shown in Table 1 for representative chimeric thermostable DNA polymerases and thermostable DNA polymerases from exemplary thermophilic species.

[0183] Table 1: Amino Acid Positions Corresponding to Motif Positions Xs (e.g., of SEQ ID NOs: 8, 9, 10, and 11), Xs (of SEQ ID NO:38) and X3 (of SEQ ID NO:29) in Exemplary Polymerases.

[0184]

[0185] In some embodiments, the DNA polymerase of the present invention is derived from Thermits sp. Z05 DNA polymerase (SEQ ID NO: 1) or a variant thereof (e.g. , carrying the D580G mutation or the like). As referred to above, in Thermus sp. Z05 DNA polymerase, position Xs corresponds to Isoleucine (I) at position 616; position Xs corresponds to Aspartate (D) at position 580, and position X3 corresponds to Isoleucine (I) at position 709. Thus, in certain variations of the invention, the mutant polymerase comprises at least one amino acid substitution, relative to a Thermus sp. Z05 DNA polymerase (or a DNA polymerase that is substantially identical, e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1), at 1616, D580 and / or 1709. Thus, typically, the amino acid at position 616 of SEQ ID NO:1 is not I. In some embodiments, the amino acid at position 616 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 certain embodiments, the amino acid residue at position 616 of SEQ ID NO:1 is M. In certain embodiments, amino acid residues 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). Thus, 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. Exemplary Thermus sp. Z05 DNA polymerase mutants include those comprising the amino acid substitution(s) I616M, and / or I709K (or I709R, I709S, I709G, I709A), and / or D580G. In some embodiments, the mutant Thermus sp. Z05 DNA polymerase comprises, e.g., amino acid residue substitutions I616M and D580G. In some embodiments, the mutant Thermus sp. Z05 DNA polymerase comprises, e.g., amino acid residue substitutions I616M and I709K. In some embodiments, the mutant Thermus sp. Z05 DNA polymerase comprises, e.g., amino acid residue substitutions I616M, I709K, and D580G. In certain embodiments, the mutant Thermus sp. Z05 DNA polymerase comprises, e.g., amino acid residue substitutions independently selected from I616M, I709K, and / or D580G.

[0186] In some embodiments, the amino acid corresponding to position 604 of SEQ ID NO:1 is Ala (A). In some embodiments, the amino acid corresponding to position 604 of SEQ ID NO:1 is not Glu (E). In some embodiments, the amino acid corresponding to position 604 of SEQ ID NO: 1 is not Vai (V). In some embodiments, the amino acid corresponding to position 610 of SEQ ID NO: 1 is Ala (A). In some embodiments, the amino acid corresponding to position 610 of SEQ ID NO:1 is not Asp (D) or Vai (V). In some embodiments, the amino acid corresponding to position 616 of SEQ ID NO: 1 is not Lys (K), Asn (N), Gin (Q) or Thr (T). In some embodiments, the amino acid corresponding to position 617 of SEQ ID NO:1 is Glu (E). In some embodiments, the amino acid corresponding to position 617 of SEQ ID NO: 1 is not Gly (G) or Asp (D).The inventors have shown that substitutions at the amino acid corresponding to position 709 of SEQ ID NO:1 described above can result in DNA polymerases having improved (i.e., increased) reverse transcription efficiency, increased RT-PCR activity (e.g., more efficient amplification of an RNA template without compromising PCR efficiency on a DNA template), increased RT-PCR efficiency in the presence of Mg2+, increased reverse transcriptase activity in the presence of inhibitors (e.g., breakdown products of hemoglobin such as hemin, and / or heparin), increased extension rate and improved 3 ’-mismatch tolerance compared to a control polymerase (see U.S. Patent Application No.

[0187] 61 / 474,160). Thus, it is expected that the improved polymerases that comprise substitutions at the amino acid corresponding to position 709 of SEQ ID NO:1 described herein will also have the improved properties described above.

[0188] In addition to the mutations and substitutions described herein, the DNA polymerases of the present invention can also include other, non-substitutional modification(s). Such modifications can include, for example, covalent modifications known in the art to confer an additional advantage in applications comprising polynucleotide extension. For example, one such modification is a thermally reversible covalent modification that inactivates the enzyme, but which is reversed to activate the enzyme upon incubation at an elevated temperature, such as a temperature typically used for polynucleotide extension. Exemplary reagents for such thermally reversible modifications are described in U.S. Patent Nos. 5,773, 258 and 5,677,152.

[0189] The DNA polymerases of the present invention can be constructed by mutating the DNA sequences that encode the corresponding unmodified polymerase (e.g. , a wild-type polymerase or a corresponding variant from which the polymerase of the invention is derived), such as by using techniques commonly referred to as site-directed mutagenesis. Nucleic acid molecules encoding the unmodified form of the polymerase can be mutated by a variety of polymerase chain reaction (PCR) techniques well-known to one of ordinary skill in the art (see, e.g., PCR Strategies (M. A. Innis, D. H. Gelfand, and J. J. Sninsky eds., 1995, Academic Press, San Diego, CA) at Chapter 14; PCR Protocols : A Guide to Methods and Applications (M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White eds., Academic Press, NY, 1990)).

[0190] By way of non-limiting example, the two primer system, utilized in the Transformer Site-Directed Mutagenesis kit from Clontech, may be employed for introducing site-directed mutants into a polynucleotide encoding an unmodified form of the polymerase. Following 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, the other contains a mutation at another point in the plasmid resulting in elimination of a restriction site. Second strand synthesis is then carried out, tightly linking these two mutations, and the resulting plasmids are transformed into a mutS strain of E. coli. Plasmid DNA is isolated from the transformed bacteria, restricted with the relevant restriction enzyme (therebylinearizing the unmutated plasmids), and then retransformed into E. coli. This system allows for generation of mutations directly in an expression plasmid, without the necessity of subcloning or generation of single-stranded phagemids. The tight linkage of the two mutations and the subsequent linearization of unmutated plasmids result in high mutation efficiency and allow minimal screening. Following synthesis of the initial restriction site primer, this method requires the use of only one new primer type per mutation site. Rather than prepare each positional mutant separately, a set of "designed degenerate" oligonucleotide primers can be synthesized in order to introduce all of the desired mutations at a given site simultaneously. Transformants can be screened by sequencing the plasmid DNA through the mutagenized region to identify and sort mutant clones. Each mutant DNA can then be restricted and analyzed by electrophoresis, such as for example, on a Mutation Detection Enhancement gel (Mallinckrodt Baker, Inc., Phillipsburg, NJ) to confirm that no other alterations in the sequence have occurred (by band shift comparison to the unmutagenized control). Alternatively, the entire DNA region can be sequenced to confirm that no additional mutational events have occurred outside of the targeted region.

[0191] DNA polymerases with more than one amino acid substituted can be generated in various ways. In the case of amino acids located close together in the polypeptide chain, they may be mutated simultaneously using one oligonucleotide that codes for all of the desired amino acid substitutions. If however, the amino acids are located some distance from each other (separated by more than ten amino acids, for example) it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed. In the first method, a separate oligonucleotide is generated for each amino acid to be substituted. The oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions. An alternative method involves two or more rounds of mutagenesis to produce the desired mutant. The first round is as described for the single mutants: DNA encoding the unmodified polymerase is used for the template, an oligonucleotide encoding the first desired amino acid substitution(s) is annealed to this template, and the heteroduplex DNA molecule is then generated. The second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template. Thus, this template already contains one or more mutations. The oligonucleotide encoding the additional desired amino acid substitution(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis. This resultant DNA can be used as a template in a third round of mutagenesis, and so on. Alternatively, the multi-site mutagenesis method of Seyfang & Jin (Anal. Biochem. 324:285-291. 2004) may be utilized.

[0192] Accordingly, also provided are recombinant nucleic acids encoding any of the DNA polymerases of the present invention. Using a nucleic acid of the present invention, encoding a DNA polymerase, avariety of vectors can be made. Any vector containing replicon and control sequences that are derived from a species compatible with the host cell can be used in the practice of the invention. Generally, expression vectors include transcriptional and translational regulatory nucleic acid regions operably linked to the nucleic acid encoding the DNA polymerase. The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. In addition, the vector may contain a Positive Retroregulatory Element (PRE) to enhance the half-life of the transcribed mRNA (see Gelfand et al. U.S. Patent No. 4,666,848). The transcriptional and translational regulatory nucleic acid regions will generally be appropriate to the host cell used to express the polymerase. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells. In general, the transcriptional and translational regulatory sequences may include, e.g., promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. In typical embodiments, the regulatory sequences include a promoter and transcriptional start and stop sequences. Vectors also typically include a polylinker region containing several restriction sites for insertion of foreign DNA. In certain embodiments, “fusion flags” are used to facilitate purification and, if desired, subsequent removal of tag / flag sequence, e.g., “His-Tag”. However, these are generally unnecessary when purifying a thermoactive and / or thermostable protein from a mesophilic host (e.g., E. coli) where a “heat-step” may be employed. The construction of suitable vectors containing DNA encoding replication sequences, regulatory sequences, phenotypic selection genes, and the polymerase of interest are prepared using standard recombinant DNA procedures. Isolated plasmids, viral vectors, and DNA fragments are cleaved, tailored, and ligated together in a specific order to generate the desired vectors, as is well-known in the art (see, e.g. , Sambrook et al. , Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, NY, 2nd ed. 1989)).

[0193] In certain embodiments, the expression vector contains a selectable marker gene to allow the 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 coding for ampicillin and / or tetracycline resistance, which enables cells transformed with these vectors to grow in the presence of these antibiotics.

[0194] In one aspect of the present invention, a nucleic acid encoding a DNA polymerase is introduced into a cell, either alone or in combination with a vector. By "introduced into" or grammatical equivalents herein is meant that the nucleic acids enter the cells in a manner suitable for subsequent integration, amplification, and / or expression of the nucleic acid. The method of introduction is largely dictated by the targeted cell type. Exemplary methods include CaPOi precipitation, liposome fusion,LIPOFECTIN®, electroporation, viral infection, and the like.

[0195] In some embodiments, prokaryotes are typically used as host cells for the initial cloning steps of the present invention. They are particularly useful for rapid production of large amounts of DNA, for production of single-stranded DNA templates used for site-directed mutagenesis, for screening many mutants simultaneously, and for DNA sequencing of the mutants generated. 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), andE. 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 bacilli such as Bacillus subtilis, other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species 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 etal., supra. Alternatively, electroporation can be used for transformation of these cells. Prokaryote transformation techniques are set forth in, for example Dower, in Genetic Engineering, Principles and Methods 12:275-296 (Plenum Publishing Corp., 1990); Hanahan et al., Meth. Enzymol., 204:63, 1991. Plasmids typically used for transformation of E. coli include pBR322, pUCI8, pUCI9, pUCI18, pUCl 19, 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 available as well.

[0196] The DNA polymerases of the present invention are typically produced by culturing a host cell transformed with an expression vector containing a nucleic acid encoding the DNA polymerase, under the appropriate conditions to induce or cause expression of the DNA polymerase. Methods of 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 production of the polymerases from lambda pL promotor-containing plasmid vectors include E. coli strain DG116 (ATCC No. 53606) (see US Patent No. 5,079,352 and Lawyer, F.C. et al., PCR Methods and Applications 2:275-87, 1993). Following expression, the polymerase can be harvested and isolated. Methods for purifying the thermostable DNA polymerase are described in, for example, Lawyer et al., supra. Once purified, the ability of the DNA polymerases to have improved RT efficiency, increased mis-match tolerance, extension rate and / or tolerance of RT and polymerase inhibitors can be tested (e.g., as described in the examples). The improved DNA polymerases of the present invention may be used for any purpose in which such enzyme activity is necessary or desired. Accordingly, in another aspect of the invention, methods of polynucleotide extension (e.g., PCR) using the polymerases are 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). Generally, 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 the DNA polymerase and nucleoside triphosphates in a suitable environment to permit the addition of one or more nucleotides to the 3' end of the primer, thereby producing an extended primer complementary to the target nucleic acid. The primer can include, e.g., one or more nucleotide analog(s). In addition, the nucleoside triphosphates can be conventional nucleotides, unconventional nucleotides (e.g., ribonucleotides or labeled nucleotides), or a mixture thereof. In some variations, the polynucleotide extension reaction comprises amplification of a target nucleic acid. Conditions suitable 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. eds., Academic 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, the improved polymerases find use in 454 sequencing (Margulies, M et al. 2005, Nature, 437, 376-380).

[0197] Optionally, the primer extension reaction comprises an actual or potential inhibitor of a reference or unmodified polymerase. The inhibitor can inhibit, for example, the nucleic acid extension rate and / or the reverse transcription efficiency 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 breakdown product, such as hemin, hematoporphyrin, or bilirubin. In some embodiments, the inhibitor is an iron-chelator or a purple pigment. In other 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, e.g, Ekhardt, et al.,Biochem Biophys Res Commun. 271(3):726-30 (2000)). The DNA polymerases of the present invention can be used to extend templates in the presence of polynucleotide templates isolated from samples comprising polymerase inhibitors, e.g., such as blood. For example, the DNA polymerases of the present invention can be used to extend templates in the presence of hemoglobin, a major component of blood, or in the presence of a hemoglobin degradation product. Hemoglobin can be degraded to various heme breakdown products, such as hemin, hematin, hematoporphyrin, and bilirubin. Thus, in certain embodiments, the DNA polymerases of the present invention can be used to extend templates in the presence of hemoglobin degradation products, including but not limited to, hemin, hematin, hematoporphyrin, and bilirubin. In certain embodiments, the hemoglobin degradation product is hemin. In some embodiments, the DNA polymerases of the present invention can be used to extend templates in the presence of about 0.5 to 20.0pM, about 0.5 to lO.OpM, about 0.5 to 5.0pM, about 1.0 to lO.OpM, about 1.0 to 5.0pM, about 2.0 to 5.0pM, or about 2.0 to 3.0pM hemin. In other embodiments, the DNA polymerases of the present invention can be used to extend templates 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, orgreater than 20pM hemin. The breakdown products of hemoglobin include iron-chelators and purple pigments. Thus, in some embodiments, the DNA polymerases of the present invention can be used to extend templates in the presence of iron-chelators and / or purple pigments. In other embodiments, the DNA polymerases of the present invention can be used to extend templates in the presence of amounts of hemoglobin degradation products that would inhibit extension of the same template by a reference or control DNA polymerase.

[0198] The DNA polymerases of the present invention can be used to extend templates in the presence of heparin. Heparin is commonly present as an anticoagulant in samples isolated from blood. In some embodiments, the DNA polymerases of the present invention can be used to extend templates in the presence of about 1.0 to 400 ng / pl, 1.0 to 300 ng / pl, 1.0 to 200 ng / pl, 5.0 to 400 ng / pl, 5.0 to 300 ng / pl, 5.0 to 200 ng / pl, 10.0 to 400 ng / pl, 10.0 to 300 ng / pl, or 10.0 to 200 ng / pl heparin. In some embodiments, the DNA polymerases of the present invention can be used to extend templates 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 / pl, or greater than 400 ng / pl of heparin. In other embodiments, the DNA polymerases of the present invention can be used to extend templates in the presence of amounts of heparin that would inhibit extension of the same template by a reference or control DNA polymerase. In some embodiments, an improved polymerase of the invention is used in a reverse transcription reaction. In some embodiments, the reverse transcription reaction is carried out in a mixture containing the RNA template, one or more primer(s), and a thermostable DNA polymerase of the invention. The reaction mixture typically contains all four standard deoxyribonucleoside triphosphates (dNTPs) and a buffer containing a divalent cation and a monovalent cation. Exemplary cations include, e.g., Mg2+, although other cations, such as Mn2+or Co2+can activate DNA polymerases. In other embodiments, the reverse transcription reaction is carried out with a thermo-active DNA polymerase of the invention. In particular embodiments, the improved polymerase of the invention allows for more efficient amplification of RNA templates without compromising the efficient amplification of a DNA template in the presence of Mn2+or Mg2+, as described in the examples.

[0199] In some embodiments, the improved polymerase has increased reverse transcription efficiency compared to a control polymerase. It was not previously appreciated that substitutions at the amino acid corresponding to position 616 of SEQ ID NO:1 could result in increased RT efficiency. Thus, in some embodiments, DNA polymerases having an Ile(I) to Met (M) substitution at the amino acid corresponding to position 616 of SEQ ID NO: 1 have increased RT efficiency. In some embodiments, the DNA polymerase having increased reverse transcription efficiency comprises an Ile(I) to Met (M) substitution at the amino acid corresponding to position 616 of SEQ ID NO:1, and has at least 80%, preferably at least 90%, more preferably at least 95%, more preferably at least 98% amino acid sequence identity to SEQ ID NOs: 1-7, 32-37, or 39.In some embodiments, the improved polymerase has increased reverse transcription efficiency using an RNA template without a substantial decrease in polymerase activity using a DNA template. Thus, in some embodiments, the improved DNA polymerase has increased RT efficiency without a substantial decrease in DNA-dependent polymerase activity when compared to a control polymerase. In some embodiments, the improved DNA polymerase described herein has DNA-dependent polymerase activity that is substantially the same as a control polymerase. Thus, in some embodiments, the improved DNA polymerase described herein has DNA-dependent polymerase activity that is at least about 90% of the activity of a control polymerase, for example, at least about 90%, 91%, 92%, 93%, 94%, 95%, or more (e.g., 98%) of the activity of a control polymerase. The DNA-dependent polymerase activity can be measured, for example, by amplifying a DNA template and determining Cp values as described herein. Thus, in some embodiments, the DNA polymerase has improved RT efficiency measured as a decreased Cp value compared to a control polymerase when RNA is used as a template, but has 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, the DNA-dependent polymerase activity is determined as described in the Examples.

[0200] In some embodiments, an improved polymerase of the invention increases reverse transcription efficiency by reducing the reaction time required for extending an RNA template. For example, an improved polymerase described herein can significantly shorten the reaction time required to transcribe RNA to cDNA as compared to a control polymerase, thereby increasing the reverse transcriptase efficiency. Without being limited by theory, the improved polymerase can increase RT efficiency by, for example, increasing the activity of the enzyme on an RNA template, such as increasing the rate of nucleotide incorporation and / or increasing the processivity of the polymerase, thereby effectively shortening the extension time of an RNA template or population of RNA templates. Reaction times for the initial RT step are typically on the order of 30 minutes or longer at 65 degrees C when using an unmodified or control polymerase. Thus, in some embodiments, the improved polymerase can transcribe an RNA template into cDNA in 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 C. In some embodiments, the improved polymerase can transcribe an RNA template derived from Hepatitis C Virus (HCV) transcript JP2-5, containing the first 800 bases of HCV genotype lb 5’NTR, into cDNA in less time or faster than a control polymerase. For example, the improved polymerase can transcribe 240 bases of the HCV JP2-5 RNA template into full-length cDNA in about 15 seconds less, 30 seconds less, one minute less, two minutes less, 3 minutes less, 4 minutes less, 5 minutes less, or about 10 minutes lessthan a control polymerase under identical reaction conditions. In some embodiments, the improved polymerase can transcribe 240 bases of the HCV JP2-5 RNA template into full-length cDNA faster than a control polymerase, for example, about 5 seconds, 10 seconds, 15 seconds, 30 seconds, 45 seconds, or 60 seconds or more faster than a control polymerase under identical reaction conditions. In some embodiments, the reaction conditions are those described in the Examples. In some embodiments, an improved polymerase described herein is contacted with an RNA template at 65 degrees C for about 2 minutes in the reaction mixture described above. The extension step can be followed by PCR amplification of the extended template, as described in the examples.

[0201] The most efficient RT activity in thermostable DNA polymerases has been achieved using Mn2+as the divalent metal ion activator. However, it is well known that when Mn2+is present in reactions the fidelity of DNA polymerases is lower. Unless one is trying to generate mutations, it is generally favored to maintain a higher fidelity. Fortunately, most conventional sequencing, PCR and RT-PCR applications do not require high fidelity conditions because the detection systems generally are looking at a population of products. With the advent of next generation sequencing, digital PCR, etc., the fidelity of the product is more important and methods that allow for higher fidelity DNA synthesis are critical. Achieving efficient RT activity using Mg2+as the divalent metal ion activator is an excellent way to substantially increase the fidelity of the DNA polymerase and allow for more reliable copying of the nucleic acid target. Accordingly, in some embodiments, the improved polymerase of the invention allows for efficient extension and / or amplification of RNA templates using Mg2+as the divalent metal ion activator, as described in the examples.

[0202] Because the polymerases described herein can also have increased mismatch tolerance, the polymerases find use in methods where variation of the target template is likely and yet the template is nevertheless desired to be amplified regardless of the variation at the target template. An example of such templates can include, for example, viral, bacterial, or other pathogen sequences. In many embodiments, it is desirable to determine simply whether an individual (human or non-human animal) has a viral or other infection, regardless of the precise viral variant that has infected the individual. As an example, one can use a primer pair to amplify HCV using a polymerase of the invention and detect the presence of the HCV even if the particular virus infecting the individual has a mutation resulting in a mismatch at the primer hybridization site.

[0203] Target nucleic acids can come from a biological or synthetic source. The target can be, for example, DNA or RNA. Generally, where amplicons are generated, the amplicons will be composed of DNA, though ribonucleotides or synthetic nucleotides can also be incorporated into the amplicon. Where one wishes to detect an RNA, the amplification process will typically involve the use of reverse transcription, including for example, reverse transcription PCR (RT-PCR).

[0204] Specific target sequences can include, e.g., viral nucleic acids (e.g., human immunodeficiency virus(HIV), hepatitis virus B (HBV), (cytomegalovirus (CMV), parvo B19 virus, Epstein-Barr virus, hepatitis virus C (HCV), human papilloma virus (HPV), Japanese encephalitis virus (JEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV), Murray Valley encephalitis virus, and Kunjin virus), bacterial nucleic acids (e.g., S. aureus, Neisseria meningitidis, Plasmodium falciparum, Chlamydia muridarum, Chlamydia trachomatis), mycobacteria, fungal nucleic acids, or nucleic acids from animals or plants. In some embodiments, the target nucleic acids are animal (e.g., human) nucleic acids or are derived from an animal (e.g., human) sample (i.e., viral or other pathogenic organism nucleic acids may be present in a sample from an animal biopsy, blood sample, urine sample, fecal sample, saliva, etc.). In some embodiments, the target nucleic acids are, for example, human genetic regions that may include variants associated with 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 a diversity of related sequences could be in a target sequence. As an example, the invention can 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 in cancer or other disease genetic markers where variation in sequence is known or likely to occur.

[0205] Other methods for detecting extension products or amplification products using the improved polymerases described herein include the use of fluorescent double-stranded nucleotide binding dyes or fluorescent double-stranded nucleotide intercalating dyes. Examples of fluorescent double-stranded DNA binding dyes include SYBR-green (Molecular Probes). The double stranded DNA binding dyes can be used in conjunction with melting curve analysis to measure primer extension products and / or amplification products. The melting curve analysis can be performed on a real-time PCR instrument, such as the ABI 5700 / 7000 (96 well format) or ABI 7900 (384 well format) instrument with onboard software (SDS 2.1). Alternatively, the melting curve analysis can be performed as an end point analysis. Exemplary methods of melting point analysis are described in U.S. Patent Publication No.

[0206] 2006 / 0172324.

[0207] In another aspect of the present invention, kits are provided for use in primer extension methods described herein. In some embodiments, the kit is compartmentalized for ease of use and contains at least one container providing an improved DNA polymerase in accordance with the present invention. One or more additional containers providing additional reagent(s) can also be included. In some embodiments, the kit can also include a blood collection tube, container, or unit that comprises heparin or a salt thereof, or releases heparin into solution. The blood collection unit can be a heparinized tube. Such additional containers can include any reagents or other elements recognized by the skilled artisan for use in primer extension procedures in accordance with the methods described above, including reagents for use in, e.g., nucleic acid amplification procedures (e.g., PCR, RT-PCR), DNA sequencingprocedures, or DNA labeling procedures. For example, in certain embodiments, the kit further includes a container providing a 5' sense primer hybridizable, under primer extension conditions, to a predetermined polynucleotide template, or a primer pair comprising the 5' sense primer and a corresponding 3' antisense primer. In other, non-mutually exclusive variations, the kit includes one or more containers providing nucleoside triphosphates (conventional and / or unconventional). In specific embodiments, the kit includes alpha-phosphorothioate dNTPs, dUTP, diTP, and / or labeled dNTPs such as, e.g., fluorescein- or cyanin-dye family dNTPs. In still other, non-mutually exclusive embodiments, the kit includes one or more containers providing a buffer suitable for a primer extension reaction.

[0208] In another aspect of the present invention, reaction mixtures are provided comprising the polymerases with increased reverse transcriptase efficiency, mismatch tolerance, extension rate and / or tolerance of RT and polymerase inhibitors as described herein. The reaction mixtures can further comprise reagents for use in, e.g., nucleic acid amplification procedures (e.g., PCR, RT-PCR), DNA sequencing procedures, or DNA labeling procedures. For example, in certain embodiments, the reaction mixtures comprise a buffer suitable for a primer extension reaction. The reaction mixtures can also contain a template nucleic acid (DNA and / or RNA), one or more primer or probe polynucleotides, nucleoside triphosphates (including, e.g., deoxyribonucleotides, ribonucleotides, labeled nucleotides, unconventional nucleotides), salts (e.g., Mn2+, Mg2+), labels (e.g., fluorophores). In some embodiments, the reaction mixtures contain a 5 '-sense primer hybridizable, under primer extension conditions, to a predetermined polynucleotide template, or a primer pair comprising the 5'-sense primer and a corresponding 3' antisense primer. In some embodiments, the reaction mixtures contain alpha-phosphorothioate dNTPs, dUTP, diTP, and / or labeled dNTPs such as, e.g., fluorescein- or cyanin-dye family dNTPs. In some embodiments, the reaction mixtures comprise an iron chelator or a purple dye. In certain embodiments, the reaction mixtures comprise hemoglobin, or a degradation product of hemoglobin. For example, in certain embodiments, the degradation products of hemoglobin include heme breakdown products such as hemin, hematin, hematophoryn, and bilirubin. In other embodiments, the reaction mixtures comprise heparin or a salt thereof. In certain embodiments, the reaction mixture contains a template nucleic acid that is isolated from blood. In other embodiments, the template nucleic acid is RNA and the reaction mixture comprises heparin or a salt thereof.

[0209] In some embodiments, the reaction mixture comprises two or more polymerases. For example, in some embodiments, the reaction mixture comprises a first DNA polymerase having increased reverse transcriptase efficiency compared to a control polymerase, and a second DNA polymerase having DNA-dependent polymerase activity. The second DNA polymerase can be a wild-type or unmodified polymerase, or can be an improved polymerase having increased DNA- dependent polymerase activity. Such reaction mixtures are useful for amplification of RNA templates (e.g., RT-PCR) by providingboth a polymerase having increased reverse transcriptase activity and a polymerase having DNA-dependent polymerase activity.

[0210] EXAMPLES

[0211] The following examples are offered to illustrate, but not to limit the claimed invention.

[0212] Example 1: Library Generation

[0213] In brief, the steps in this screening process included library generation, expression and partial purification of the mutant enzymes, screening of the enzymes for the desired properties, DNA sequencing, clonal purification, and further characterization of selected candidate mutants. Each of these steps is described further below.

[0214] Clonal Library generation: A nucleic acid encoding the polymerase domain of Z05 D580G I709K DNA polymerase was subjected to error-prone (mutagenic) PCR between Blp I and Bgl II restriction sites of a plasmid including this nucleic acid sequence. The primers used for this are given below: Forward Primer: 5’- CTACCTCCTGGACCCCTCCAA-3 ’ (SEQ ID NO:30); and,

[0215] Reverse Primer: 5’- ATAACCAACTGGTAGTGGCGTGTAA-3 ’ (SEQ ID NO:31)

[0216] PCR was performed using a Mg2+concentration of 1.8 mM, in order to generate a library with a desired mutation rate. Buffer conditions were 50mM Bicine pH 8.2, 115mM KOAc, 8% w / v glycerol, and 0.2mM each dNTPs. A GeneAmp® AccuRT Hot Start PCR enzyme was used at 0.15 U / pL. Starting with 5xl05copies of linearized Z05 D580G I709K plasmid DNA per reaction volume of 50pL, reactions were denatured using a temperature of 94°C for 60 seconds, then 30 cycles of amplification were performed, using a denaturation temperature of 94°C for 15 seconds, an annealing temperature of 60°C for 15 seconds, an extension temperature of 72°C for 120 seconds, and followed by a final extension at a temperature of 72°C for 5 minutes.

[0217] The resulting amplicon was purified with a QIAquick PCR Purification Kit (Qiagen, Inc., Valencia, CA, USA) and cut with Blp I and Bgl II, and then re-purified with a QIAquick PCR Purification Kit. A Z05 D580G_I709K vector plasmid was prepared by cutting with the same two restriction enzymes and treating with alkaline phosphatase, recombinant (RAS, cat# 03359123001) and purified with a QIAquick PCR Purification Kit. The cut vector and the mutated insert were mixed at a 1:3 ratio and treated with T4 DNA ligase for 5 minutes at room temperature (NEB Quick Ligation™ Kit). The ligations were purified with a QIAquick PCR Purification Kit and transformed into an E. coli host strain by electroporation.

[0218] Aliquots of the expressed cultures were plated on ampicillin-selective medium in order to determine the number of unique transformants in each transformation. Transformations were pooled and stored at -70°C to -80°C in the presence of glycerol as a cryo-protectant.

[0219] The library was then spread on large format ampicillin-selective agar plates. Individual colonies weretransferred to 384- well plates containing 2X Luria broth with ampicillin and 10% w / v glycerol using an automated colony picker (QPix2, Genetix Ltd). These plates were incubated overnight at 30°C to allow the cultures to grow and then stored at -70°C to -80°C. The glycerol added to the 2X Luria broth was low enough to permit culture growth and yet high enough to provide cryo-protection. Several thousand colonies were prepared in this way for later use.

[0220] Extract library preparation Part 1 - Fermentation: From the clonal libraries described above, a corresponding library of partially purified extracts suitable for screening purposes was prepared. The first step of this process was to make small-scale expression cultures of each clone. These cultures were grown in 96-well format; therefore, there were 4 expression culture plates for each 384-well library plate. 0.5 pL was transferred from each well of the clonal library plate to a well of a 96 well seed plate, containing 150pL of Medium A (see Table 2 below). This seed plate was shaken overnight at 1150 rpm at 30°C, in an iEMS plate incubator / shaker (ThermoElectron). These seed cultures were then used to inoculate the same medium, this time inoculating 20pL into 250pL Medium A in large format 96 well plates (Nunc # 267334). These plates were incubated overnight at 37°C with shaking. The expression plasmid contained transcriptional control elements, which allow for expression at 37°C but not at 30°C. After overnight incubation, the cultures expressed the clone protein at typically 1-10% of total cell protein. The cells from these cultures were harvested by centrifugation. These cells were either frozen (-20°C) or processed immediately, as described below.

[0221] Table 2: Medium A (Filter-sterilized prior to use)

[0222]

[0223] Extract library preparation Part 2 - Extraction: Cell pellets from the fermentation step were resuspended in 25pL Lysis buffer (Table 3 below) and transferred to 384-well thermocycler plates and sealed. Note that the buffer contained lysozyme to assist in cell lysis, and DNase to remove DNA from the extract. To lyse the cells the plates were incubated at 37°C for 15 minutes, frozen overnight at -20°C, and incubated again at 37°C for 15 minutes. Ammonium sulfate was added (1.5pL of a 2M solution) and the plates incubated at 75°C for 15 minutes in order to precipitate and inactivate contaminating proteins, including the exogenously added nucleases. The plates were centrifuged at 3000 x g for 15 minutes at 4°C and the supernatants transferred to a fresh 384-well thermocycler plate.These extract plates were frozen at -20°C for later use in screens. Each well contained about 0.5-3pM of the mutant library polymerase enzyme.

[0224] Table 3: Lysis Buffer

[0225]

[0226] Example 2: Identification of mutant DNA polymerases with improved reverse transcription efficiency

[0227] Screening extract libraries for improved reverse transcription efficiency: The extract library was screened by comparing Cp (Crossing Point) values from growth curves generated by fluorescent 5’ nuclease (TaqMan) activity of crude enzyme extracts in a RT-PCR system from amplification of a 240 base pair amplicon from Hepatitis C Virus (HCV) transcript JP2-5, containing the first 800 bases of HCV genotype lb 5’NTR in pSP64 poly(A) (Promega).

[0228] Reactions were carried out on the Roche LC 480 kinetic thermocycler in 384 well format with each well containing 3 pL of an individual enzyme extract diluted 10-fold with buffer containing 20 mM Tris-HCl, pH 8, 100 mM KC1, 0.1 mM EDTA, and 0.1% Tween-20 added to 12 pL of RT-PCR master mix described in Table 4. The thermocycling conditions were: 2 minute at 50°C (“UNG” step); 2 minute at 65°C (“RT” step); 5 cycles of 94°C for 15 seconds followed by 62°C for 30 seconds; and 45 cycles of 91°C for 15 seconds followed by 62°C for 30 seconds.

[0229] Table 4: PCR master mix

[0230]

[0231] 5,000 clones were screened using the above protocol. Forty clones were chosen from the original pool for rescreening based on earliest Crossing Point (Cp) values and fluorescent plateau values above an arbitrary cut off as calculated by the Abs Quant / 2ndderivative max method. Culture wells corresponding to the top extracts were sampled to fresh growth medium and re-grown to produce new culture plates containing the best mutants, as well as a number of parental Z05 D580G_ I709K cultures to be used for comparison controls. These culture plates were then used to make fresh crude extracts which were rescreened with the same RNA target and conditions as previously described for the original screen. Table 5 shows average Cp values obtained from the fluorescent signal increase due to 5’ hydrolysis of a FAM labeled probe. Results show that clone 0686-C21 amplifies the RNA target with higher efficiency than the Z05 D580G I709K parental.

[0232] Table 5: average Cp values

[0233]

[0234] The DNA sequence of the mutated region of the polymerase gene was sequenced to determine the mutation(s) that were present in any single clone. Clone 0686-C21 was chosen for further testing, so mutant polymerase protein was expressed in flask culture, purified to homogeneity, and quantified.

[0235] Use of Z05_D580G_I709K mutant in Mg2+-based RT-PCR: Sequencing results revealed that the polymerase expressed by clone 0686-C21 carried the I616M mutation in addition to the parental D580G and I709K mutations. Purified mutant Z05 D580G I709K I616M was compared to parental Z05_ D580G I709K in TaqMan Mg2+-based RT-PCR. Reverse transcription and PCR efficiencies were measured by comparing Cp values from amplifications of JP2-5 RNA transcript and pJP2-5 DNA linear plasmid digested with the restriction endonuclease EcoRI. Oligonucleotides and Master Mix conditions (Table 4) were the same as used in the original screen. Each reaction had either 100,000 copies of JP2-5 transcript RNA, 100,000 copies of pJP2-5 linear plasmid DNA, or 1000 copies of pJP2-5 linear plasmid DNA. All targets were amplified with Primer 1 and Primer 2, as described above, in duplicate reactions to generate a 240 base pair amplicon. All reactions were performed on the Roche Light Cycler 480 thermal cycler with a reaction volume of 15pL. Crossing Point (Cps) were calculated by the Abs Quant / 2ndderivative max method and averaged. Amplifications were carried out using a range of DNA Polymerase concentrations from 5nM- 40nM. The thermocycling conditions were: 2 minute at 50°C (“UNG” step); 2 minute at 65°C (“RT” step); 5 cycles of 94°C for 15 seconds followed by 62°C for 30 seconds; and 45 cycles of 91°C for 15 seconds followed by 62°C for 30 seconds. Table 6 shows Cp values obtained from fluorescent signal increase due to cleavage of the TaqMan probe at 20nM enzyme condition.Table 6

[0236]

[0237] The results indicate that mutant Z05 D580G I709K I616M allows for more efficient amplification of RNA target without compromise of PCR efficiency on a DNA target, as compared to the D580G I709K parental enzyme.

[0238] Example 3: Blending Z05 D580G I709K I616M mutant DNA Polymerase with AmpliTaq Gold® DNA polymerase in Mg2+-based RT-PCR

[0239] Purified mutant Z05 D580G I709K I616M (0686-C21) was blended with AmpliTaq Gold® DNA Polymerase (“TaqGold”) in a modified Tris-HCl buffered TaqGold master mix (Table 7) and used to amplify RNA and DNA templates in TaqMan® Mg2+- based RT-PCR.

[0240] Table 7

[0241]

[0242] Reverse transcription and PCR efficiencies were measured by comparing Cp values from amplifications of JP2-5 RNA transcript and pJP2-5 DNA linear plasmid digested with the restriction endonuclease EcoRI. Each reaction had either 100,000 copies of JP2-5 transcript RNA, 100,000 copies of pJP2-5 linear plasmid DNA, or 1000 copies of pJP2-5 linear plasmid DNA. All targets were amplified with Primer 1 and Primer 2, as described above, in duplicate reactions to generate a 240 base pair amplicon. All reactions were performed on the Roche Light Cycler 480 thermal cycler with a reaction volume of 15pL. Crossing Point (Cps) were calculated by the Abs Quant / 2ndderivative max method and averaged. Amplification of each of the RNA and DNA templates were carried out with the following separate enzyme conditions: lOnM of Z05 D580G I709K I616M blended with 0.5 U / pL ofTaqGold; 0.5 U / pL of TaqGold; 20nM of Z05 D580G I709K I616M. The thermal cycling conditions were: 2 minute at 50°C (“UNG” step); 2 minute at 55°C, 4 minute at 60°C, 60 minute at 65°C (three temperature “RT” step); 10 minute at 95°C (“TaqGold” activation step); 5 cycles of 94°C for 15 seconds followed by 62°C for 30 seconds; and 45 cycles of 91°C for 15 seconds followed by 62°C for 30 seconds. Table 8 shows Cp values obtained from fluorescent signal increase due to cleavage of the TaqMan probe for the three different enzyme conditions.

[0243] Table 8

[0244]

[0245] N / S= no signal

[0246] The results indicate that blending mutant Z05 D580G I709K I616M with TaqGold allows for efficient amplification of RNA target without compromise of PCR efficiency on DNA target. The TaqGold control condition affirms the commonly known fact that Taq polymerase amplifies RNA templates with poor efficiency. The Z05 D580G I709K I616M control suggests that the mutant DNA polymerase cannot amplify RNA or DNA targets in this Tris-based buffered master mix with this modified thermal profile.

[0247] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art.

[0248] Example 4". Library Generation

[0249] Clonal Library generation: A nucleic acid encoding 380 residues of the polymerase domain of Z05 D580G_I709K DNA polymerase was subjected to mutagenesis by site specific variant library (SSVL) performed by TWIST Biosciences. The library consisted of 19 different amino acids at each position with a total theoretical diversity of 7220 variants. The library was delivered as linear DNA on plates with one codon position per well (19 variants). All wells were pooled and cloned into our expression plasmid by overlap assembly using protocols and components supplied in NEBuilder® HiFi DNA Assembly Cloning Kit (New England Biolabs catalogue #E5520S). The primers used for this are given below:

[0250] SSVL Vector fwd: 5’- cgcgaagggtTAAGGTTGACCCCAAGGG -3 ’

[0251] SSVL Vector rev: 5’- accacagcagTTTTTCCTCGCCCTTCAG -3 ’

[0252] SSVL Fragmenfyfwd: 5’- cgaggaaaaaCTGCTGTGGTTGTACCAAGAGGTC -3’

[0253] SSVL Fragment rev: 5’- gtcaaccttaACCCTTCGCGCTCAGCCA -3’

[0254] Assembly products were transformed into an E.coli host strain by electroporation.Aliquots of the expressed cultures were plated on ampicillin-selective medium in order to determine the number of unique transformants in each transformation. Transformations were pooled and stored at -70°C to -80°C in the presence of glycerol as a cryo-protectant.

[0255] The library was then spread on large ampicillin-selective agar plates. Individual colonies were transferred to 384-well plates containing 2X Luria broth with ampicillin and 10% w / v glycerol using an automated colony picker (QPix2, Genetix Ltd). These plates were incubated overnight at 30°C to allow the cultures to grow and then stored at -70°C to -80°C. The glycerol added to the 2X Luria broth was low enough to permit culture growth and yet high enough to provide cryo-protection. Several thousand colonies were prepared in this way for later use.

[0256] Extract library preparation Part 1 - Fermentation: From the clonal libraries described above, a corresponding library of partially purified extracts suitable for screening purposes was prepared. The first step of this process was to make small-scale expression cultures of each clone. These cultures were grown in 96-well format; therefore there were 4 expression culture plates for each 384-well library plate. 5pL was transferred from each well of the clonal library plate to a well of a large format 96 well plates (Nunc # 267334), containing 500pL of Magic Media (Invitrogen Cat #: K6803) supplemented with lOOpg / ml carbenicillin. This expression plate was shaken for 24 hours at 850 rpm at 28°C, in a benchtop plate incubator / shaker. The cells from these cultures were harvested by centrifugation. These cells were either frozen (-20°C) or processed immediately, as described below.

[0257] Extract library preparation Part 2 - Extraction: Cell pellets from the fermentation step were resuspended in 150pL Lysis buffer (Table 9 below) shaken for 2 hours at 650 rpm at 37°C, in a benchtop plate incubator / shaker. Lysates are frozen overnight at -20°C and incubated at 37°C for 1 hour after addition of 15pL DNase Buffer (Table 10 below). Note that the buffers contained lysozyme to assist in cell lysis, and DNase to remove DNA from the extract. Lysate harvested by centrifugation and lOOpL of the supernatant was transferred to a new 96 well plate. Ammonium sulfate was added (6pL of a 2M solution) and the plates incubated at 75°C for 15 minutes in order to precipitate and inactivate contaminating proteins, including the exogenously added nucleases. The plates were centrifuged at 3000x g for 15 minutes at 4°C and the supernatants transferred to a fresh 384-well thermocycler plate. These extract plates were frozen at -20°C for later use in screens.

[0258] Table 9: Lysis Buffer

[0259]

[0260] Table 10: DNase Buffer

[0261]

[0262] Example 5: Identifying alternative DNA polymerases, which produce less non-specific amplification products (NSA), while retaining RT and PCR efficiency

[0263] The goal is to identify DNA polymerases, which produce less non-specific amplification products (NSA), while retaining the reverse transcriptase (RT) and polymerase chain reaction (PCR) efficiency of “gold standard” polymerases, such as the Z05D polymerase. To that end, a mutagenic library was generated and screened with increasingly complex multiplexed model systems tuned for challenging specificity and sensitivity.

[0264] A number of polymerase candidates were considered to form the basis of the mutagenic library. For example, the Z05 D polymerase is a popular polymerase known for its improved reverse transcription efficiency, as compared to wild type Z05, and mismatch tolerance. However, Z05 D is also known to generate off-target amplification products. Another example is the Z05 C21 polymerase, which is the polymerase of choice for Mg-based RT-PCR systems, due to its improved reverse transcription efficiency with Mg2+as a co-factor, its property of being less thermostable, and that it can be blended with other PCR enzymes (e.g., Z05). However, one disadvantage of the Z05 C21 polymerase is that, not unlike the Z05 D polymerase, it generates increased levels of off-target amplification products. The goal was to generate enzymes with improved or altered activity for specific applications. To that end, a cycle of directed evolution was employed, mimicking the process of biological evolution. This cycle is depicted in FIG. 4, which is taken from Packer and Liu, “Method of the directed evolution of proteins,” Nature Reviews: Genetics 16:379-394 (2015). This method involves the following steps: (1) step 1 is a divarication step, which involves the generation of a diverse library of a single gene, (2) step 2 is a translation step, which involves translating the library into corresponding gene products, (3) step 3 is a screening step, which involves isolating members with the desired properties, and (4) step 4 is a replication steps, wherein the best variants undergo subsequent rounds of diversification and screening / selection. The Z05 library screening workflow is depicted in FIG. 5.

[0265] A polymerase used as a reference is the C21 polymerase, which is recited as SEQ ID NO:43. As compared to wild type Z05 polymerase the C21 polymerase exhibits better RT activity. The sequence for C21 is the same sequence as the Z05 polymerase but with the following three (3) position changes: I616M, I709K, and D580G (as shown in SEQ ID NO:43).

[0266] The parental type polymerase employed is Z05 I616M I709K, which is recited as SEQ ID NO: 42. Theparental type polymerase is a modified version of the C21 polymerase. Relative to the C21 polymerase the parental type is the same as the C21 polymerase but without the D580G substitution. Relative to the Z05 polymerase, the C21 polymerase is the same sequence as the Z05 polymerase, but with the following two (2) position changes: I616M and I709K. The parental type polymerase provides the basis or template upon which modifications or mutations will be introduced, and the activity of the parental type polymerase will be the basis upon which to assess and compare enzymatic activity of the modified mutant polymerase. TheZ051616MI709K parental type has improved specificity for reverse transcription in the presence of Mg2+.

[0267] To generate mutants / modified polymerases based on the Z05 I616M I709K parental, a site saturation variant library of the polymerase domain was obtained from TWIST Bioscience. A total of 380 positions were targeted, resulting in 19 different amino acids / positions, yielding 7,220 variants. These were delivered as linear DNA on plates with one amino acid position per well (19 variants), and all wells were pooled and cloned into expression vectors by two piece assembly.

[0268] Plate-based expression and purification were conducted. Lysates were active in MS2 (bacteriophage virus RNA genome template) Mg-based singleplex RT-PCR.

[0269] The screening strategy was to try to detect variants having improved specificity and improved reverse transcription (RT) efficiency. The variables detected to assess specificity include Mg-based RT-PCR, 5’ probe hydrolysis signal (RFI), and increased complexity with each round of screening. The variables detected to assess RT efficiency were enzyme concentration and RT incubation time.

[0270] Under primary screening, roughly 6,000 clones were expressed and screened by primary MS2 singleplex Mg-based RT-PCR with 1 minute RT incubation time. Hits or results with early Cps and high RFI were sequenced, re-arrayed, and screened by secondary assays for RT efficiency and specificity. Sequencing hits from the primary screen are depicted in FIG. 6.

[0271] The substitution at the 542 position (i.e., K542F) (SEQ ID NO:41) emerged as one of the leading candidates with good RT efficiency and specificity (i.e., RFI). K542F (SEQ ID NO:41) was purified and characterized in Roche cobas® LIAT “RAB” multiplex system (CRAB-COVID19) with C21 and parental Z05 I616M I709K controls. FluB Wild Type and mutant RNA templates (mismatch under RT primer) were employed in four-plex with RSV, FluB, FluA, and internal control (IC) oligonucleotides. Mg-based RT-PCR was performed on Roche LC 480 with unmodified primers. Results show that that the K542F variant (SEQ ID NO:41) was the only polymerase to pick up the mutant FluB target, as shown in FIG. 7A and FIG. 7B. The results also show that the K542F variant (SEQ ID NO:41) shows a higher RFI than the C21 polymerase and the parental polymerase (see, FIG. 7B).

[0272] Thus, after the extensive screening, the K542F variant (SEQ ID NO:41), Z05 I616M I709K K542F emerged as a promising candidate, exhibiting excellent and improved RT efficiency and excellent and improved specificity. In particular, the K542F variant (SEQ ID NO:41), Z05 I616M I709K K542Fexhibited better specificity than the C21 polymerase and the parental, exhibited comparable sensitivity to the C21 polymerase, and exhibited better inclusivity than the C21 polymerase.

[0273] Example 6: Identifying variant DNA polymerases, with substitutions at position K542 having increased RT efficiency and increased specificity

[0274] Under primary screening, roughly 6,000 clones were expressed and screened by primary MS2 singleplex Mg-based RT-PCR with 1 minute RT incubation time. Hits or results with early Cps and high RFI were sequenced, re-arrayed, and screened by secondary assays for RT efficiency and specificity. Sequencing hits from the primary screen are depicted in FIG. 6.

[0275] As described above, position 542 of the parental type (Z05 I616M I709K) (SEQ ID NO: 42) is a position attractive for mutating. In particular, the K542F variant (SEQ ID NO: 41), Z05 I616M I709K K542F emerged as a promising candidate, exhibiting excellent RT efficiency and excellent specificity. The K542F variant (SEQ ID NO:41) (z.e., Z05 I616M I709K K542F), exhibited improved RT efficiency as compared to the parental type (Z05 I616M I709K) (SEQ ID NO: 42), and improved specificity as compared to the parental type (SEQ ID NO:42) and as compared to the C21 polymerase (SEQ ID NO:43).

[0276] An effort was made to identify other improved variant polymerases of the parental type polymerase with changes at position 542. To that end, position 542 of the parental type polymerase was mutated to all of the 19 other amino acids (apart from wild type K) to find the best substitution at this position. Lysates were randomly arrayed in a 384 well plate.

[0277] Primary screening was conducted with HCV RT PCR assays (to assess RT efficiency) and a SYBR test (to assess specificity).

[0278] First, to measure RT efficiency, the variants were screened by Hepatitis C Virus (HCV) RT-PCR. An HCV RT-PCR was established with 0.5X RAB oligos, 3,000 copies of genomic DNA, 105copies of HCV RNA, with 75 seconds of RT reaction (see, FIG. 8). The HCV RT-PCR results for all samples are shown in FIG 9 A, and the results for the top samples are shown in FIG. 9B.

[0279] Next, to measure specificity, the variants were screened by a SYBR test without template RNA (see, FIG. 10). SYBR binds double-stranded DNA non-specifically. When this PCR is run without a template, SYBR signal is generated by the amplification of non-specific products (e.g., primer dimers, etc.). In this screen, enzyme variants that generate more non-specific products will have earlier Cps in the PCR. Results of the SYBR test are shown in FIG. 11.

[0280] Thus, based on the primary screen, ideal variant enzymes exhibit earlier Cps in the HCV RT-PCR assay (which would evince increased RT efficiency), and later Cps in the SYBR no template condition (which would evince increased specificity). The results of these assays show that the K542Y substitution (SEQ ID NO:40) exhibited both increased RT efficiency and increased specificity. That is, the polymerase with the K542Y substitution (i.e., SEQ ID NO:40) exhibited an earlier Cp in theHCV RT-PCR test, indicating better RT efficiency, and exhibited a later Cp in the SYBR test, indicating improved specificity.

[0281] The samples (38 in total) were sequenced via Sanger sequencing to uncover and ascertain which amino acid, at the 542 position, was most common in the top performing samples. Sanger sequencing results show that all six (6) top samples exhibit the K542Y substitution, as shown in the bar graph of FIG.

[0282] 12A, and the JMP graph of FIG. 12B. Additionally, the lysate concentrate was assessed for the top samples and controls. These results show that the lysate concentrations of the top samples was higher than the controls (by roughly 1.5-2 times higher), as shown in FIG. 13, suggesting, perhaps, better and higher expression of the 542F and 542Y mutations.

[0283] The HCV RT-PCR assay and SYBR tests were conducted with a 6 minutes RT time and a 75 second RT time. A table showing all of the data from the HCV RT-PCR tests and SYBR tests results with 6 minutes and 75 seconds RT time as shown in FIG. 14. These results show that from the K542X library, the K542Y (SEQ ID NO: 40) is shown to be an excellent mutation, in terms of RT efficiency and specificity.

[0284] Thus, after the extensive screening, the K542Y variant (SEQ ID NO:40), Z05 I616M I709K K542Y emerged as a promising candidate, exhibiting excellent RT efficiency and excellent specificity The results of these assays show that the K542Y substitution (SEQ ID NO: 40) exhibited both increased specificity and increased RT efficiency. Thus, after the extensive screening, the K542Y variant (SEQ ID NO:40), Z05 I616M I709K K542Y emerged as a promising candidate, exhibiting excellent RT efficiency and excellent specificity.

[0285] Example 7: Reverse Transcription (RT) and PCR Efficiency of pure ZY (SEQ ID NO:40) A Single Site Variant Library consisting of Z05_I616M_I709K (Z05DM) backbone (SEQ ID NO:42) with one additional amino acid in the polymerase domain was cloned and transformed into E. coli BL21 expression strain. Clonal mutant library glycerol culture stocks were arrayed in 384 well plates. Plate-based expression and partial purification was done to make corresponding clone lysates. Screening of diluted lysates was conducted in Mg-based MS2 RT-PCR. The primary screening workflow is shown in FIG. 15. On plate 1910, the top clones were chosen for early Cps and high fluorescence signal, and were in wells H19 and E13, as shown in FIG. 16.

[0286] Secondary screening was conducted on the top clones from the primary screen, by way of Sanger sequencing. The top clones were Sanger sequenced, quantified, and re-arrayed on a new plate, and screened under more stringent conditions. The sanger results revealed that the top two clones had the following substitutions: K542F (SEQ ID NO:41) and K542Y (SEQ ID NO:40). That is, the K542F (SEQ ID NO:41) and K542Y (SEQ ID NO:40) mutants displayed the highest RFIs. In particular, the K542Y (SEQ ID NO:40) mutant had a similar RFI as K542F (SEQ ID NO:41) but had an earlier Cps. The K542F (SEQ ID NO:41) and K542Y (SEQ ID NO:40) mutants were purified and characterizedfurther in an HCV RT-PCR system. In particular, an HCV Mg-based RT-PCR reaction was conducted, comparing the purified K542Y (SEQ ID NO:40) against the Z05DM parental (SEQ ID NO:42). In this reaction, 50nM enzyme was used, with a 2 minute RT incubation at 65°C, with RNA and DNA as templates. The results show that for the K542Y mutant (SEQ ID NO:40), RNA Cps are 5 cycles earlier than the Z05DM parental (SEQ ID NO:42), that the RT efficiency for the K542Y (SEQ ID NO:40) is ~ 60 times higher than the Z05DM parental (SEQ ID NO:42), and that the PCR Efficiencies are comparable between the two enzymes, but the Z05DM parental (SEQ ID NO:42) DNA Cps come up 1 cycle earlier. These results are shown in FIG. 17.

[0287] Additionally, the RT and PCR efficiency of pure K542Y (SEQ ID NO: 40) was assessed in a SYBR-NTC assay. In particular, the specificity was assessed in an HCV Mg-based RT-PCR multiplex (MMx) assay was conducted comparing the following three (3) enzymes: (i) K542Y mutant (SEQ ID NO:40), (ii) the Z05DM parental (SEQ ID NO:42), and (iii) the Z05 wild type polymerase (SEQ ID NO: 1). The reaction conditions were 50 nM enzyme, 2 minute RT incubation at 65°C, no template, 0.2X SYBR Green, with four replicates for each enzyme. Cps represent non-specific amplification product (NSA), and the earlier the Cp, the more NSA present. The results show that the K542Y mutant (SEQ ID NO:40) SYBR Cps are ~3 cycles delayed compared to the Z05DM parental (SEQ ID NO:42), and the Z05 wild type polymerase (SEQ ID NO: 1). Additionally, the K542Y mutant (SEQ ID NO:40) exhibits improved specificity compared to the Z05DM parental (SEQ ID NO:42), and the Z05 wild type polymerase (SEQ ID NO:1). The results are depicted in FIG. 18.

[0288] These studies demonstrate that the K542Y mutant (SEQ ID NO: 40) exhibits improved efficiency and specificity as compared to the Z05DM parental (SEQ ID NO:42), and the Z05 wild type polymerase (SEQ ID NO: 1).

[0289] Collectively, these examples and studies show that certain modified DNA polymerases, with mutations at positions 616, 709, and 542, relative to a Z05 polymerase (SEQ ID NO: 1) or a parental polymerase (SEQ ID NO:42), exhibit improved RT efficiency and improved specificity, relative to a Z05 polymerase (SEQ ID NO:1) or a parental polymerase (SEQ ID NO:42). In particular, certain modified DNA polymerases, with the following modifications: (i) 1616M; (ii) I709K; and / or (iii) K542Y or K542F, relative to a Z05 polymerase (SEQ ID NO:1), exhibit improved RT efficiency and improved specificity, relative to a Z05 polymerase (SEQ ID NO:1) or a parental polymerase (SEQ ID NO:42). In particular, certain modified DNA polymerases, with the following modification: K542Y or K542F, relative to the parental type polymerase (SEQ ID NO: 42), exhibit improved RT efficiency and improved specificity, relative to a Z05 polymerase (SEQ ID NO: 1) or a parental polymerase (SEQ ID NO:42). In particular, certain modified polymerases, for example: (a) aK542Y polymerase (SEQ ID NO:40), and / or (b) a K542F (SEQ ID NO:41), exhibit improved RT efficiency and improved specificity, relative to a Z05 polymerase (SEQ ID NO: 1) or a parental polymerase (SEQ ID NO:42).

Claims

CLAIMS1. A modified DNA polymerase, wherein the modified DNA polymerase is modified relative to a reference DNA polymerase, wherein the reference DNA polymerase has an amino acid sequence of SEQ ID NO: 42, and wherein the amino acid corresponding to position 542 is an amino acid other than K.

2. The modified DNA polymerase of claim 1, wherein the modified DNA polymerase comprises an amino acid sequence at least 90% identical to SEQ ID NO: 42.

3. The modified DNA polymerase of any one of claims 1 to 2, wherein the amino acid corresponding to position 542 is an amino acid selected from a group of amino acids, wherein the group of amino acids consists of F and Y.

4. The modified DNA polymerase of claim 3, wherein the amino acid corresponding to position 542 isF.

5. The modified DNA polymerase of any one of claims 1 to 4, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:41.

6. The modified DNA polymerase of claim 3, wherein the amino acid corresponding to position 542 is Y.

7. The modified DNA polymerase of any one of claims 1 to 3 and 6, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:40.

8. The modified polymerase of any one of claims 1 to 7, wherein the modified DNA polymerase has increased reverse transcriptase efficiency as compared to the reference polymerase.

9. The modified polymerase of any one of claims 1 to 8, wherein the modified DNA polymerase has improved specificity compared to the reference DNA polymerase.

10. A modified DNA polymerase, wherein: (i) the modified DNA polymerase is modified relative to a reference DNA polymerase, wherein the reference DNA polymerase has an amino acid sequence of SEQ ID NO:1; and (ii) the amino acid corresponding to position 616 is an amino acid other than I, the amino acid corresponding to position 709 is an amino acid other than I, and the amino acid corresponding to position 542 is an amino acid other than K.

11. The modified DNA polymerase of claim 10, wherein the modified DNA polymerase comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1.

12. The modified DNA polymerase of any one of claims 10 to 11, wherein the amino acid corresponding to position 616 is M.

13. The modified DNA polymerase of any one of claims 10 to 12, wherein the amino acid corresponding to position 709 is K.

14. The modified DNA polymerase of any one of claims 10 to 13, wherein the amino acid corresponding to position 542 is an amino acid selected from a group of amino acids, whereinthe group of amino acids consists of F and Y.

15. The modified DNA polymerase of claim 14, wherein the amino acid corresponding to position 542 isF.

16. The modified DNA polymerase of any one of claims 10 to 15, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:41.

17. The modified DNA polymerase of claim 14, wherein the amino acid corresponding to position 542 is Y.

18. The modifiedDNA polymerase of any one of claims 10 to 14 and 17, wherein the modifiedDNA polymerase comprises the amino acid sequence of SEQ ID NO:40.

19. The modified DNA polymerase of any one of claims 10 to 16, wherein the amino acid corresponding to position 616 is M, the amino acid corresponding to position 709 is K, and the amino acid corresponding to position 542 is F.

20. The modified DNA polymerase of claim 19, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:41.

21. The modified DNA polymerase of any one of claims 10 to 16, wherein the amino acid corresponding to position 616 is M, the amino acid corresponding to position 709 is K, and the amino acid corresponding to position 542 is Y.

22. The modified DNA polymerase of claim 21, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:40.

23. The modified polymerase of any one of claims 10 to 12, wherein the modified DNA polymerase has increased reverse transcriptase efficiency as compared to the reference polymerase.

24. The modified polymerase of any one of claims 10 to 23, wherein the modified DNA polymerase has improved specificity as compared to the reference DNA polymerase.

25. A modified DNA polymerase, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:41.

26. A modified DNA polymerase, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:40.

27. A method of conducting primer extension, wherein the method comprising contacting a modified DNA polymerase with a primer, a polynucleotide template, and a nucleoside triphosphate under conditions suitable for extension of the primer, thereby producing an extended primer, and whereinthe modified DNA polymerase is modified relative to a reference DNA polymerase, wherein the reference DNA polymerase has an amino acid sequence of SEQ ID NO: 42, and wherein the amino acid corresponding to position 542 is an amino acid other than K.

28. The method of claim 27, wherein the modified DNA polymerase comprises an amino acidsequence at least 90% identical to SEQ ID NO:42.

29. The method of any one of claims 27 to 28, wherein the amino acid corresponding to position 542 is an amino acid selected from a group of amino acids consisting of F and Y.

30. The method of claim 29, wherein the amino acid corresponding to position 542 is F.

31. The method of any one of claims 27 to 30, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:41.

32. The method of claim 29, wherein the amino acid corresponding to position 542 is Y.

33. The method of any one of claims 27 to 29 and 32, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:40.

34. The method of any one of claims 27 to 33, wherein the modified DNA polymerase has increased reverse transcriptase efficiency as compared to the reference polymerase.

35. The method of any one of claims 27 to 34, wherein the modifiedDNA polymerase has improved specificity as compared to the reference DNA polymerase.

36. The method of any one of claims 27 to 35, wherein the polynucleotide template is RNA.

37. The method of any one of claims 27 to 35, wherein the polynucleotide template is DNA.

38. The method of any one of claims 27 to 37, wherein the method for conducting primer extension comprises a polymerase chain reaction (PCR).

39. A method of conducting primer extension, the method comprising contacting a modified DNA polymerase with a primer, a polynucleotide template, and a nucleoside triphosphate under conditions suitable for extension of the primer, thereby producing an extended primer, wherein: (i) the modified polymerase is modified relative to a reference DNA, wherein the reference DNA polymerase has an amino acid sequence of SEQ ID NO: 1 ; and (ii) the amino acid corresponding to position 616 is an amino acid other than I, the amino acid corresponding to position 709 is an amino acid other than I, and the amino acid corresponding to position 542 is an amino acid other than K.

40. The method of claim 39, wherein the modified DNA polymerase comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1.

41. The method of any one of claims 39 to 40, wherein the amino acid corresponding to position 616 is M.

42. The method of any one of claims 39 to 41, wherein the amino acid corresponding to position 709 is K.

43. The method of any one of claims 39 to 42, wherein the amino acid corresponding to position 542 is selected from a group of amino acids consisting of F and Y.

44. The method of claim 43, wherein the amino acid corresponding to position 542 is F.

45. The method of any one of claims 39 to 44, wherein the modified DNA polymerase comprisesthe amino acid sequence of SEQ ID NO:41.

46. The method of claim 43, wherein the amino acid corresponding to position 542 is Y.

47. The method of any one of claims 39 to 43 and 46, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:40.

48. The method of any one of claims 39 to 42, wherein the amino acid corresponding to position 616 is M, the amino acid corresponding to position 709 is K, and the amino acid corresponding to position 542 is F.

49. The method of claim 48, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:41.

50. The method of any one of claims 39 to 42, wherein the amino acid corresponding to position 616 is M, the amino acid corresponding to position 709 is K, and the amino acid corresponding to position 542 is Y.

51. The method of claim 50, wherein the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO:40.

52. The method of any one of claims 39 to 51 , wherein the modified DNA polymerase has increased reverse transcriptase efficiency as compared to the reference polymerase.

53. The method of any one of claims 39 to 52, wherein the modifiedDNA polymerase has improved specificity as compared to the reference DNA polymerase.

54. The method of any one of claims 39 to 53, wherein the polynucleotide template is RNA.

55. The method of any one of claims 39 to 53, wherein the polynucleotide template is DNA.

56. The method of any one of claims 39 to 55, wherein the method for conducting primer extension comprises a polymerase chain reaction (PCR).

57. A reaction mixture for performing a polynucleotide extension method comprising a DNA polymerase according to any one of claims 1 to 26.

58. The reaction mixture of claim 57 further comprising one or more additional reagents selected from the group of nucleoside triphosphates, a buffer suitable for polynucleotide extension, and / or one or more primer or probe polynucleotides suitable for hybridizing to a predetermined polynucleotide template under polynucleotide extension conditions.

59. A kit for performing a polynucleotide extension method comprising a DNA polymerase according to any one of claims 1 to 26.

60. The kit of claim 59 further comprising one or more additional containers providing one or more additional reagents selected from the group of nucleoside triphosphates, a buffer suitable for polynucleotide extension, and / or one or more primer or probe polynucleotides suitable for hybridizing to a predetermined polynucleotide template under polynucleotide extension conditions.

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