DNA polymerase mutants having enhanced template discrimination activity

Abstract

To provide mutant DNA polymerases having enhanced primer and / or template discrimination activity, and uses of the same for polymerase-based assays for genetic diagnostic analysis.SOLUTION: This invention provides mutant DNA polymerases having an enhanced template discrimination activity compared with unmodified DNA polymerases, where amino acid sequences of the mutant DNA polymerases include substitutions at specific residue positions of unmodified Taq DNA polymerases.SELECTED DRAWING: None

Description

Technical Field

[0001] This application was filed on November 14, 2013, and claims the benefit of priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 61 / 904,335, entitled "DNA Polymerase Mutants Having Increased Template Discrimination Activity," the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to mutant DNA polymerases having increased primer and / or template discrimination activity, and their use for polymerase-based assays for genetic diagnostic analysis.

Background Art

[0003] The ability to accurately diagnose a given genetic condition and predictably treat diseases caused by genetics requires a reliable method for accurately determining gene sequence information. A number of diseases caused by genetics are associated with single nucleotide polymorphisms (SNPs) within genes that encode proteins. The presence of SNPs associated with diseases caused by genetics, such as cancer, can be difficult to detect by a small number of genetically modified cells in a population encoding one or more alleles ("low frequency alleles").

[0004] Polymerase-based assays, such as polymerase chain reaction (PCR), have important implications and wide use in genetic diagnosis and molecular medicine. Polymerases synthesize DNA sequences by adding nucleotides to the 3’ end of short oligonucleotides (hereinafter abbreviated as “primers”). Primers are hybridized to the single-stranded sequence (“template”) to be amplified. DNA polymerase catalyzes the formation of a phosphodiester bond between the 3’-oxygen at the 3’ end of the primer and the incoming deoxynucleoside triphosphate (“dNTP”). This chemical reaction (“primer extension”) adds nucleotides to the primer (e.g., the newly growing DNA strand). Primer extension is base-specific in that deoxynucleoside triphosphates complementary to the bases in the template are added to the primer. The fidelity of DNA polymerase enzymes is very high, and the mutation rate introduced into the replicated DNA strand is low; however, the exact error rates vary among different DNA polymerase enzymes, and these rates are well characterized. The extension reaction can be repeated until the end of the template is reached.

[0005] Most polymerase-based assays for detecting SNPs rely on there being polymerase enzyme discrimination between at least two different substrates. The first substrate contains the desired SNP to be detected; the second substrate contains the normal nucleotide that is not to be detected. Polymerase-based discrimination can be achieved by providing the first substrate as a preferred polymerase-compatible substrate for the assay. This discrimination can be maximized to the extent that the first substrate is simply a polymerase-compatible substrate for the assay.

[0006] Many strategies for establishing conditions to support polymerase-based discrimination in substrates with minimal nucleotide differences, such as those containing only a single nucleotide difference, are known in the art. One strategy relies on the ability of a polymerase that does not efficiently initiate synthesis on a substrate lacking the 3'-paired nucleotide of the primer. Allele-specific primer design is a primer in which the 3'-nucleotide forms a perfect match with the complementary base at the location containing the SNP-containing allele and forms a mismatch pair when annealing to the allele lacking the SNP. The primer for the SNP-containing allele:template serves as a preferred polymerase-compatible substrate because the polymerase can efficiently initiate primer synthesis from such a substrate. Examples of these strategies are described in Chen et al., "Single nucleotide polymorphism genotyping: biochemistry, protocol, cost and throughput," Pharmacogenomics J., 3(2):77-96 (2003); Kwok et al., "Detection of single nucleotide polymorphisms," Curr. Issues Mol. Biol. 5(2):43-60 (April 2003); Shi, "Technologies for individual genotyping: detection of genetic polymorphisms in drug targets and disease genes," Am. J. Pharmacogenomics 2(3):197-205 (2002); and Kwok, "Methods for genotyping single nucleotide polymorphisms," Annu. Rev. Genomics Hum. Genet., 2:235-58 (2001). A strategy for improving the selectivity of this class of allele-specific PCR primers is to introduce a second mutation at the penultimate base adjacent to the nucleotide at the 3'-end of the primer (i.e., adjacent to the SNP site).As described above, the 3'-terminal residue is either a match or a mismatch to the base being examined in the sample nucleic acid (SNP), where the primer has either two adjacent mismatches to the target (both the 3'-terminal base and the second-to-last base) or one mismatch to the target (only the second-to-last base, with the 3'-terminal base being a match). See, for example, Newton et al., "Analysis of any point mutation in DNA. The amplification refractory mutation systems (ARMS)", Nucleic Acids Res., 17(7):2503-15 (1989). Yet another strategy for improving the selectivity of this class of allele-specific PCR primers is to employ nucleic acid residues chemically modified at the 3'-terminus of the primer, such as locked nucleic acids (LNAs), which reduce the ability of DNA polymerase to initiate DNA synthesis from a 3'-terminal mismatch. See, for example, Latorra et al., "SNP genotyping using 3’ locked nucleic acid (LNA) primers", Human Mut., 22(1):79-85 (2003).

[0007] Template substrate identification can be enhanced in polymerase-based assays by requiring the formation of a second nuclease-catalyzed nucleic acid from one or more primers for use in polymerase-based assays. In one such assay, the ligase chain reaction assay, DNA ligase is used with polymerase to detect a template containing an SNP. Since polymerase-based assays require primers with a minimum length that hybridizes to the template substrate, DNA ligase can be used to generate primers for polymerase from oligonucleotides of sub-optimal length. This assay relies on directly hybridizing two probes across the SNP polymorphic site, such that ligation can occur if the probe is identical to the target DNA. Design two probes: an allele-specific probe that hybridizes to the target DNA such that the 3'-base is positioned directly across the SNP nucleotide, and a second probe that hybridizes to the template downstream of the complementary strand of the SNP polymorphic site that provides the 5'-end for the ligation reaction. If the allele-specific probe matches the target DNA, it hybridizes completely to the target DNA and ligation can occur. Ligation generally does not occur in the presence of a mismatched 3'-base. Once the oligonucleotide product is formed, it can serve as a primer or a template for polymerase-based assays. Examples of this strategy are described in Barany F., "Genetic disease detection and DNA amplification using cloned thermostable ligase.", Proc Natl Acad Sci USA., 1991 Jan 1;88(1):189-93, and Wiedmann M., Wilson W.J., Czajka J., Luo J., Barany F., Batt C.A., "Ligase chain reaction (LCR)--overview and applications.", PCR Methods and Applications 1994 Feb;3(4):S51-64.

[0008] A polymerase-compatible substrate is a primer having an available 3'-hydroxyl group on the primer, which is another strategy known in the art. Since RNase H-based PCR (rhPCR) requires a template, it can be used to improve polymerase-based discrimination. The rhPCR method uses an RNase H enzyme to convert a primer lacking a 3'-hydroxyl group ("blocked primer") or otherwise non-functional and unable to support PCR into a primer containing a 3'-hydroxyl group that can support PCR ("unblocked primer"). Blocked primers in rhPCR include oligonucleotides that function as cleavage sites and oligonucleotides with a blocked 3'-end or other modifications that prevent either the priming or templating function of the oligonucleotide and internal RNA residues. Type II RNase H recognizes the annealed primer:template duplex containing these blocked primers and cleaves the 5' of the primer strand of the RNA residue to generate a 3'-hydroxyl group with the adjacent DAN residue. Since the RNase H enzyme does not cleave substrates containing RNA residues that do not pair at mismatch sites, allele-specific template discrimination can be achieved by placing RNA residues at positions complementary to SNPs on the selected allele template. The resulting type II RNase H cleavage product can function as a polymerase-compatible substrate.This enzyme cleavage strategy, similar RNase H strategies, and methods of blocking primer extension or inhibiting template function, thereby rendering PCR non-functional, are described in U.S. Patent No. 7,112,406 to Behlke et al. entitled "POLYNOMIAL AMPLIFICATION OF NUCLEIC ACIDS"; U.S. Patent No. 5,763,181 to Han et al. entitled "CONTINOUS FLUOROMETRIC ASSAY FOR DETECTING NUCLEIC ACID CLEAVAGE"; U.S. Patent No. 7,135,291 to Sagawa et al. entitled "METHOD OF DETECTING NUCLEOTIDE POLYMORPHISM"; U.S. Patent Application No. 20090068643 to Behlke and Walder entitled "DUAL FUNCTION PRIMERS FOR AMPLIFYING DNA AND METHODS OF USE"; and U.S. Patent Application No. 20100167353 to Walder et al. entitled "RNASE H-BASED ASSAYS UTILIZING MODIFIED RNA MONOMERS".

[0009] This region focuses on substrate-based approaches such as those exemplified above to improve the detection of genetic differences and low-frequency alleles. Currently, the sensitivity of polymerase-based assays is limited by the formation of non-specific amplification products resulting from ectopic or aberrant primer-related extension products independent of the desired template. The inherent reactivity of the polymerase appears to be a major cause of the generation of such artifacts during amplification.

[0010] Accordingly, any further improvement in mismatch discrimination may, when using one or more of the described strategies, require a modified polymerase enzyme that is inherently better endowed with 3'-nucleotide discrimination. Modified polymerase enzymes having activities different from the unmodified form can be prepared by chemical or enzymatic modification of the protein or by mutagenesis of the corresponding gene encoding the protein. In general, the latter approach is preferred, since a genetically modified gene encoding a given mutant protein can be stably maintained, expressed and purified to obtain an enzyme preparation having well-characterized properties.

[0011] Unbiased mutagenesis strategies can be used to generate libraries of mutant polymerase genes, but this approach has certain drawbacks. Millions of mutant enzymes have to be screened for activity and success often depends on the likelihood that effective mutations are present within the limited pool generated by random mutagenesis. Direct genetic selection methods are not sensitive enough to identify mutations related to a second function in addition to the essential polymerase activity. Most of the mutant polymerase genes in a positive selection assay probably encode proteins that retain the functional attributes of the normal polymerase enzyme. Therefore, a secondary screening procedure using biochemical assays has to be carried out to identify whether any of the mutant polymerases have the desired activity. Despite the technical difficulties in setting up the initial selection process, the associated costs of performing secondary screening using biochemical assays are high if more than 100 clones need to be purified and assayed.

[0012] Another approach is to apply a biased mutagenesis strategy that specifically targets a preselected region of a gene involved in a function. In this approach, the gene region is first identified by a selection method. One such selection method is the comparative phylogenetic analysis of a specific gene required by organisms of diverse origins. The principle of comparative phylogenetic analysis is based on the hypothesis that diverse organisms do not share protein-coding sequences in essential genes if they are not evolutionarily constrained for reasons related to essential protein functions.

[0013] Phylogenetic comparative analysis of genes encoding DNA polymerases can provide insights into potentially important amino acid residues for polymerase function. The overall folding pattern of DNA polymerases resembles that of the human right hand and includes three different subdomains: the palm, the fingers, and the thumb. (See, for example, Beese et al., Science 260:352-355, 1993; Patel et al., Biochemistry 34:5351-5363, 1995.) The structures of the finger and thumb subdomains vary greatly among polymerases that differ in size and cellular function, but the catalytic palm subdomains can all be superimposed. For example, motif A, which interacts with incoming dNTPs and stabilizes the transition state during the chemical catalytic reaction, can be superimposed with an average root mean square deviation of about 1 angstrom in mammalian polα and prokaryotic polI family DNA polymerases (Wang et al., Cell 89:1087-1099, 1997). Motif A structurally begins with an antiparallel β-strand containing mainly hydrophobic residues, followed by an α-helix. The primary amino acid sequence of the DNA polymerase active site is exceptionally conserved.

[0014] In addition to being well conserved, the active site of DNA polymerase has also been shown to be relatively mutable and to tolerate certain amino acid substitutions without significantly reducing DNA polymerase activity. (See, e.g., U.S. Patent No. 6,602,695). Such mutant DNA polymerases can provide various selective advantages in diagnostic and research applications, including, for example, nucleic acid synthesis reactions. The inventors identify mutations in protein sequences using the single letter amino acid code and an integer indicating the position of the mutation from the beginning of the protein sequence. The single letter amino acid code is well known in the art and is described, for example, in Stryer et al., Biochemistry, 5 th ed., Freeman and Company (2002). As an example, aspartic acid ("D") is changed to glycine ("G") in the D580G mutant, and the mutation is located at amino acid 580 from the beginning of the protein sequence.

[0015] Reichert et al. performed a comparative phylogenetic analysis of thermoactive DNA polymerases from thermophilic bacteria. Therein, the protein coding sequences of DNA polymerase I enzymes were aligned for 13 phylogenetically distinct species. The analysis revealed that eight amino acid positions within a 15 amino acid long motif located at amino acid positions 645 - 685 of Thermus sp. Z05 DNA polymerase ("Z05 DNA polymerase") can tolerate changes without impairing the function of the core enzyme.

[0016] Comparative phylogenetic analysis does not provide specific functional information regarding non-conserved amino acids, rather than suggesting that non-conserved amino acids do not appear to be important for coenzyme function. Therefore, specific mutations were introduced into the recombinant gene encoding a mutant of Z05 DNA polymerase (the "Z05 D580G polymerase"), and the resulting Z05 D580G polymerase mutants were screened for their ability to confer a decrease in the ability to extend oligonucleotide primers having a 3'-mismatch to the template. Reichert et al. found that one such mutant, the Z05 D580G V667E polymerase, exhibits better discrimination (by about two-fold) than the parental Z05 D580G polymerase. See U.S. Patent Application No. 2012 / 0015405 by Reichert et al., entitled "DNA POLYMERASES WITH INCREASED 3’-MISMATCH DISCRIMINATION".

[0017] Comparative phylogenetic analysis has limitations with respect to the identification of DNA polymerase activity showing improved 3'-nucleotide discrimination. This is due to the fact that all DNA polymerases of a given enzyme class are faced with similar template substrates and nucleotide pools across a spectrum of phylogenetically diverse organisms. Given the fact that all DNA polymerases must exhibit 3'-nucleotide mismatch discrimination to preserve high-fidelity replication of the daughter template strand, it is not surprising that comparative phylogenetic analysis can be applied to identify amino acid positions that may affect mismatch discrimination. It is necessary to identify DNA polymerases having improved 3'-nucleotide discrimination for template substrates having different 3'-terminal modifications that are shown for the polymerase only in biochemical assays, such as those used in some PCR-based assays for low-frequency allele detection.

[0018] Taq DNA polymerase is an enzyme discovered in the bacterium Thermus aquaticus (Chien, A. et al., J Bacteriol., 1976, 127:1550 - 1557). The enzyme is classified as a deoxyribonucleic acid polymerase, class I (enzyme code, EC2.7.7.7). Its catalytic activity is to amplify DNA sequences. The peptide sequence and gene sequence of the Taq DNA polymerase enzyme isolated from nature are well - known in the art and are listed in Table 1 (Lawyer, F.C. et al., J.Biol.Chem., 1989, 264:6427 - 6437; Genbank database ID J04639.1).

[0019]

Table 1

[0020] Taq DNA polymerase extends primers composed of deoxyribonucleotides, however, some chemical modifications of the primers are tolerated and do not reduce the efficiency of the primer - extension reaction. For example, when the nucleotide at the 3' primer end is a ribonucleotide instead of a deoxyribonucleotide, Taq DNA polymerase can extend such primers with considerable efficiency and speed.

Prior Art Documents

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Summary of the Invention

Means for Solving the Problems

[0023] (Gist of the Invention) In one aspect, a mutant Taq DNA polymerase having enhanced template discrimination activity as compared to unmodified Taq DNA polymerase is provided. The amino acid sequence of the mutant Taq DNA polymerase contains at least one substitution at residue position 783 or 784 of the unmodified Taq DNA polymerase.

[0024] In another aspect, a mutant thermostable DNA polymerase having increased template discrimination activity as compared to unmodified thermostable DNA polymerase is provided. The amino acid sequence of the mutant thermostable DNA polymerase contains at least one substitution at an orthologous residue position relative to positions 783 or 784 of the unmodified Taq DNA polymerase.

[0025] In another aspect, a mutant DNA polymerase having increased template discrimination activity as compared to the corresponding unmodified DNA polymerase is provided, and the mutant DNA polymerase includes a thermostable polymerase. The amino acid sequence of the mutant DNA polymerase peptide contains at least one substitution at an orthologous residue position relative to positions 783 or 784 of the unmodified Taq DNA polymerase, where the mutant DNA polymerase is selected from the group of species consisting of Escherichia coli, Eubacterium siraeum, Clostridium leptum, Enterococcus, Facklamia hominis, Bacillus anthracis, and Bacillus cereus ATCC 10987.

[0026] In another aspect, a mutant non-VH related DNA polymerase having increased template discrimination activity compared to an unmodified non-VH related DNA polymerase counterpart is provided, and the mutant non-VH related DNA polymerase includes a thermostable polymerase. The amino acid sequence of the mutant non-VH related DNA polymerase includes at least one substitution at an orthologous residue position relative to residue positions 783 and / or 784 of the unmodified Taq DNA polymerase.

[0027] In another aspect, a recombinant nucleic acid encoding any of the above-described mutant DNA polymerases is provided.

[0028] In another aspect, a method for performing primer extension is provided. The method includes contacting the above-described mutant DNA polymerase with a primer, a polynucleotide template, and nucleoside triphosphates under conditions suitable for a primer extension method, thereby generating an extended primer.

[0029] In another aspect, a kit for generating an extended primer is provided, which includes at least one container containing the above-described mutant DNA polymerase.

[0030] In another aspect, a reaction mixture is provided that includes the above-described mutant DNA polymerase, at least one primer, a polynucleotide template, and nucleoside triphosphates.

[0031] In another aspect, a method for performing rhPCR is provided, which includes the step of performing primer extension using the above-described mutant DNA polymerase.

[0032] In another aspect, mutant Taq DNA polymerases are provided that have increased template discrimination activity compared to unmodified Taq DNA polymerase. The amino acid sequence of the mutant Taq DNA polymerase contains one of the following selected substitutions: (1) A661E; I665W; F667L [SEQ ID NO: 87]; (2) V783F [SEQ ID NO: 83]; (3) H784Q [SEQ ID NO: 85]; (4) V783L; H784Q [SEQ ID NO: 89]; (5) H784A [SEQ ID NO: 147]; (6) H784S [SEQ ID NO: 149]; (7) H784I [SEQ ID NO: 155]; (8) H784T [SEQ ID NO: 151]; (9) H784V [SEQ ID NO: 153]; (10) H784M [SEQ ID NO: 157]; (11) H784F [SEQ ID NO: 159]; or (12) H784Y [SEQ ID NO: 161].

[0033] In another aspect, mutant Taq DNA polymerases are provided that have a deleted 5' exonuclease domain (KlenTaq), contain additional mutations, and have increased template discrimination activity compared to unmodified Taq DNA polymerase. The amino acid sequence of the mutant Taq DNA polymerase contains one of the following selected substitutions: (1) A661E; I665W; F667L [SEQ ID NO: 170]; (2) V783F [SEQ ID NO: 172]; (3) H784Q [SEQ ID NO: 174]; (4) V783L; H784Q [SEQ ID NO: 176]; (5) H784S [SEQ ID NO: 178]; or (6) H784Y [SEQ ID NO: 180].

Brief Description of the Drawings

[0034]

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BRIEF DESCRIPTION OF THE INVENTION

[0035] The present invention provides novel thermostable DNA polymerases, including specific examples derived from Thermus aquaticus (Taq) DNA polymerase. These polymerases present improvements over existing methods for nucleic acid amplification, genotyping, and the detection of low-frequency alleles. Also provided are novel assay formats that include the use of these novel thermostable DNA polymerases.

[0036] Definitions To aid in the understanding of the present invention, several terms are defined below.

[0037] The terms used herein are intended as "open" terms (e.g., the term "comprising" should be intended as "including but not limited to", the term "having" should be intended as "having at least", the term "including" should be intended as "including but not limited to", etc.).

[0038] The articles "a" and "an" mean one or more than one of the grammatical object to which the article is attached (e.g., at least one).

[0039] The terms "about" and "approximately" generally mean the degree of tolerance of error of a measured quantity that gives the nature or accuracy of the measurement. Examples of the degree of error are within 20 - 25 percent (%) of a given value or range of values, typically within 10%, more typically within 5%.

[0040] Furthermore, in cases where a conventional expression similar to "at least one of A, B, and C" is used, generally, such syntax is intended in the sense that one of ordinary skill in the art would understand the conventional expression (e.g., "a system having at least one of A, B, and C" includes, without limitation, a system having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It is further understood by one of ordinary skill in the art that virtually any disjunctive words and / or phrases presenting two or more alternative terms should be understood to contemplate including one, any, or both of the terms in the specification or drawings. For example, the phrase "A or B" is understood to include the possibilities of "A" or "B" or "A and B".

[0041] All turns of phrase such as "from", "to", "up to", "at least", "more than", "less than", etc. refer to a range that includes the recited numerical value and can be divided into sub-ranges thereafter.

[0042] Ranges include their respective individual members. Thus, for example, a group having 1 - 3 members means a group having 1, 2, or 3 members. Similarly, a group having 6 members means a group having 1, 2, 3, 4, or 6 members, etc.

[0043] The modal verb "can" means the preferred use or selection of one or more alternatives, or the selection among several described embodiments or features contained within the same. With respect to a particular embodiment or feature contained within the same, where no alternatives or selections are disclosed, the modal verb "can" refers to an affirmative act of how to make or use the aspect of the described embodiment or feature contained within the same, or a definitive determination to use a particular technique with respect to the described embodiment or feature contained within the same. In this latter context, the modal verb "can" has the same meaning and implication as the auxiliary verb "able to".

[0044] When referring to nucleobases, nucleoside triphosphates or nucleotides, the terms "normal" or "natural" mean those that occur naturally in the polynucleotide described (i.e., for DNA, these are dATP, dGTP, dCTP and dTTP). Further, dITP and 7-deaza-dGTP are frequently utilized in place of dGTP, and 7-deaza-dATP can be used in place of dATP in in vitro DNA synthesis reactions such as sequencing. Collectively, these can be referred to as dNTPs.

[0045] When referring to nucleobases, nucleosides or nucleotides, the terms "abnormal" or "modified" include modifications, derivations or analogs of the normal bases, nucleosides or nucleotides that occur naturally in a particular polynucleotide. Certain abnormal nucleotides are modified at the 2'-position of the ribose sugar compared to normal dNTPs. In this way, for RNA, the naturally occurring nucleotides are ribonucleotides (i.e., ATP, GTP, CTP, UTP, collectively referred to as rNTPs), but as described herein, these nucleotides have a hydroxyl group at the 2'-position of the sugar and, in comparison, no dNTPs are present, so when used herein, ribonucleotides are abnormal nucleotides as substrates for DNA polymerases. As used herein, abnormal nucleotides include, but are not limited to, compounds used as terminators for nucleic acid sequencing. Exemplary terminator compounds include, but are not limited to, compounds having a 2',3'-dideoxy structure and called dideoxynucleoside triphosphates. The dideoxynucleoside triphosphates ddATP, ddTTP, ddCTP and ddGTP are collectively referred to as ddNTPs.

[0046] As used herein, the term "nucleotide" is understood to refer to naturally occurring ribonucleotide or deoxyribonucleotide monomers, as well as related structural variants thereof, and includes derivatives and analogs that are functionally equivalent with respect to the particular context in which the nucleotide is being used (e.g., hybridization to complementary bases), unless the context clearly indicates otherwise.

[0047] As used herein, the terms "nucleic acid" and "oligonucleotide" refer to polydeoxyribonucleotides (including 2-deoxy-D-ribose), polyribonucleotides (including D-ribose), and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base. No distinction in length is intended between the terms "nucleic acid", "oligonucleotide", and "polynucleotide". These terms are used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double-stranded DNA and single-stranded DNA, as well as double-stranded RNA and single-stranded RNA. For use in the present invention, oligonucleotides can also include nucleotide analogs in which the base, sugar, or phosphate backbone is modified, as well as non-purine or non-pyrimidine nucleotide analogs.

[0048] Oligonucleotides can be prepared by any suitable method, including direct chemical synthesis by methods such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90-99, incorporated herein by reference; the phosphodiester method of Brown et al., 1979, Meth. Enzymol. 68:109-151, incorporated herein by reference; the diethylphosphoramidite method of Beaucage et al., 1981, Tetrahedron Lett., 22:1859-1862, incorporated herein by reference; the solid support method of U.S. Patent No. 4,458,066, incorporated herein by reference. A review of the synthesis methods for conjugates of oligonucleotides and modified nucleotides is described in Goodchild, 1990, Bioconjugate Chemistry 1(3):165-187, incorporated herein by reference.

[0049] As used herein, the term "primer" refers to an oligonucleotide that can act as a starting point for DNA synthesis under appropriate conditions. Such conditions include inducing the synthesis of a primer extension product complementary to a nucleic acid strand in the presence of four different nucleoside triphosphates and an agent for extension (e.g., DNA polymerase or reverse transcriptase) in a suitable buffer at an appropriate temperature. Primer extension can also be carried out in the absence of one or more nucleoside triphosphates, in which case an extension product of limited length is produced. As used herein, the term "primer" is intended to include oligonucleotides used in ligation-mediated reactions where one oligonucleotide is "extended" by ligation to a second oligonucleotide that hybridizes at an adjacent position. Thus, the term "primer extension" as used herein refers to both the polymerization of individual nucleoside triphosphates using a primer as a starting point for DNA synthesis and the ligation of two oligonucleotides to form an extension product.

[0050] Primers are preferably single-stranded DNA. The appropriate length of a primer depends on its intended use but typically ranges from 6 to 50 nucleotides, preferably 15 to 35 nucleotides. Shorter primer molecules generally require a lower temperature to form a sufficiently stable hybrid complex with the template. A primer need not reflect the exact sequence of the template nucleic acid but must be sufficiently complementary to hybridize with the template. The design of appropriate primers for the amplification of a desired target sequence is well known in the art and is described in the references cited herein.

[0051] Primers can incorporate additional features that allow for primer detection or immobilization, but do not alter the basic properties of the primer that act as the starting point for DNA synthesis. For example, a primer may contain an additional nucleic acid sequence at the 5' end that does not hybridize to the target nucleic acid, but facilitates the cloning or detection of the amplification product. The region of the primer that is sufficiently complementary to the hybridizing template is referred to herein as the hybridization region. Primers may incorporate modified residues other than DNA, so long as the modification does not interfere with the priming or template function.

[0052] The term "3'-nucleotide discrimination" refers to the property of a DNA polymerase to catalyze primer extension reactions with higher specificity and lower efficiency with respect to deoxyribonucleotides when the nucleotide at the 3' end of the primer is chemically modified. For example, a mutant Taq DNA polymerase that exhibits 3'-nucleotide discrimination shows selectivity for deoxyribonucleotide primers and suppressed catalytic activity when the primer is modified, for example, with ribonucleotides.

[0053] The term "3'-mismatch discrimination" refers to the property of a DNA polymerase to distinguish a perfectly 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 3' end of the nucleic acid compared to the template to which the nucleic acid hybridizes. In some embodiments, the nucleic acid to be extended contains a mismatch at the 3' end relative to a perfectly complementary sequence.

[0054] The term "low-frequency allele discrimination" refers to the property of a DNA polymerase for preferentially replicating a first nucleic acid in a nucleic acid population containing a plurality of second nucleic acids, wherein the first nucleic acid is underrepresented in the nucleic acid population relative to the plurality of second nucleic acids. Typically, the first nucleic acid may be underrepresented in a nucleic acid population containing a plurality of second nucleic acids at a ratio of the first nucleic acid to the second nucleic acid in the range of about 1:10 to about 1:1,000,000, including ratios of 1:100, 1:1,000, and 1:100,000 among other ratios. Typically, but not exclusively, a polymerase having low-frequency allele discrimination can be used to detect SNP differences between the first nucleic acid and the second nucleic acid, as will be described in more detail herein.

[0055] The phrase "template discrimination activity" refers to a DNA polymerase having at least one of 3'-nucleotide discrimination, 3'-mismatch discrimination, low-frequency allele discrimination, and combinations thereof.

[0056] The phrase "enhanced discrimination activity" refers to a DNA polymerase having at least one of 3'-nucleotide discrimination, 3'-mismatch discrimination, and low-frequency allele discrimination, or combinations thereof, wherein the DNA polymerase exhibits higher activity than a reference DNA polymerase. For example, a DNA polymerase mutant having "enhanced template discrimination activity" exhibits at least one of 3'-nucleotide discrimination, 3'-mismatch discrimination, low-frequency allele discrimination, and combinations thereof that is higher than the corresponding activity of the naturally occurring wild-type DNA polymerase from which the DNA polymerase mutant is derived.

[0057] A "template discrimination activity assay" refers to an assay for evaluating the ability of a polymerase to discriminate between two templates that differ in one or more variables. Assays designed to reveal 3'-nucleotide discrimination, 3'-mismatch discrimination, or low-frequency allele discrimination are examples of template discrimination activity assays.

[0058] The term "quantification cycle value" is described as Cq and refers to the number of amplification cycles at which a positive signal is first detected.

[0059] The term "discrimination quantification cycle value" is described as ΔCq and refers to the calculated difference between a first reference state and a second reference state, where both the first and second reference states differ in only one variable. For example, the first and second reference states can differ in a polymerase such as a wild-type polymerase and a polymerase mutant, in a primer-template nucleotide sequence such as a mismatched primer-template and a matched primer-template, or in a primer-template 3'-nucleotide ribose structure such as a primer-template containing a 3'-deoxyribose moiety and a primer-template containing a 3'-ribose moiety, and can refer to the same polymerase reaction.

[0060] The term "differential discrimination quantification cycle value" is described as ΔΔCq and refers to the calculated difference between a first discrimination quantification cycle value and a second discrimination quantification cycle value for a polymerase reaction that differs in two variables. In the context of the present disclosure, the ΔΔCq value is a measure of the improvement shown by a given polymerase mutant compared to the wild-type polymerase in a template discrimination activity assay. The preferred ΔΔCq value depends on the nature of the assay, but generally, a preferred ΔΔCq value is at least 1.0 and typically greater than 1.0.

[0061] The terms "target", "target sequence", "target region", and "target nucleic acid" are synonymous when used herein and refer to the region or sequence of a nucleic acid that is to be amplified, sequenced, or detected.

[0062] The term "template" refers to a nucleic acid that includes at least one single-stranded region. The term "template" when modifying "substrate" refers to the nucleic acid used in a hybridization reaction that anneals using a primer and / or an extension reaction using a polymerase.

[0063] As used herein, the term "hybridization" refers to the formation of a double-stranded structure by two single-stranded nucleic acids for complementary base pairing. Hybridization can occur between completely complementary nucleic acid strands or between "substantially complementary" nucleic acid strands containing minor regions of mismatch. The hybridization of completely complementary nucleic acid strands under very favorable conditions is referred to as "stringent hybridization conditions" or "sequence-specific hybridization conditions". Stable double-strands of substantially complementary sequences can be achieved under less stringent hybridization conditions; the degree of allowable mismatch can be controlled by appropriately adjusting the hybridization conditions. One skilled in the art of nucleic acid technology can empirically determine duplex stability according to guidelines provided by the art (see, for example, Sambrook et al., 1989, Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., which is incorporated herein by reference; Wetmur, 1991, Critical Review in Biochem. and Mol. Biol. 26(3 / 4):227-259; and Owczarzy et al., 2008, Biochemistry, 47:5336-5353), taking into account several variables including, for example, the length and base pair composition of the oligonucleotide, the ionic strength, and the frequency of mismatched base pairs).

[0064] The term "amplification reaction" refers to any chemical reaction that results in more copies of a template nucleic acid sequence or that results in transcription of a template nucleic acid. Amplification reactions include polymerase chain reaction (PCR) (see U.S. Pat. Nos. 4,683,195 and 4,683,202; see also PCR Protocols: A Guide to Methods and Applications (Innis et al., eds., 1990)), which includes reverse transcription, and ligase chain reaction (LCR) (see Barany et al., U.S. Pat. No. 5,494,810). Exemplary "amplification reaction conditions" or "amplification conditions" typically include either a two-step or a three-step cycle. A two-step cycle includes a high-temperature denaturation step followed by a hybridization / extension (or ligation) step. A three-step cycle includes a denaturation step, followed by a hybridization step, and then a separate extension or ligation step.

[0065] "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 20 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 (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). When an "X" residue is not defined, these are defined as "any amino acid." The structures of these 20 natural amino acids are described, for example, in Stryer et al., Biochemistry, 5, which is incorporated by reference. thIt is shown in Stadtman, T.C., ed., Freeman and Company (2002). Additional amino acids such as selenocysteine and pyrrolidine can also be genetically encoded (Stadtman, T.C. (1996), “Selenocysteine,” Annu Rev Biochem., 65:83–100, and Ibba, M. et al. (2002), “Genetic code: introducing pyrrolidine,” Curr Biol., 12(13):R464–R466, both incorporated by reference). The term “amino acid” also includes non-natural amino acids, modified amino acids (e.g., having modified side chains and / or backbone), and amino acid analogs. For example, Zhang, X. 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, J.C. et al. (2004), “An expanded genetic code with a functional quadruplet codon,” Proc. Natl. Acad. Sci. U.S.A., 101(20):7566–7571, Ikeda, T. et al. (2003), “Synthesis of a novel histidine analogue and its efficient incorporation into a protein in vivo,” Protein Eng. Des. Sel., 16(9):699–706, Chin, J.W. et al. (2003), “An Expanded Eukaryotic Genetic Code,” Science, 301(5635):964–967, James, C.M. et al. (2001), “Kinetic characterization of ribonuclease S mutants containing photoisomerizable phenylazophenylalanine residues,” Protein Eng. Des. Sel., each incorporated by reference., see Kohrer et al., (2001), Proc. Natl. Acad. Sci. U.S.A., 98(25):14310-14315, pages 14(12):983-991, "Import of amber and ochre suppressor tRNAs into mammalian cells: A general approach to site-specific insertion of amino acid analogues into proteins"; Bacher et al., (2001), J. Bacteriol., 183(18):5414-5425, "Selection and Characterization of Escherichia coli Variants Capable of Growth on an Otherwise Toxic Tryptophan Analogue"; Hamano-Takaku et al., (2000), J. Biol. Chem., 275(51):40324-40328, "A Mutant Escherichia coli Tyrosyl-tRNA Synthetase Utilizes the Unnatural Amino Acid Azatyrosine More Efficiently than Tyrosine"; and Budisa et al., (2001), Protein Sci., 10(7):1281-1292, "Proteins with {beta}-(thienopyrrolyl)alanines as alternative chromophores and pharmaceutically active amino acids".

[0066] The term "residue" is synonymous with "amino acid" or "nucleotide" depending on the context and is substitutable.

[0067] As used herein, "polymerase" refers to an enzyme that catalyzes the polymerization of nucleotides. Generally, the enzyme initiates synthesis at the 3' end of a primer annealed to a nucleic acid template sequence. "DNA polymerase" catalyzes the polymerization of deoxyribonucleotides.Examples of well-known DNA polymerases include, for example, Pyrococcus furiosus (Pfu) DNA polymerase (Lundberg et al., 1991, Gene, 108:1), E. coli DNA polymerase (Lecomte and Doubleday, 1983, Nucleic Acids Res., 11:7505), T7 DNA polymerase (Nordstrom et al., 1981, J. Biol. Chem., 256:3112), Thermus thermophilus (Tth) DNA polymerase (Myers and Gelfand, 1991, Biochemistry, 30:7661), Bacillus stearothermophilus DNA polymerase (Stenesh and McGowan, 1977, Biochim Biophys Acta 475:32), Thermococcus litoralis (Tli) DNA polymerase (also called Vent DNA polymerase, Cariello et al., 1991, Nucleic Acids Res, 19:4193), Thermotoga maritima (Tma) DNA polymerase (Diaz and Sabino, 1998, Braz, J. Med. Res., 31:1239), Thermus aquaticus (Taq) DNA polymerase (Chien et al., 1976, J. Bacteoriol., 127:1550), Pyrococcus kodakaraensis KOD DNA polymerase (Takagi et al., 1997, Appl. Environ. Microbiol., 63:4504), JDF-3 DNA polymerase (International Publication No. WO0132887), and Pyrococcus sp. GB-D (PGB-D) DNA polymerase (Juncosa-Ginesta et al., 1994, Biotechniques, 16:820). The polymerase activity of any of the above enzymes can be determined by means well known in the art.

[0068] The term "thermostable polymerase" refers to an enzyme that is stable to heat, heat-resistant, retains sufficient activity to achieve subsequent polynucleotide extension reactions, and does not irreversibly denature (inactivate) when exposed to high temperatures for the time required to achieve denaturation of double-stranded nucleic acids. The heating conditions required for nucleic acid denaturation are well known in the art and are exemplified, for example, in U.S. Patent Nos. 4,683,202, 4,683,195, and 4,965,188, which are incorporated herein by reference. As used herein, a thermostable polymerase is suitable for use in temperature cycling reactions such as polymerase chain reaction ("PCR"). For the purposes herein, irreversible denaturation refers to the permanent and complete loss of enzyme activity. With respect to a thermostable polymerase, enzyme activity refers to the catalytic action of combining nucleotides in an appropriate manner to form a polynucleotide extension product complementary to a template nucleic acid strand. Examples of thermostable DNA polymerases derived from thermophilic bacteria include, among others, DNA polymerase derived from Thermus aquaticus.

[0069] The term "thermophilic" refers to an enzyme that maintains catalytic properties at the temperatures normally used for reverse transcription or annealing / extension steps in RT-PCR and / or PCR reactions (i.e., 45 - 80°C). Some thermostable enzymes are not irreversibly inactivated and denatured when exposed to the high temperatures required for nucleic acid denaturation. Thermophilic enzymes may or may not be thermostable. Thermophilic DNA polymerases may be DNA- or RNA-dependent and may be derived from thermophilic or mesophilic species including, but not limited to, Escherichia coli, Moloney murine leukemia virus, and avian myeloblastosis virus.

[0070] As used herein, a primer is "specific" for a target sequence if it hybridizes predominantly to the target nucleic acid when used in an amplification reaction under sufficiently stringent conditions. Typically, a primer is specific for a target sequence if the stability of the primer-target duplex is greater than the stability of any duplex formed between the primer and any other sequence found in the sample. Those skilled in the art will recognize that various factors such as salt conditions and the base composition and mismatch positions of the primer affect primer specificity, and that routine experimental confirmation of primer specificity is required in many cases. Hybridization conditions can be selected under which the primer can form a stable duplex only with the target sequence. Thus, the use of a target-specific primer under appropriately stringent amplification conditions enables the selective amplification of the target sequence containing the target primer binding site.

[0071] As used herein, the term "non-specific amplification" refers to the amplification of a nucleic acid sequence other than the target sequence that hybridizes to a sequence other than the target sequence and then results from a primer that functions as a substrate for primer extension. Hybridization of a primer to a non-target sequence is referred to as "non-specific hybridization" and occurs particularly during low temperature, reduced stringency, pre-amplification conditions, or in the presence of variant alleles in a sample having a sequence very closely related to the true target in the case of a single nucleotide polymorphism (SNP).

[0072] As used herein, the term "primer dimer" refers to a template-independent non-specific amplification product that is thought to result from primer extension in which another primer functions as a template. Primer dimers often appear to be two primers, i.e., concatamers of the dimer, but concatamers of more than two primers can also occur. The term "primer dimer" is used herein to comprehensively encompass template-independent non-specific amplification products.

[0073] As used herein, the term "reaction mixture" refers to a solution containing the reagents necessary to carry out a given reaction. An "amplification reaction mixture", which refers to a solution containing the reagents necessary to carry out an amplification reaction, typically contains oligonucleotide primers and DNA polymerase or ligase in a suitable buffer. A "PCR reaction mixture" typically contains oligonucleotide primers, DNA polymerase (most typically, a thermostable DNA polymerase), dNTPs and divalent metal cations in a suitable buffer. A reaction mixture is termed complete if it contains all of the reagents necessary to enable the reaction, and incomplete if it contains only some of the necessary reagents. It will be understood by those skilled in the art that the reaction components are usually stored as separate solutions, each containing a portion of the total components, for reasons of convenience, storage stability or use-dependent adjustment of the component concentrations, and that the reaction components are combined prior to the reaction to make up the complete reaction mixture. Furthermore, it will be understood by those skilled in the art that the reaction components are packaged separately for commercialization, and that useful commercially available kits may contain some portion of the reaction components, including the blocked primers of the present invention.

[0074] As used herein, the terms "non-activated" or "inactivated" refer to a primer or other oligonucleotide that cannot participate in a primer extension reaction or ligation reaction because either DNA polymerase or DNA ligase cannot interact with its oligonucleotide for its intended purpose. In some embodiments, when the oligonucleotide is a primer, the primer is blocked at or near the 3' end so as to prevent primer extension, resulting in a non-activated state. When a particular group is attached to or near the 3' end of the primer, DNA polymerase cannot bind to the primer and extension cannot occur. However, a non-activated primer can hybridize to a substantially complementary nucleotide sequence.

[0075] As used herein, the term "activated" refers to a primer or other oligonucleotide that can participate in a reaction with a DNA polymerase or DNA ligase. A primer or other oligonucleotide becomes activated after hybridizing to a substantially complementary nucleic acid sequence, is cleaved, and generates a functional 3' or 5' end capable of interacting with a DNA polymerase or DNA ligase. For example, when the oligonucleotide is a primer and the primer hybridizes to a template, the 3'-blocking group can be removed from the primer by a cleaving enzyme so that, for example, a DNA polymerase can bind to the 3' end of the primer and promote primer extension.

[0076] As used herein, the terms "cleavage domain" or "domain that cleaves" are synonymous and refer to the region located between the 5' and 3' ends of a primer or other oligonucleotide that is recognized by a cleavage compound, such as a cleaving enzyme, that cleaves the primer or other oligonucleotide. For the purposes of the present invention, the cleavage domain is designed such that the primer or other oligonucleotide is cleaved only when hybridized to a complementary nucleic acid sequence and not when single-stranded. The cleavage domain or the sequence adjacent to the cleavage domain may include a) a portion that prevents or inhibits the extension or ligation of the primer or other oligonucleotide by a polymerase or ligase, b) a portion that increases discrimination for detecting a mutant allele, or c) a portion that suppresses an undesired cleavage reaction. One or more such portions can be included in the cleavage domain or the sequence adjacent to the cleavage domain.

[0077] As used herein, the term "RNase H cleavage domain" is a type of cleavage domain containing one or more ribonucleic acid residues or alternative analogs that provide a substrate for RNase H. The RNase H cleavage domain can be located anywhere within a primer or oligonucleotide, and preferably is located at or near the 3' or 5' terminus of the molecule.

[0078] An "RNase H1 cleavage domain" generally contains at least three contiguous RNA residues. An "RNase H2 cleavage domain" can contain one RNA residue, a sequence of continuously linked RNA residues, or RNA residues separated by DNA residues or other chemical groups. For example, an RNase H2 cleavage domain can contain, among other things, 2'-fluoronucleoside residues.

[0079] As used herein, the term "cleavage compound" or "cleaving agent" refers to any compound that can recognize a cleavage domain within a primer or other oligonucleotide and selectively cleave the oligonucleotide based on the presence of the cleavage domain. The cleavage compounds utilized in the present invention selectively cleave a primer or other oligonucleotide containing a cleavage domain only when hybridized to a substantially complementary nucleic acid sequence, and do not cleave a primer or other oligonucleotide when single-stranded. The cleavage compound cleaves a primer or other oligonucleotide within or adjacent to the cleavage domain. As used herein, the term "adjacent" means that the cleavage compound cleaves a primer or other oligonucleotide at either the 5' or 3' terminus of the cleavage domain. The preferred cleavage reaction in the present invention generates 5'-phosphate and 3'-OH groups.

[0080] In a preferred embodiment, the cleavage compound is a "cleavage enzyme". A cleavage enzyme can recognize a cleavage domain when a primer or other oligonucleotide is hybridized to a substantially complementary nucleic acid sequence, but does not cleave the complementary nucleic acid sequence (i.e., introduces a single-strand break within the double strand). A cleavage enzyme also does not cleave a primer or other oligonucleotide containing a cleavage domain when it is single-stranded. Examples of cleavage enzymes are RNase H enzymes and other nick-forming enzymes.

[0081] As used herein, the term "nick-forming" refers to cleaving only one strand of the double-stranded portion of a completely or partially double-stranded nucleic acid. The position at which a nick is formed in a nucleic acid is referred to as the "nick-forming site" (NS). A "nick-forming agent" (NA) is an agent that forms a nick in a partially or completely double-stranded nucleic acid. A nick-forming agent can be an enzyme or any other compound or composition. In certain embodiments, a nick-forming agent can recognize a specific nucleotide sequence of a completely or partially double-stranded nucleic acid and cleave only one strand of the completely or partially double-stranded nucleic acid at a specific position (i.e., NS) with respect to the position of the recognition sequence. Such nick-forming agents (referred to as "sequence-specific nick-forming agents") include, but are not limited to, nick-forming endonucleases (e.g., N.BstNB1).

[0082] Accordingly, a "nick-forming endonuclease" (NE), as used herein, refers to an endonuclease that recognizes the nucleotide sequence of a completely or partially double-stranded nucleic acid molecule and cleaves only one strand of the nucleic acid molecule at a position specific with respect to the recognition sequence. In such cases, the entire sequence from the recognition site to the cleavage point constitutes the "cleavage domain".

[0083] As used herein, the term "blocking group" refers to a chemical moiety that is attached to a primer or other oligonucleotide such that an amplification reaction does not occur. For example, primer extension and / or DNA ligation do not occur. When the blocking group is removed from the primer or other oligonucleotide, the oligonucleotide can participate in the assay (such as PCR, ligation, sequencing, etc.) for which it was designed. Thus, a "blocking group" can be any chemical moiety that inhibits recognition by polymerase or DNA ligase. The blocking group can be incorporated into the cleavage domain, but is generally located either 5' or 3' to the cleavage domain. A blocking group can be composed of more than one chemical moiety. In the present invention, the "blocking group" is typically removed after hybridization of the oligonucleotide to its target sequence.

[0084] The term "blocked cleavage primer" refers to a primer that is inactive or inactivated with respect to priming DNA synthesis from polymerase due to the presence of a blocking group at or near the 3'-end of the primer. A blocked cleavage primer can be converted to a competent primer by a cleavage compound or agent (such as a cleavage enzyme) that results in an active primer or an activated primer by removing the blocking group at or near the 3'-end of the primer.

[0085] The RDDDDx blocked cleavage primer (also known as the "generation 1" or "Gen 1" blocked cleavage primer) refers to a blocked cleavage primer having the sequence RDDDDx (where R is an RNA base, D is a DNA base, and x is a C3 spacer group) at its 3'-end.

[0086] The RDxxD block-cleavable primer (also known as the "Generation 2" or "Gen 2" block-cleavable primer) refers to a block-cleavable primer having the sequence RDxxD at its 3' end, where R is an RNA base, D is a DNA base, and x is a C3 spacer group.

[0087] The term "fluorescent generating probe" refers to either a) an oligonucleotide to which a fluorophore and a quencher are attached and optionally a minor groove binder is attached, or b) a DNA binding reagent such as SYBR (trademark) Green dye.

[0088] The term "fluorescent label" or "fluorophore" refers to a compound having a maximum fluorescence emission of about 350 to 900 nm. A wide variety of fluorophores can be used, including but not limited to: 5-FAM (also referred to as 5-carboxyfluorescein; spiro(isobenzofuran-1(3H),9'-(9H)xanthene)-5-carboxylic acid, also referred to as 3',6'-dihydroxy-3-oxo-6-carboxyfluorescein); 5-hexachloro-fluorescein; ([4,7,2',4',5',7'-hexachloro-(3',6'-dipivaloyl-fluoresceinyl)-6-carboxylic acid]); 6-hexachloro-fluorescein; ([4,7,2',4',5',7'-hexachloro-(3',6'-dipivaloylfluoresceinyl)-5-carboxylic acid]); 5-tetrachloro-fluorescein; ([4,7,2',7'-tetra-chloro-(3',6'-dipivaloylfluoresceinyl)-5-carboxylic acid]); 6-tetrachloro-fluorescein; ([4,7,2',7'-tetrachloro-(3',6'-dipivaloylfluoresceinyl)-6-carboxylic acid]); 5-TAMRA (5-carboxytetramethylrhodamine); xanthylium, 9-(2,4-dicarboxyphenyl)-3,6-bis(dimethyl-amino); 6-TAMRA (6-carboxytetramethylrhodamine); 9-(2,5-dicarboxyphenyl)-3,6-bis(dimethylamino); EDANS (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid); 1,5-IAEDANS (5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid); Cy5 (indodicarbocyanine-5); Cy3 (indo-dicarbocyanine-3); and BODIPY FL (2,6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid); Quasar®-670 dye (Biosearch Technologies); Cal Fluor® Orange dye (Biosearch Technologies); Rox dye; Max dye (Integrated DNA Technologies), and appropriate derivatives thereof.

[0089] As used herein, the term "quencher" refers to a molecule or a portion of a compound that, when bound or in proximity to a fluorescent donor, can reduce the emission from that donor. Quenching can occur by any of several mechanisms, such as fluorescence resonance energy transfer, photoinduced electron transfer, paramagnetic enhancement of intersystem crossing, Dexter exchange coupling, and exciton coupling, such as formation of a dark complex. Fluorescence is "quenched" when the fluorescence emitted by a fluorophore is reduced by at least 10%, for example, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9% or more as compared to the fluorescence in the absence of a quencher. Several commercially available quenchers are known in the art, including, but not limited to, DABCYL, Black Hole™ Quenchers (BHQ-1, BHQ-2, and BHQ-3), Iowa Black® FQ, and Iowa Black® RQ. These are so-called dark quenchers. These do not have intrinsic fluorescence in the wavelength range of 300 to 900 nm, and in fact, eliminate the background problems seen with other quenchers that are intrinsically fluorescent, such as TAMRA.

[0090] As used herein, the term "ligation" refers to the covalent joining of two polynucleotide ends. In various embodiments, ligation involves the covalent joining of the 3' end of a first polynucleotide (acceptor) and the 5' end of a second polynucleotide (donor). Ligation results in the formation of a phosphodiester bond between the polynucleotide ends. In various embodiments, ligation can be mediated by any enzyme, chemical, or process that results in the covalent joining of polynucleotide ends. In certain embodiments, ligation is mediated by a ligase enzyme.

[0091] As used herein, "ligase" refers to an enzyme capable of covalently bonding the 3'-hydroxyl group of one polynucleotide to the 5'-phosphate group of a second polynucleotide. Examples of ligases include E. coli DNA ligase, T4 DNA ligase, and the like.

[0092] The ligation reaction can be used in DNA amplification methods, for example, in the "ligase chain reaction" (LCR) (also referred to as the "ligase amplification reaction" (LAR), see Barany, Proc. Natl. Acad. Sci., 88:189 (1991); and Wu and Wallace, Genomics 4:560 (1989), which are incorporated herein by reference). In LCR, a mixture of four oligonucleotides, two adjacent oligonucleotides (which individually hybridize to one strand of the target DNA) and a complementary set of two adjacent oligonucleotides (which hybridize to the opposite strand), is combined, and DNA ligase is added to the mixture. In the presence of the target sequence, DNA ligase covalently bonds each of the sets of hybridized molecules. Importantly, in LCR, two oligonucleotides are ligated to each other only when they base pair with a gapless sequence. Short segments of DNA are amplified by repeated cycles of denaturation, hybridization, and ligation. Mismatches at the junction between adjacent oligonucleotides inhibit ligation. As with other oligonucleotide ligation assays, this property allows LCR to be used for discrimination between variant alleles such as SNPs. LCR is also used in combination with PCR to obtain improved detection of single-base changes. See Segev, International Publication No. WO 90 / 01069 (1990).

[0093] The term "unmodified form" is used herein for the purpose of defining a host cell-specific codon-optimized Taq DNA polymerase gene that expresses Taq DNA polymerase in a host cell, in the context of Taq DNA polymerase. The term "unmodified form" refers to a functional DNA polymerase having the amino acid sequence of a naturally occurring polymerase. The term "unmodified form" includes recombinant forms of functional DNA polymerases.

[0094] The term "mutant" means, in the context of the disclosed DNA polymerases, a polypeptide, typically recombinant, that contains one or more amino acid substitutions compared to the corresponding naturally occurring or unmodified form of the DNA polymerase.

[0095] "Recombinant", as used herein, refers to an amino acid sequence or nucleotide sequence that has been intentionally modified by recombinant methods. The term "recombinant nucleic acid" as used herein generally means a nucleic acid that has been generated in vitro, usually in a form not found naturally, by manipulation of nucleic acids by endonucleases. Thus, a linear isolated mutant DNA polymerase nucleic acid, or an expression vector formed in vitro by ligating DNA molecules that normally do not associate, are both considered recombinants for the purposes of the present invention. When a recombinant nucleic acid is made and reintroduced into a host cell, it replicates non-recombinantly, i.e., using the in vivo cellular machinery of the host cell rather than in vitro manipulation; however, such a nucleic acid is understood to be considered a recombinant for the purposes of the present invention even though it replicates non-recombinantly after being recombinantly generated. A "recombinant protein" is a protein made using recombinant techniques, i.e., through the expression of the recombinant nucleic acids described above.

[0096] A nucleic acid is "operably linked" when 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.

[0097] The term "vector" typically refers to a double-stranded DNA fragment into which an exogenous DNA fragment may be inserted. Alternatively, the vector may be, for example, plasmid-derived. The vector contains a "replicon" polynucleotide sequence that promotes autonomous replication of the vector within a host cell. Exogenous DNA is defined as heterologous DNA that is not normally present in the host cell and that, for example, encodes a molecule for replicating the vector, a selectable or screenable marker, or a transgene. Vectors are used to transport exogenous or heterologous DNA into a suitable host cell. Once inside the host cell, the vector can replicate independently of or simultaneously with the host's chromosomal DNA, generating multiple copies of the vector and its inserted DNA. In addition, the vector can contain the necessary factors to enable transcription of the inserted DNA into mRNA molecules or to replicate multiple copies of RNA from the inserted DNA. Some expression vectors further contain sequence elements adjacent to the inserted DNA that extend the half-life of the expressed mRNA and / or enable translation of the mRNA into protein molecules. Thus, large numbers of molecules of mRNA and polypeptide encoded by the inserted DNA can be rapidly synthesized.

[0098] The term "affinity tag" refers to a short polypeptide sequence that enables the detection and / or selection of a polypeptide sequence. For the purposes of the present disclosure, a recombinant gene encoding a recombinant DNA polymerase may include an affinity tag. Specifically, an affinity tag is typically located at either the N-terminus or the C-terminus of the coding sequence of the DNA polymerase through the use of recombinant techniques. Exemplary affinity tags include, among others, polyhistine (e.g., (His 6 )), glutathione-S-transferase (GST), HaloTag®, AviTag, calmodulin tag, polyglutamic acid tag, FLAG tag, HA tag, Myc tag, S tag, SBP tag, Softag3, V5 tag, Xpress tag.

[0099] The term "host cell" refers to both single-celled prokaryotes and eukaryotes (e.g., bacteria, yeast, and actinomycetes) when grown in cell culture, as well as single cells derived from higher plants or animals. Exemplary suitable host cells include E. coli, S. cerevisiae, and S. frugiperda.

[0100] 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 may include additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences (which does not include additions or deletions). 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, determining the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0101] The terms "identical" or "identity," in reference to two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same. When sequences are compared and aligned to maximize correspondence over a comparison window, or a specified region measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection, the sequences are "substantially identical" to each other if they have a specified percentage of nucleotide 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). These definitions also refer to the complements of test sequences. In some cases, identity exists over a region of at least about 50 nucleotides, or more typically over a region of 100 to 500 or 1000 or more nucleotides in length.

[0102] The terms "similarity" or "percent similarity," in reference to two or more polypeptide sequences, refer to two or more sequences or subsequences that have amino acid residues that are the same or similar as defined by conserved amino acid substitutions, when the sequences are compared and aligned to maximize correspondence over a comparison window, or a specified region measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection, at a specified ratio (e.g., 60% similarity over a specified region, in some cases 65%, 70%, 75%, 80%, 85%, 90% or 95% similarity). Sequences are "substantially similar" to each other if 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% are similar to each other. In some cases, this similarity exists over a region of at least about 50 amino acids, more typically over a region of at least about 100 to 500 or 1000 or more amino acids in length.

[0103] For array comparison, typically, one array is used as a reference array and a test array is compared to that reference array. When using an array comparison algorithm, the test and reference arrays are input into a computer, partial array coordinates are specified as needed, and array algorithm program parameters are specified. Default program parameters are typically used, or alternative parameters can be specified. Subsequently, based on the program parameters, the array comparison algorithm calculates the percent of array identity or similarity of the test array to the reference array.

[0104] A "comparison window" as used herein includes any one segment of a number of contiguous positions selected from the group consisting of from 20 to 600, generally about 50 to about 200, and more generally about 100 to about 150, where after optimally aligning the two arrays, the arrays may be compared to a reference array of the same number of contiguous positions. Methods of aligning arrays for comparison are well known in the art. Optimal alignment of arrays for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math., 2:482, 1970), the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol., 48:443, 1970), the similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA, 85:2444, 1988), computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.) or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 Supplement)).

[0105] Suitable algorithms for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, 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 for Biotechnology Information in the National Library of Medicine of the National Institutes of Health (http: / / www.ncbi.nlm.nih.gov / ) and on the Internet and in public databases.

[0106] Rational Design of Taq DNA Polymerase Mutants As outlined above, many strategies have been developed to improve the discrimination of polymerase chain reactions that selectively amplify specific nucleic acid sequences, based on the identity of single nucleotide polymorphisms with the modifications most frequently introduced into primers in the past, while using naturally occurring DNA polymerases. The ability of DNA polymerases to discriminate between matches and mismatches at the 3' end of primer nucleic acids is limited and varies greatly depending on the identity of the specific base pairs present. Another strategy for improving the selectivity of PCR amplification is to modify the properties of DNA polymerases to improve discrimination between primers that are a match to the template nucleic acid and primers that have a terminal mismatch to the template nucleic acid. The present invention provides DNA polymerase mutants with improved mismatch discrimination for base pairing at the 3' end of primers, resulting in improved specificity of the subsequent amplification reaction.

[0107] The rhPCR method employs block-type cleavage primers that must be unblocked by the action of RNase H2 before amplification can begin. The enzymatic unblocking step typically requires cleavage at a single internal RNA base within the primer, which is located at the SNP site. RNase H2 cleaves the RNA on the 5' side, leaving a primer with a 3'-hydroxyl that can prime PCR. Cleavage by RNase H2 occurs with high efficiency when the primer matches the template and with low efficiency when a mismatch is present due to the SNP. Thus, the matched template is amplified with higher efficiency than the mismatched template. The main mechanism that allows amplification of the mismatched template begins with alternative cleavage of the substrate (i.e., the block-type cleavage primer) on the 3' side of the RNA residue, which leads to inappropriate priming, retention of the RNA base in the primer, and conversion of the PCR product to the primer sequence in the presence of a mismatch, and is then thought to be faithfully replicated as a match in subsequent PCR cycles. The fidelity of the rhPCR process can be improved through the improvement of DNA polymerase that limits the ability to initiate DNA synthesis from primers with 3'-RNA residues. The present invention provides a DNA polymerase mutant with a reduced ability to initiate DNA synthesis from a 3'-RNA-containing primer, resulting in improved specificity of the next amplification reaction.

[0108] The present invention includes novel DNA polymerase mutants having improved discrimination for base identity at the 3' end of the primer nucleic acid and / or DNA polymerase mutants with reduced priming efficiency from 3' RNA residues.

[0109] The novel design strategy was developed to rationally design DNA polymerase mutants with improved discrimination at the 3'-terminal base of the primer, compared to the discrimination of natural DNA polymerases, and to limit the ability to initiate DNA synthesis in the presence of a mismatch or an RNA residue. The method described herein employed Taq DNA polymerase as the parental enzyme; this approach can be applied to other DNA polymerases, particularly when the crystal structure is known. The design strategy includes a first component based on the theoretical analysis of biophysical, biochemical, and genetic information regarding natural DNA polymerases and, to a lesser extent, related polymerases with different amino acid sequences. The design strategy includes a second component based on the molecular biological and biochemical analysis of known genetically engineered mutant polymerases to assist as a guide in predicting the effects of novel mutations in attempts to rationally engineer new properties into the mutant polymerase, in this case to improve 3'-nucleotide discrimination.

[0110] In the first step, the enzymatic reaction mechanism of Taq DNA polymerase was analyzed based on published structure-activity relationship (SAR) studies of mutations. When known, it was correlated with the protein structure, and when not known, it was predicted using molecular dynamics simulations. The enzymatic catalytic mechanism has been reported in the prior art (Patel, P.H. et al., J. Mol. Biol., 2001, 308: 823-837; Li, Y. & Waksman, G., Protein Sci 2001, 10: 1225-1233). Amino acid residues located at the C-terminus from position 424 to 832 are involved in the primer extension catalytic activity of the protein. Taq DNA polymerase binds to the primer-template duplex for forming a two-component complex. This allows the incoming substrate dNTP to bind in a pocket at the 3'-end of the primer, forming an open ternary complex. When the dNTP is complementary to the template nucleotide, the active site changes conformation. In this case, the α-helix formed by residues 659 to 671 rotates towards that site, and the template base rotates towards the incoming dNTP, promoting the formation of Watson-Crick base pairs. This event "closes" the ternary complex, bringing the α-phosphate group of the dNTP close to the 3'-OH group of the primer. The oxygen of this hydroxyl group makes a nucleophilic attack on the phosphorus, forming a covalent bond and releasing pyrophosphate. The catalytic activity of Taq DNA polymerase requires the presence of magnesium ions, which are assumed to facilitate the deprotonation of the attacking hydroxyl group.

[0111] One criterion for the rational design of Taq DNA polymerase mutants with improved 3'-nucleotide discrimination is to provide novel polymerase enzyme mutants that have normal or near-normal polymerase processing capabilities compared to native DNA polymerases. For this reason, the first step in the analysis serves to narrow the sequence space of the amino acids available for changes that should not impair the core enzyme function. For example, residues D610, D785, and E786 form the catalytic core. These carboxylic acid groups are assumed to bind to divalent metal ions and, in turn, to bind to and stabilize the incoming dNTP and the terminal nucleotide of the primer. Mutations of these three essential residues can inactivate the polymerase. Thus, mutant polymerases containing changes in residues D610, D785, and E786 were excluded from consideration. Similarly, mutants that affect the fidelity of complementary base recognition, such as residues that promote opening to triple complex formation closed with complementary dNTPs, and template bases were excluded from consideration.

[0112] The additional criterion for the first stage of the analysis was to identify the amino acid residues of the polymerase in the vicinity of the 3'-terminal nucleotide of the primer. For this purpose, the atomic three-dimensional structure of Taq DNA polymerase available from the prior art (Eom, S.H. et al., Nature, 1996, 382:278-281; Li, Y. et al., EMBO J., 1998, 17:7514-7525; Doublie, S. et al., Structure 1999, 7:R31-R35; Li, Y. et al., Protein Sci 1998, 7:1116-1123) was selected for analysis. The structure was downloaded from the Protein Data Bank (H.M. Berman et al., Nucleic Acids Research, 2000, 28:235-242). The structures of PDB ID 2KTQ and 3KTQ were thoroughly analyzed to show the open-closed triple complex of a large fragment of Taq DNA polymerase cocrystallized with the primer and the template nucleic acid (Li, Y. et al., EMBO J., 1998, 17:7514-7525). This structure shows the interaction between the position of the 3'-end of the primer at the active site and the major amino acid residues (Figure 1).

[0113] For visualization of the structure, the hydrogen atoms attached to the 2'-carbon are replaced with hydroxyl groups for the primer modified with a 3'-ribonucleotide. These amino acid residues in proximity to the 2'-carbon of the nucleotide at the 3'-end of the primer were selected for further analysis. These amino acid residues are listed in Table 2 and are likely to interact with the 3'-terminal nucleotide of the primer. When primer modifications such as OH are attached to the 2'-carbon atom of the ribose, mutations at these sites can affect the catalytic activity of the polymerase.

[0114]

Table 2

[0115] A further aspect of this criterion relates to an approach for increasing specificity while retaining the catalytic activity of the polymerase. One approach for increasing the specificity of Taq DNA polymerase is to decrease the size of the binding pocket, such that the modified primer will not fit therein. Any additional chemical groups increase the steric volume occupied by the 3'-nucleotide. The active site pocket must be flexible to accommodate the oxygen of the OH group of an additional one or more atoms, e.g., an RNA residue, for effective catalysis and nucleophilic attack. The size of the active site can be decreased by substituting adjacent amino acids with larger amino acids. Further considerations are given to the amino acid properties involved in electrostatic and van der Waals interactions and the ability of these side chains. Amino acids can be classified into groups of positively charged side chains (R, H, K), negatively charged side chains (D, E), uncharged polar side chains (S, T, N, Q), hydrophobic side chains (A, V, I, L, M, F, Y, W) and special side chains (C, G, P). Mutations within a group are conservative and are likely to maintain existing properties, while mutations across groups or within the amino acids of special groups are likely to result in substantial changes in enzyme activity and / or specificity.

[0116] Another approach to increasing the 3'-nucleotide discrimination of Taq DNA polymerase is to employ residue substitutions that reduce the flexibility of the binding pocket. Examples include substitution of amino acid aliphatic side chains with aromatic side chains, which results in a higher energy barrier to change the rotamer conformation. As described above, the residues of the catalytic core are preferably not altered, and residues that are spatially close to the catalytic core are given the greatest care for change. For example, it is proposed herein that the three non-catalytic core residues H784, V783, and R573 in Table 2 be substituted with more or less flexible amino acids while maintaining the general physical characteristics of the side chains. These residues show major interactions with the ribose portion of the primer via a water-mediated hydrogen bond network. Also, R573 binds to the primer base in the minor groove of the primer-template duplex. Using this strategy, mutants ID1 to ID4 were designed (Table 3). The next mutant, ID 5, Q582K, was designed to alter the interaction with the important H784 residue and its position. This can be seen from the known crystal structure where Q582 is located on the opposite side of H784 from the oligonucleotide primer. Substitution of Q582H can shift H784 towards the nucleotide of the terminal primer, resulting in a more constrained binding pocket. Also, the interaction of residue 582 with the second nucleotide from the end may also be affected.

[0117] In addition, the residues that stack on the incoming dNTP molecule can affect the size of the binding pocket in the polymerase active site. For example, substitution of F667 at this position is known to change the selectivity for the incoming dNTP. For example, the F667Y substitution significantly improves the incorporation of dideoxynucleoside triphosphates by Taq DNA polymerase (Tabor, S. & Richardson, C. C., Proc. Nat. Acad. Sci. USA, 1995, 92: 6339-6343), which is a useful property of DNA polymerases employed in Sanger method terminator DNA sequencing. Mutant ID6 increases the size of the aromatic side chain of F667 from phenylalanine to tryptophan in an attempt to push the dNTP towards the primer-terminal ribonucleotide and reduce the ability of the binding pocket to accommodate the 2'-hydroxyl group, thereby biasing this mutant containing the 3'-RNA residue. Mutant ID7 was designed based on a similar concept framework. H639 interacts with the amino acid of F667, and the H639W mutant may also push F667 towards the incoming dNTP.

[0118] Additional mutants were thought to be able to effectively reduce the size of the polymerase binding pocket. Mutants ID8 to 16 were designed from negative inferential analysis based on published studies of "relaxed specificity" mutant polymerases. It has been reported that mutants can alter polymerase specificity towards the ribose of incoming dNTPs. Prior art in which Taq DNA polymerase mutants have been reported evolved from large random libraries either through selection or screening. Chen et al. reported a mutation that enables Taq DNA polymerase to combine dNTP with a large substitution on the ribose 3'-carbon atom (Chen, F. et al., Proc. Nat. Acad. Sci. USA, 2010, 107: 1948-1953). This residue was found to be important because it also interacts with F667. The substitution L616A reduces specificity by giving the phenylalanine residue more space. Mutant ID8 (L616M) was designed to have the opposite effect. The methionine substitution may slightly increase steric hindrance at this site compared to leucine. This limitation may also reduce the likelihood that the active site can accommodate additional substituents in dNTP or primer nucleotides, which can reduce the activity of primers with mismatches to primers containing 3'-RNA or templates, and probably occupy more space than primers with complete mismatches to template nucleic acids.

[0119] A framework of a similar concept was applied to design the Taq DNA polymerase mutant ID9. The mutations I614E and E615G were reported to relax the active site pocket so that the polymerase could extend primers using 2'-O-methyl ribonucleoside triphosphates (Fa, M. et al., J. Am. Chem. Soc., 2004, 126: 1748-1754). The nature of these mutations is essentially a shift of glutamic acid from residue 615 to 614. Therefore, the reverse shift, E615L and L616E, can impose constraints at the active site and generate a Taq DNA polymerase mutant that does not accept ribonucleotide residues.

[0120] Another approach to increasing 3'-nucleotide discrimination is to focus on the site of interest identified in Taq DNA polymerase studies that reported amino acid substitutions (i.e., mutations that improve replication fidelity) that increase the fidelity of base selection and decrease the incorporation of mismatched base pairs. These changes may similarly potentially affect the selectivity of Taq DNA polymerase with respect to the modification of the terminal primer nucleotide. One position reported to improve fidelity involves the F667 residue and adjacent amino acids (Suzuki, M. et al., J. Biol. Chem., 2000, 275:32728-32735). Another potential site of interest includes residues 782 to 784 adjacent to the essential aspartic acid residue (Strerath, M. et al., Chem. Bio. Chem., 2007, 8:395-401). Mutants ID10 through ID13 were designed to alter the amino acid characteristics at these positions. F667 affects specificity because it interacts with the terminal base of the primer and stacks on the base of the incoming dNTP; this residue is in the O-helix. Residues I665 and A661 are located on the opposite side of the helix. Mutations to larger amino acids from themselves (A661E, I665W) can move the O-helix toward the active site, limit the size of the active pocket, and limit the ability of the polymerase to accept mismatched bases or RNA residues (mutants ID10: A661E, I665W, F667L).

[0121] In addition, data obtained from mutagenesis studies of different polymerases can be used to help select positions for modification, but the use of this data is more difficult due to the absence of crystal structures or the possibility of different effects between polymerases. Polymerase I from Escherichia coli ("E. coli Pol") exhibits a somewhat similar structure in the active site and maintains the same essential catalytic residues when compared to Taq DNA polymerase. Both protein sequences show a high degree of homology (Li, Y. et al., EMBO J., 1998, 17:7514-7525). Thus, mutations reported for Escherichia coli DNA polymerase were also considered by extrapolating the amino acid positions to the corresponding positions in Taq DNA polymerase. For example, the triple amino acid substitution Q879P, V880L, H881Q improved the base fidelity of E. coli DNA polymerase (Summerer, D. et al., Angew. Chem. Int. Ed., 2005, 44:4712-4715). The substitutions in mutant ID14 include substitutions at Q782, V783, H784 in the Taq DNA polymerase active site that appear to correspond to the triple of these E. coli residues.

[0122] Several additional substitutions in E. coli DNA polymerase are known and decrease or increase the specificity of primer extension (Minnick, D.T. et al., J. Biol. Chem., 1999, 274:3067-3075). Mutants Q849A and R754A improved fidelity. These have positions equivalent to Q754 and R659 in the Taq DNA polymerase active site, respectively. Arginine 659 has a major impact on the selection of the base complementary to the template base. This seems to be a common feature in the polymerase A family. For example, in Thermotoga neapolitana polymerase I, the equivalent residue is R722. Mutation of this residue to histidine increases the fidelity of this polymerase (Yang, S.W. et al., Nucleic Acids Res., 2002, 30:4314-4320). These two residues were also selected for study (mutants ID15 and 16 in Table 3). Mutant ID17 represents a combination of mutations studied in mutants ID2 and 3. Mutant ID18 represents the modification of triple mutant ID14 (Q782P, V783L, H784Q) reduced to a double mutant (V783L, H784H) by elimination of the Q782 mutation; substitution of the less flexible P for the Q residue may cause significant structural perturbations that alter function, and mutant ID18 can avoid this problem. Initial tests indicated that mutations at positions beyond 1 at the H784 position showed improved mismatch discrimination, suggesting that this position is generally important for determining primer specificity. Therefore, an extensive study of amino acid substitutions at this site was conducted, including mutants ID19-36.

[0123]

Table 3

[0124] The second component of the design strategy involves the molecular biological and biochemical analysis of genetically engineered Taq DNA polymerase mutants to identify novel enzymes with improved 3'-nucleotide discrimination. This requires the expression of native Taq DNA polymerase and a series of designed mutants in a suitable host such as the bacterium E. coli. To maximize expression, the codons of the native gene sequence encoding Taq DNA polymerase are altered and optimized for expression in E. coli using the standard codon usage frequency table for this organism (see Codon usage tabulated from the international DNA sequence databases: status for the year 2000, Nakamura, Y., Gojobori, T. and Ikemura, T., (2000), Nucleic Acids Res., 28:292). Codon optimization does not alter the amino acid sequence of the expressed protein. The recombinant form of the codon-optimized gene encoding the unmodified Taq DNA polymerase peptide is incorporated into a plasmid vector as an artificial gene made from synthetic oligonucleotides using standard methods and cloned (Example 1). The plasmid vector for this purpose can be any plasmid vector routinely available in the art. The synthetic recombinant forms of a series of specified desired Taq DNA polymerase mutants (Table 3, Mutant IDs 1-36) were prepared by site-directed mutagenesis (SDM) of the previously incorporated codon-optimized recombinant native Taq DNA polymerase using techniques well known to those skilled in the art (Example 1). Unmodified and mutant Taq DNA polymerases were prepared from E. coli host cells after introduction of an expression vector containing the corresponding recombinant forms of the gene operably linked to appropriate transcriptional and translational control elements.

[0125] The enzyme properties of unmodified Taq DNA polymerase and mutant Taq DNA polymerase were evaluated for primer extension assay, thermal stability, PCR assay, allele-specific PCR assay, ability to use primers with 3'-ribonucleotides, and their suitability for use in the rhPCR assay. The mutant Taq DNA polymerase exhibited one of four categories of enzyme properties: (1) inactivated polymerase activity; (2) normal polymerase activity; (3) improved 3'-nucleotide discrimination activity but with decreased activity (e.g., decreased throughput); and (4) improved 3'-nucleotide discrimination and normal or near-normal activity (e.g., throughput equivalent to that of the native polymerase).

[0126] Mutant Taq DNA polymerases having a fourth category of enzyme characteristics exhibit equivalent or increased 3'-mismatch discrimination (i.e., equivalent or increased performance in standard primer extension assays and allele-specific PCR assays when compared to wild-type Taq DNA polymerase); increased 3'-nucleotide discrimination (i.e., decreased primer extension activity from templates containing RNA primers when compared to wild-type Taq DNA polymerase), and increased low-frequency allele discrimination (e.g., improved specificity in rhPCR assays when compared to wild-type Taq DNA polymerase). These mutant Taq DNA polymerases contain mutations at one of the following residue positions: (1) A661E; I665W; F667L triple substitution mutant peptide (mutant ID10 in Table 3); (2) V783F single substitution mutant peptide (mutant ID2 in Table 3); H784Q single substitution mutant peptide (mutant ID3 in Table 3); and V783L; H784Q double substitution mutant peptide (mutant ID18 in Table 3), H784A, single substitution mutant peptide (mutant ID20 in Table 3); H784S, single substitution mutant peptide (mutant ID21 in Table 3); H784T, single substitution mutant peptide (mutant ID22 in Table 3); H784V, single substitution mutant peptide (mutant ID24 in Table 3); H784I, single substitution mutant peptide (mutant ID26 in Table 3); H784M, single substitution mutant peptide (mutant ID27 in Table 3); H784F, single substitution mutant peptide (mutant ID29 in Table 3); and H784Y single substitution mutant peptide (mutant ID30 in Table 3).

[0127] Thus, the novel design algorithm provides a robust approach for predicting mutant DNA polymerases with improved 3'-nucleotide discrimination, as considered by polymerase activity in allele-specific PCR, low-frequency allele detection assays, and rhPCR assays that utilize a template regardless of the presence or absence of 3'-RNA residues in the primer. Specifically, residues V783 and H784 have been identified as important residues that affect the polymerase's ability to examine the state of the 3'-base of the primer oligonucleotide (e.g., whether this residue matches or does not match the template, and / or whether this residue is DNA or RNA). The importance of these residues in polymerase function has not been recognized heretofore. In addition to the mutations directly tested in the examples, the present invention also contemplates other amino acid substitutions at these two positions, or double mutations that affect both the V783 and H784 sites.

[0128] The properties of these mutants are further described in the examples presented herein. Importantly, however, the design strategy employed herein provides access to a functional space for novel Taq DNA polymerase mutants that has not been previously recognized or anticipated, or obtained using other approaches (e.g., previous attempts using phylogenetic comparative analysis or random mutagenesis).

[0129] Evaluation of Taq DNA Polymerase Mutants at Residue Positions 783 and 784 The present disclosure demonstrates that mutations at residue positions 783 and / or 784 result in active Taq DNA polymerase mutants having increased template discrimination activity as compared to unmodified Taq DNA polymerase. Accordingly, the entire sequence space including all possible single amino acid substitutions at the individual positions of 783 or 784, as well as all possible double amino acid substitutions at both positions of 783 and 784, is within the scope of the present disclosure related to Taq DNA polymerase. Accordingly, active Taq DNA polymerase mutants selected from a set of mutants including 19 single residue 783 mutants, 19 single residue 784 mutants (Table 3, mutant IDs 19 - 30) and 361 double residue 783 / 784 mutants having increased template discrimination activity are within the scope of the present disclosure.

[0130] Since Taq DNA polymerase is a thermostable enzyme, one easy approach to screening a collection of 399 single and double replacement mutants at residue positions 783 and 784 is to perform a PCR assay using pre-treated samples encoding candidate Taq DNA polymerase mutant enzymes. The samples can be selected individual colonies or corresponding microcultures (e.g., 50 μL to 1.0 mL cultures) obtained from individual colonies transformed with recombinant DNA expressing the desired recombinant Taq DNA polymerase mutant gene. The pre-treatment regimen can include the step of pre-incubating the sample at 70 - 95 °C, followed by the step of clarifying the supernatant to remove denatured cell debris. For samples expressing thermostable polymerase activity under standard PCR assay conditions, the corresponding recombinant DNA can be further characterized to confirm the sequence of the polymerase protein purified for the desired recombinant Taq DNA polymerase mutant genotype and further biochemical analysis. For the purposes of the present disclosure, Taq DNA polymerase mutants expressing thermostable polymerase activity at a level of at least 0.01, expressed by wild-type Taq DNA polymerase, under equivalent PCR assay conditions can be considered to have thermostable polymerase activity.

[0131] Evaluation of other alternative polymerase candidate mutants that are functionally homologous to Taq DNA polymerase at residue positions 783 and 784 Comparative phylogenetic analysis tools can be used to identify the sequence space of other thermoactive polymerases that have sequence information homologous to unmodified Taq DNA polymerase at residue positions corresponding to V783 and H784. As explained above, a powerful prediction of comparative phylogenetic analysis is that structural sequences shared among DNA polymerases across phylogenetically diverse species are conserved for functional reasons. If the identified V783 / H784 residues of Taq DNA polymerase are invariant in sequence identity among wild-type polymerases from diverse species, that observation strongly supports the conclusion that the properties selected against specific mutations of amino acid substitutions at those positions result in the observed increased template discrimination activity of the engineered Taq DNA polymerase mutants disclosed herein.

[0132] Example 11 provides an exemplary BLAST search using the Taq DNA polymerase sequence encompassing positions V783 and H784 as a comparison window for identifying candidate wild-type DNA polymerases from other species that widely share sequence identity using Taq DNA polymerase. As further detailed in Example 11, the BLAST results revealed that virtually all of the identified DNA polymerases from diverse species maintained Val and His at positions orthologous to V783 and H784 of Taq DNA polymerase. Thus, the BLAST results confirm natural selection against DNA polymerases with increased template discrimination activity, and the disclosed, engineered Taq DNA polymerase mutants having these properties provide strong evidence that they are novel and non-obvious. Each of the identified non-Taq DNA polymerases, like those observed with the engineered Taq DNA polymerase mutants, represents a sequence space in which engineered mutant enzymes can arise and have increased template discrimination activity compared to their respective unmodified counterparts.

[0133] In cases where comparative phylogenetic analysis cannot access the sequence space of more evolutionarily distant organisms, comparative biophysical crystallographic analysis can provide clues to related sequence residues having functions homologous to Taq DNA polymerase V783 and H784. As described above, the Q782, V783, and H784 residue triple of Taq DNA polymerase was selected for analysis based on the corresponding triple amino acid substitutions Q879P, V880L, and H881Q of E. coli DNA polymerase, which have improved base fidelity and an active site structure similar to Taq DNA polymerase. Conversely, based on the significantly increased template discrimination activity of Taq DNA polymerase mutants at V783 and H784 compared to wild-type Taq DNA polymerase, the present invention contemplates that the corresponding substitutions at V880 and H881 of E. coli DNA polymerase have increased template discrimination activity compared to wild-type E. coli DNA polymerase.

[0134] Identification and Characterization of Non-VH Related Polymerase Mutants with Increased Template Discrimination Activity The aforementioned collection of DNA polymerases share sequences that are widely conserved in the region corresponding to V783 and H784 of Taq DNA polymerase (the "VH-related polymerase"). Comparative biophysical analysis is useful for the identification of wild-type DNA polymerases having different amino acid sequences at positions that are functionally homologous to V783 and H784 of Taq DNA polymerase (the "non-VH related DNA polymerase"). This disclosure contemplates engineering mutant polymerases with increased template discrimination activity from these non-VH related DNA polymerases in the same manner as that disclosed for VH-related DNA polymerases. Candidate non-VH residues for site-directed mutagenesis and analysis by the increased template discrimination activity assay include those residues within 0.40 - 0.45 nm of C2' of the primer terminal residue, as revealed in the polymerase:template co-crystal structure.

[0135] Combinations of Site-Specific Taq DNA Mutants with Deletions in the 5'-Exonuclease Domain The present invention discloses a novel Taq DNA polymerase mutant that exhibits improved discrimination of mismatches located at the 3' residue of a primer oligonucleotide and / or discrimination against the presence of RNA residues at the 3' end of a primer oligonucleotide. The improved mismatch discrimination has been described for the "KlenTaq" deletion mutant of Taq DNA polymerase that completely removes the domain having 5'-exonuclease activity (Barnes, W.M. et al., Gene, 112: 29-35, 1992). The combination of the novel mutant of the present invention with the KlenTaq 5'-exonuclease domain deletion results in further improvement in mismatch discrimination (Examples 18 to 22). However, this combination has resulted in a decrease in enzyme activity that can reduce the utility of this family of double mutants. In particular, in some situations, especially when the amplicon size is small and limited processing capacity can be tolerated, the increased discrimination of these mutants has advantages.

[0136] Reaction mixture In another aspect, a reaction mixture is provided that contains a polymerase having increased 3'-nucleotide discrimination activity. The reaction mixture can further contain, for example, reagents for use in nucleic acid amplification methods (e.g., PCR, RT-PCR, rhPCR), DNA sequencing procedures, or DNA labeling procedures. For example, in certain embodiments, the reaction mixture contains a buffer suitable for primer extension reactions. The reaction mixture also contains template nucleic acid (DNA and / or RNA), one or more primer or probe polynucleotides, nucleoside triphosphates (e.g., including deoxyribonucleotides, ribonucleotides, labeled nucleotides, non-conventional nucleotides), salts (e.g., Mn 2+ , Mg 2+) and may contain a label (e.g., a fluorophore). In some embodiments, the reaction mixture further comprises a double-stranded DNA binding dye, such as SYBR Green, or a double-stranded DNA intercalating dye, such as ethidium bromide. In some embodiments, the reaction mixture contains a primer pair that can hybridize to a given polynucleotide template under primer extension conditions, the primer pair including a 5' sense primer or a 3' antisense primer corresponding to the 5' sense primer. In certain embodiments, the reaction mixture further comprises a fluorescent FRET hydrolysis probe, such as a Taqman® or PrimeTime® probe, for detection of the amplified template nucleic acid. In some embodiments, the reaction mixture contains two or more primers that are completely complementary to a single nucleotide polymorphism or multiple nucleotide polymorphisms. In some embodiments, the reaction mixture contains alpha-phosphorothioate dNTPs, dUTP, dITP, and / or labeled dNTPs, such as fluorescein or cyanine dye family dNTPs. In some embodiments, the reaction mixture contains a block-type cleavable primer and RNase H2.

[0137] Kit In another aspect, a kit for use in the primer extension methods described herein is provided. In some embodiments, the kit is intended for ease of use and includes at least one container providing a DNA polymerase of the invention with increased 3'-nucleotide discrimination, according to the present disclosure. It can also include one or more additional containers providing additional reagent(s). Such additional containers can include any reagent or other element recognized by those skilled in the art for use in a primer extension procedure according to the methods described above, including, for example, reagents for use in nucleic acid amplification procedures (e.g., PCR, RT-PCR, rhPCR), DNA sequencing procedures, or DNA labeling procedures. For example, in certain embodiments, the kit further includes a container providing a 5' sense primer capable of hybridizing to a given polynucleotide template under primer extension conditions, or a primer pair including a 5' sense primer and a corresponding 3' antisense primer. In some embodiments, the kit includes one or more containers containing one or more primers that are fully complementary to a single nucleotide polymorphism or multiple nucleotide polymorphisms, where the primers are useful for multiple reactions as described above. In some embodiments, the reaction mixture contains one or more containers containing block-type cleavable primers. In some embodiments, the reaction mixture contains one or more containers containing RNase H2. In other non-mutually exclusive variations, the kit includes one or more containers providing (conventional and / or non-conventional) nucleoside triphosphates. In a specific embodiment, the kit includes alpha-phosphorothioate dNTPs, dUTP, dITP, and / or labeled dNTPs, such as fluorescein or cyanine dye family dNTPs. In yet other non-mutually exclusive embodiments, the kit includes one or more containers providing a buffer suitable for the primer extension reaction. In some embodiments, the kit includes one or more labeled or unlabeled probes. Examples of probes include dual-labeled FRET (fluorescence resonance energy transfer) probes and molecular beacon probes.In another embodiment, the kit contains an aptamer, for example, an aptamer for a hot start PCR assay.

[0138] The present disclosure contemplates kits that provide novel DNA polymerases having increased template discrimination activity. As shown in more detail in the examples, each DNA polymerase can exhibit unique characteristics of increased template discrimination activity. Certain DNA polymerases can exhibit relatively greater 3'-nucleotide discrimination compared to other activities (3'-mismatch discrimination and low-frequency allele discrimination). On the other hand, other DNA polymerases can exhibit relatively greater low-frequency allele discrimination compared to other activities (3'-nucleotide discrimination and 3'-mismatch discrimination), and still other DNA polymerases can exhibit relatively greater low-frequency allele discrimination compared to other activities (3'-nucleotide discrimination and 3'-mismatch discrimination). Thus, the kit can include individual containers of a specific DNA polymerase having an activity profile optimally adapted to a specific increased template discrimination activity for a specific assay platform. Alternatively, the kit can include a single container containing multiple DNA polymerases having activity profiles optimally adapted to adjust the increased template discrimination activity as required for multiple assay platforms.

Examples

[0139] The present invention is further illustrated with reference to the following examples. However, it should be noted that these examples are illustrative only, as are the embodiments described above, and should in no way be construed as limiting the scope of the present invention.

[0140] [Example 1] Cloning and Expression of a Codon-Optimized DNA Polymerase from Thermus aquaticus The amino acid sequence and gene sequence of Taq DNA polymerase are known (Table 1, SEQ ID NOs: 1 and 2). Since codon usage frequencies vary among organisms, the codons of the natural gene sequence encoding Taq DNA polymerase were optimized for expression in E. coli using a standard codon usage frequency table (Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nakamura, Y., Gojobori, T. and Ikemura, T. (2000) Nucleic Acids Res. 28:292); synonymous codon changes were introduced to avoid the repeated use of the same codon throughout a 20-amino acid stretch. The recombinant codon-optimized gene encoding the unmodified Taq DNA polymerase peptide was assembled from synthetic oligonucleotides using standard methods. The gene was made in three fragments, each of which was subcloned into a plasmid vector; the sequences are shown in Table 4 (SEQ ID NOs: 3-5). Sequence identity was confirmed by Sanger DNA sequencing. The three Taq DNA polymerase subfragments were assembled together using the Gibson assembly method (Gibson, D.G. et al., Nature Methods, pages 343-345 (2009)) and cloned into the plasmid expression vector pET-27b(+) using the terminal NdeI and NotI restriction sites to generate the final full-length codon-optimized Taq DNA polymerase gene (referred to as "OptiTaq"). The sequence was confirmed by Sanger DNA sequencing; the sequence is shown in Table 4 (SEQ ID NO: 6). The translated amino acid sequence of the new codon-optimized gene is identical to that of the natural Taq DNA polymerase (Table 1, SEQ ID NO: 1).

[0141]

Table 4

[0142] [Example 2] Generation of Codon-Optimized Taq DNA Polymerase Mutants Eighteen mutant versions of Taq DNA polymerase (Table 3, MUT ID1-18) were generated by site-directed mutagenesis of the cloned OptiTaq codon-optimized Taq DNA polymerase. Specific mutations were introduced into the OptiTaq sequence using the PCR site-directed mutagenesis method (Weiner MP et al., Gene., 151(1-2):119-23 (1994)). Each mutagenesis reaction was carried out in 1× KOD PCR buffer with 10 pmol of two complementary oligonucleotides (Table 5) containing the desired base change, annealed to double-stranded OptiTaq plasmid (20 ng), 5 U KOD DNA polymerase (Novagen-EMD Chemicals, San Diego, CA), 1.5 mM MgSO 4 was employed. The thermal cycling parameters were 16 cycles of 95°C for 3 minutes (95°C for 20 seconds - 55°C for 20 seconds - 70°C for 2.5 minutes), followed by an incubation at 70°C for 4 minutes. After PCR site-directed mutagenesis, the amplification products were treated with 10 U of DpnI (NEB, Ipswish, MA) at 37°C for 1 hour and then inactivated at 80°C for 20 minutes. One-hundred-and-tenth of the digested material was transformed into XL-1 Blue competent bacteria. Bacterial clones were isolated, plasmid DNA was prepared, and individual mutations were confirmed by Sanger DNA sequencing. All mutants were retained in the pET-27b(+) expression vector suitable for expressing recombinant protein in E. coli.

[0143] [Table 5] TIFF2025081508000010.tif194170

[0144] [Example 3] Expression of Recombinant Taq DNA Polymerase The following examples demonstrate the expression of recombinant unmodified Taq DNA polymerase and mutant peptides. The synthetic gene sequences from Examples 1, 2, and 12 were cloned into the pET-27b(+) expression vector (Novagen, EMD Biosciences, La Jolla, CA). This vector places six histidine residues (collectively including the "His-tag") at the carboxy terminus of the expressed peptide, followed by a stop codon. The "His-tag" is well known to those skilled in the art for Ni 2+ affinity chromatography methods to enable rapid and easy purification of recombinant proteins. Alternatively, the synthetic genes were expressed in native form without the His-tag and could be purified using size exclusion chromatography, anion exchange chromatography, or other such methods well known to those skilled in the art.

[0145] BL21(DE3) competent E. coli cells (Novagen) were transformed with approximately 1 ng of each plasmid. Briefly, the plasmid was added to the cells on ice and gently agitated. After incubating on ice for 5 minutes, the cells were heat shocked at 42°C for 30 seconds and then returned to ice for 2 minutes. SOC (80 μL) was added to the transformed cells at room temperature, followed by growth at 37°C for 1 hour with agitation at 250 rpm. The cells were plated (20 μL) on LB / Kan plates (Luria Broth agar plates supplemented with 50 μg / mL kanamycin) pre-warmed to 37°C and left overnight at 37°C. The next morning, one colony was picked and grown to log phase (OD 600It was grown to 0.3 - 0.9 (37 °C, 250 rpm). Next, the cells were introduced with the pET-15b expression (trademark) autoinduction system 1 (Novagen) in Terrific Broth overnight at 37 °C, 250 rpm according to the protocol recommended by the manufacturer. The culture volume was 100 mL for wild-type OptiTaq and 200 mL for the mutants. Growth saturation was reached after 18 hours, and the cultures were pelleted at 10,000 × g for 10 minutes using a Beckman Avanti (trademark) J-25 centrifuge. The pellet (about 6 g) was lysed using 30 mL of BugBuster (registered trademark) protein expression reagent (Novagen), 30 kU of r-lysozyme (trademark) solution (Novagen), and 1500 U of DNase I (Life Technologies, Grand Island, NY) according to the manufacturer's instructions to release soluble proteins and degrade nucleic acids. After centrifugation at 15,000 × g for 20 minutes to remove cell debris, the lysate was heated at 75 °C for 15 minutes to inactivate DNase I and other cellular nucleases. Next, the lysate was spun at 15,000 × g for 20 minutes to precipitate denatured proteins. The heat denaturation step and the centrifugation step provide important purity enhancement of the recombinant enzyme. Both the "total" and "soluble" fractions of the bacterial lysate were analyzed using SDS 4 - 20% polyacrylamide gel electrophoresis at 125 V for 1 hour. Proteins were visualized by 1-hour Coomassie blue staining, followed by 3 - 4 rounds of destaining until the protein bands disappeared.

[0146] The recovered soluble proteins were applied to a Ni column containing His bind resin (Novagen). 2+It was passed through an affinity column and eluted using a buffer containing 200 mM imidazole (200 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH 7.9). Next, the purified protein (about 6 mL) was concentrated to about 200 μL at 3210×g in a Beckman Coulter 6R benchtop centrifuge swinging bucket rotor using an Amicon Ultra-15, PLGC Ultracel-PL Membrane, 10KDa concentrator (EMD Millipore, Billerica, MA) and stored at -20 °C until dialysis. Next, the concentrated protein was dialyzed against the storage buffer (20 mM Tris pH 7.5, 100 mM sodium chloride, 1 mM DTT, 0.1 mM EDTA, 50% glycerol, 0.1% Triton X-100) at 4 °C overnight and then three times for 2 hours each (the ratio of the protein solution to the dialysis buffer was 1000-fold each time). The finally purified protein was stored at -20 °C. Using this protocol, the yield of the automatically introduced 100 mL culture was about 1.2 mg / 67.6 μM / 12,168 pmol of the soluble protein purified for OptiTaq. A similar yield was obtained for the mutant DNA polymerase.

[0147] To determine the protein concentration, samples were examined together with known amounts of BSA (bovine serum albumin) using SDS 4-20% polyacrylamide gel electrophoresis for 1 hour. The protein was visualized by Coomassie blue staining for 1 hour, followed by 3-4 rounds of destaining until the protein bands disappeared. Band intensities were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD).

[0148] To evaluate the purity and quality of the recombinant protein preparations, 500 ng of each recombinant protein (wild-type OptiTaq and each mutant) was separated on a 4-20% SDS-PAGE gel, stained with Coomassie blue, and visualized. All recombinant proteins migrated to appropriate positions on the gel with respect to the protein having a molecular weight of 97.1 kDa. The preparations showed relatively high purity, and several additional species were detected. Gel images are shown in Figures 2A, 2B, 2C, and 2D. Similar gels were run for MUT ID22 (H784T), 24 (H784V), 30 (H784Y), 31 (H784W), and 35 (H784K), and a single band corresponding to the desired recombinant protein was visualized (data not shown).

[0149] The purified enzymes were tested for nuclease contamination using the DNaseAlert™ and RNaseAlert® nuclease detection kits (Integrated DNA Technologies, Coralville, IA) according to the protocols recommended by the manufacturer. All enzyme preparations were found to be free of nuclease contamination.

[0150] [Example 4] Characterization of the Properties of 18 Mutant Taq DNA Polymerases in PCR The 18 mutant Taq DNA polymerase enzymes described in Example 3 were characterized for polymerase activity and the ability to discriminate 3'-RNA residues in primer oligonucleotides.

[0151] The unit activity of the purified wild-type protein was determined by comparing the performance of known amounts of OptiTaq and each mutant in qPCR with that of commercially available non-hot start Taq DNA polymerase, Taq-B DNA polymerase (Enzymatics, Beverly, MA). The quantification cycle value (Cq, the number of amplification cycles at which a positive signal is first detected) and amplification curve shape were analyzed to determine the nanogram amounts at which both enzymes perform similarly within a sub-optimal range for each. Using these nanogram amounts and the known unit value of Taq-B DNA polymerase, relative activity unit values could be extrapolated for all mutant DNA polymerase enzymes having sufficient activity to support PCR.

[0152] The following reaction conditions were employed: 1× qPCR buffer (20 mM Tris pH 8.4, 50 mM KCl, 3 mM MgCl 2 , 0.01% Triton-X100) in a final volume of 10 μL, 800 μM dNTP (200 μM each), 500 nM For primer (Hs HPRT F517, SEQ ID NO: 43), 500 nM Rev primer (Hs HPRT R591, SEQ ID NO: 44), 250 nM probe (Hs HPRT P554, SEQ ID NO: 45), 2×10 3Linear, cloned plasmid template for the copy (HPRT-targ, SEQ ID NO: 46). The amount of DNA polymerase added to each reaction was varied as follows: for wild-type (OptiTaq), the reactions were set up using 10, 1, 0.1, 0.01, 0.001 U / μL (220, 22, 2.2, 0.22 or 0.022 ng per 10 μL reaction). Mutant polymerases were driven at similar concentrations. Also, those mutant enzymes showing polymerase activity were titrated more finely, testing 220, 22, 10.6, 4.8, 2.2, 1.1, 0.48 and 0.22 ng of protein per 10 μL reaction. Enzyme dilutions were made in enzyme dilution buffer (20 mM Tris pH 7.5, 100 mM NaCl, 1 mM DTT, 0.1% Triton-X100, 1 mg / mL BSA, 10% glycerol). Reactions were carried out in 384-well format on a BIO-RAD CFX384™ real-time system (BIO-RAD, Hercules, CA) using cycling parameters of 30 seconds at 95°C followed by 60 cycles of [15 seconds at 95°C followed by 1 minute at 60°C]. Detection was achieved using a fluorescence-quenched probe (5'-nuclease assay format. Note that the mutations introduced in this series of Taq mutants do not reside in the 5'-nuclease domain.). The sequences of the primers, probes and template (plasmid insert) are shown in Table 6.

[0153]

Table 6

[0154] These 18 Taq DNA polymerase mutants were characterized as outlined above. The results are summarized in Table 7. Six mutants, including mutants ID4, 5, 9, 12, 13, and 17, showed no detectable DNA polymerase activity and were not further studied. Six mutants, mutants ID6, 7, 11, 14, 15, and 16, had DNA polymerase activity; however, the processing ability was reduced 4 - 50-fold compared to the wild-type enzyme. Six mutants, mutants ID1, 2, 3, 8, 10, and 18, showed DNA polymerase activity similar to wild-type OptiTaq.

[0155]

Table 7

[0156] A subset of these mutant Taq DNA polymerases that showed DNA polymerase activity was studied for the ability to discriminate between primers with 3'-DNA as opposed to 3'-RNA residues compared to the wild-type OptiTaq enzyme. Real-time PCR was performed as described above, and an amount of each mutant DNA polymerase equal to 0.5 units of wild-type OptiTaq per 10 μL reaction was employed in the reaction. The following reaction conditions were employed: 1×qPCR buffer (20 mM Tris pH 8.4, 50 mM KCl, 3 mM MgCl 2 , 0.01% Triton-X100) in a final volume of 10 μL, 800 μM dNTP (200 μM each), 500 nM For primer (Hs SFRS9 F569rU, SEQ ID NO: 47), 500 nM Rev primer (Hs SFRS9 R712 rA, SEQ ID NO: 48), 250 nM probe (Hs SFRS9 P644, SEQ ID NO: 49), 2×10 3Linear, cloned plasmid template for copy (SFRS9-targ, SEQ ID NO: 50). The reaction was carried out in a 384-well format on a BIO-RAD CFX384™ real-time system (BIO-RAD, Hercules, CA) using cycling parameters of 30 seconds at 95°C, followed by 60 cycles of [15 seconds at 95°C, followed by 1 minute at 60°C]. Detection was achieved using a fluorescence-quenched probe (5'-nuclease assay format). The sequences of the primers, probes, and template (plasmid insert) are shown in Table 8.

[0157] [Table 8]

[0158] Twelve Taq DNA polymerase mutants that support PCR were tested for their ability to use 3'-RNA-modified primers as outlined above. The results are summarized in Table 7. Mutants ID1 and 8 showed no difference between primers with 3'-DNA compared to 3'-RNA residues. Mutants ID2, 3, 6, 7, 10, 11, 14, 15, 16, and 18 showed amplification delays using 3'-RNA primers. Thus, the rational design strategy employed herein was successful, Taq DNA polymerase mutants were identified, and discrimination against priming from 3'-RNA residues was achieved. Those mutants that showed some delay with RNA priming and high throughput were studied for improvement in primer 3'-residue mismatch discrimination.

[0159] [Example 5] Improved Mismatch Discrimination in Allele-Specific PCR Using Mutant Taq DNA Polymerase Of the 18 mutant enzymes studied in Example 4, mutant IDs 2, 3, 10, and 18 showed the ability to discriminate the 3'-RNA residue in the primer and retained high enzyme activity / processing ability. These four mutants were studied for their ability to discriminate 3'-terminal DNA mismatches compared to wild-type OptiTaq DNA polymerase using an allele-specific qPCR assay. The amplification reaction was performed on a synthetic oligonucleotide template, in which a single base (SNP) was varied that was positioned to be at the 3'-end of the reverse primer. Synthetic templates with each of the four possible bases at this position were employed. Reverse primers with each of the four possible bases at the 3'-end were employed. The relative amplification efficiency of all pairs of combinations was evaluated using qPCR.

[0160] Quantitative allele-specific real-time PCR (AS-qPCR) was performed in a 10 μL reaction volume in a 384-well format using 2 × 10 3 copies of a 103 bp synthetic template (SEQ ID NOs: 51 - 54). The final reaction conditions used were 20 mM Tris-HCL (pH 8.4 at 25°C), 50 mM KCL, and 3 mM MgCl 2, 0.01% Triton X-100, 800 μM total dNTP, and 200 nM of a universal forward primer (SEQ ID NO: 60), 200 nM of a reverse primer (separate reactions were set up for each of the allele-specific primer SEQ ID NOs: 55 - 58 or the control universal primer SEQ ID NO: 59), and 200 nM of a 5' nuclease detection probe (SEQ ID NO: 61). Each allele-specific primer was tested based on its respective SNP template. The reactions utilized either 0.5 U (10.8 ng / 11.1 nM / 111 fmol) of wild-type OptiTaq DNA polymerase or 0.5 U of one of the four Taq DNA polymerase mutants studied (MUT ID No. 2 V783F, MUT ID NO. 3 H784Q, MUT ID NO. 10 A661E I665W F667L, or MUT ID NO. 18 V783L H784Q). Amplification was performed on a CFX384™ C1000™ thermocycler system (Bio-Rad, Hercules, CA) using the following cycling parameters: an initial denaturation at 95°C for 30 seconds, followed by 60 cycles of 10 seconds at 95°C and then 30 seconds at 60°C. The oligonucleotide reagents used in this example are shown in Table 9.

[0161]

Table 9

[0162] First, all reactions were performed in triplicate. When wild-type OptiTaq was used, similar results were obtained for all replicates. However, the results showed greater variability for the mutant polymerases. Therefore, to obtain statistically significant results, each reaction was performed 96 times for the mutant polymerases and 81 times for the wild-type enzyme. The ΔCq value was calculated as the Cq value obtained for each mismatched base pair - the Cq value obtained for the matched base pair (ΔCq = Cq mismatch - Cq match). The ΔCq values for all 96 replicates were averaged and the standard deviation was calculated. The results are shown in Table 10 and summarized graphically in Figures 3A and 3B. Note that the reverse primer is an allele-specific primer. Thus, the "Syn Rev T" primer (SEQ ID NO: 55) is a perfect match to template A (SEQ ID NO: 51), etc.

[0163]

Table 10

[0164] Wild-type OptiTaq showed an average ΔCq for AS-qPCR in this synthetic amplicon system of 4.2 in the range of 1.4 to 8.0. Mutant ID2 (V783F) showed an average ΔCq of 9.4 in the range of 3.5 to 17.9. Mutant ID3 (H784Q) showed an average ΔCq of 9.9 in the range of 4.6 to 21.2. Mutant ID10 (A661E, I665W, F667L) showed an average ΔCq of 10.9 in the range of 3.3 to 19.9. Mutant ID18 (V783L, H784Q) showed an average ΔCq of 12.4 in the range of 4.9 to 26.6. Thus, in all combinations of pairs of all four template bases and four 3'-terminal primer bases, the mutant Taq DNA polymerase of the present invention showed greater discrimination against mismatches than wild-type OptiTaq DNA polymerase. The magnitude of the improvement for each mismatch pair is defined by ΔΔCq, which is the difference in discrimination between the mutant and wild-type enzymes (ΔΔCq = ΔCq mutant - ΔCq wild-type). The ΔΔCq values were calculated and are shown in Table 11.

[0165]

Table 11

[0166] Mutant ID2 (V783F) showed an average ΔΔCq of 5.2 compared to wild-type OptiTaq. Mutant ID3 (H784Q) showed an average ΔΔCq of 5.7 compared to wild-type OptiTaq. Mutant ID10 (A661E, I665W, F667L) showed an average ΔΔCq of 6.7 compared to wild-type OptiTaq. Mutant ID18 (V783L, H784Q) showed an average ΔΔCq of 8.2 compared to wild-type OptiTaq. Thus, each of the mutant Taq DNA polymerases of the present invention showed a significant improvement in mismatch discrimination compared to wild-type OptiTaq, and importantly, mismatch discrimination was improved for all possible combinations of mismatched base pairs. Overall, Mutant ID18 (V783L, H784Q) showed the best SNP discrimination within the set of four mutant enzymes studied in this example using the AS-PCR assay.

[0167] [Example 6] Discrimination of Primer 3'-RNA Residues by Taq DNA Polymerase Mutants All 18 Taq DNA polymerase mutants were screened for their ability to discriminate against priming from 3'-RNA residues in Example 4. The four mutants (MUT ID2, 3, 10, and 18) studied in the AS-PCR of Example 5 that showed good 3'-mismatch discrimination were studied in more detail in this example for their ability to discriminate against the presence of 3'-terminal RNA residues in the primer, and the possible base-specific effects were examined. The amplification reaction was performed on a synthetic oligonucleotide template, in which a single base (SNP) located at the 3'-end of the reverse primer was varied. Synthetic templates with each of the four possible bases at this position were employed. Reverse primers with each of the four possible RNA bases at the 3'-end were employed, and the results were compared to a control reaction using primers with each of the four possible DNA bases at the 3'-end. The relative amplification efficiency was evaluated using qPCR.

[0168] Quantitative real-time PCR (qPCR) was performed in a 10 μL reaction volume in a 384-well format using 2 × 10 3 copies of a 103 bp synthetic template (SEQ ID NOs: 51-54). The final reaction conditions used were 20 mM Tris-HCL (pH 8.4 at 25°C), 50 mM KCL, and 3 mM MgCl 2 , 0.01% Triton X-100, 800 μM total dNTP, and 200 nM universal forward primer (SEQ ID NO: 60), 200 nM reverse primer (separate reactions were set up for each of the four 3'-RNA primer SEQ ID NOs: 62-65, each of the four 3'-DNA primer SEQ ID NOs: 55-58, or the control universal primer SEQ ID NO: 59).) and 200 nM 5'-nuclease detection probe (SEQ ID NO: 61). Each primer was tested only based on the complementary template (mismatch conditions were not tested). The reaction was carried out using 0.5 U (10.8 ng / 11.1 nM / 111 fmol) of wild-type OptiTaq DNA polymerase or 0.5 U of one of the four Taq DNA polymerase mutants studied (MUT ID No.2 V783F, MUT ID NO.3 H784Q, MUT ID NO.10 A661E I665W F667L or MUT ID NO.18 V783L H784Q). Amplification was performed on a CFX384™ C1000™ thermocycler system (Bio-Rad, Hercules, CA) using the following cycling parameters: an initial denaturation at 95°C for 30 seconds, followed by 60 cycles of 10 seconds at 95°C and then 30 seconds at 60°C. The oligonucleotide reagents used in this example are shown in Table 12. A total of 96 replicates were performed for each pair combination.

[0169]

Table 12

[0170] The average Cq value was calculated for 96 replicate sets. The ΔCq value was calculated as the difference between the average Cq value of the 3'-RNA primer reaction and the average Cq value of the 3'-DNA primer reaction (ΔCq = Cq3’RNA - Cq3’-DNA). A higher ΔCq value indicates a greater degree of discrimination against priming from the 3'-RNA primer. The results are shown in Table 13 and summarized graphically in Figure 4.

[0171]

Table 13

[0172] Wild-type OptiTaq did not show any significant discrimination between the 3'-DNA primer and the 3'-RNA primer. However, when using the 3'-RNA primer, all four mutant Taq DNA polymerases showed a decrease in priming efficiency. Thus, the goal of creating a novel polymerase that discriminates against 3'-RNA residues in the primer was achieved using the intelligent mutagenesis design strategy described herein. Interestingly, the magnitude of discrimination was much greater for RNA pyrimidine residues (rC or rU) than for RNA purine residues (rA or rG).

[0173] [Example 7] Improved Mismatch Discrimination in rhPCR Using Mutant Taq DNA Polymerase RNase H-based PCR (rhPCR) employs the enzyme RNase H2 to convert block-type cleavable oligonucleotides that cannot prime DNA synthesis into a form that can prime DNA synthesis and initiate PCR. Block-type cleavable oligonucleotides or block-type cleavable primers contain a single RNA residue near the 3'-end of the oligonucleotide (including the cleavage site) and are modified at or near the 3'-end, such that the primer cannot prime DNA synthesis and / or has lost its template function and, thereby, is not suitable to support PCR even if primer extension can occur. This method is used for genotyping (SNP discrimination) and relies on the ability of RNase H2 to distinguish base pair matches from mismatches at the RNA base cleavage site when hybridized to the target nucleic acid. In rhPCR, SNP discrimination occurs in the primer block release step and not in the primer extension step (in AS-PCR, discrimination occurs in the primer extension step).Examples of enzyme cleavage strategies, similar RNase H strategies, and methods that block primer extension or inhibit template function and thereby invalidate PCR are described in U.S. Patent No. 7,112,406 by Behlke et al. entitled "POLYNOMIAL AMPLIFICATION OF NUCLEIC ACIDS"; U.S. Patent No. 5,763,181 by Han et al. entitled "CONTINOUS FLUOROMETRIC ASSAY FOR DETECTING NUCLEIC ACID CLEAVAGE"; U.S. Patent No. 7,135,291 by Sagawa entitled "METHOD OF DETECTING NUCLEOTIDE POLYMORPHISM"; U.S. Patent Application Publication No. 20090068643 by Behlke and Walder entitled "DUAL FUNCTION PRIMERS FOR AMPLIFYING DNA AND METHODS OF USE"; U.S. Patent Application Publication No. 20100167353 by Walder et al. entitled "RNASE H-BASED ASSAYS UTILIZING MODIFIED RNA MONOMERS"; and Dobosy et al., "RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers", BMC Biotechnology., 11:e80 (2011).

[0174] In AS-PCR, the SNP is located at the 3' end of the primer. In this configuration, a mispriming event (where DNA synthesis is initiated in the presence of a mismatch at the 3' end) results in the introduction of the base present in the primer into the nascent DNA strand and thereby into the PCR amplicon. This event converts the PCR product to the primer sequence, such that the amplified DNA now matches the primer and no longer matches the nucleic acid sequence of the originally input sample. Since the amplicon sequence now matches the primer and not the input sample, amplification proceeds with high efficiency.

[0175] In rhPCR, cleavage of the block-type cleavage primer by RNase H2 occurs on the 5'-side of the RNA residue; when the SNP is located in the RNA residue (e.g., the RNA base pair having the SNP), the first base incorporated by DNA polymerase and PCR during primer extension is the SNP site, resulting in a daughter product that remains the same as the input nucleic acid sequence. Rarely, non-standard RNase H2 cleavage occurs on the 3'-side of the RNA base leaving an RNA residue at the 3'-end of the primer positioned to cover the SNP. In this case, the rhPCR reaction proceeds like AS-PCR, with the 3'-end of the primer located at the SNP site and being either a match or a mismatch to the target nucleic acid. Similar to AS-PCR, in the case of a mismatch, the sequence of the DNA extension product and the PCR amplicon is converted to the sequence of the primer, and thus may not faithfully replicate the sequence of the sample being amplified. Any method that reduces the frequency of this undesirable mispriming event improves mismatch discrimination in the rhPCR assay. Thus, base discrimination in rhPCR is mainly mediated by RNase H2 at the primer cleavage step, but the use of a DNA polymerase with an improved ability to discriminate against 3'-terminal mismatches and / or 3'-terminal RNA residues can improve the overall mismatch discrimination ability of rhPCR by preventing extension when an undesirable 3'-cleavage event occurs. The DNA polymerase mutants described herein reduce the priming efficiency (improve mismatch discrimination) in the presence of a 3'-mismatch and reduce the priming efficiency (discriminate against primer 3'-RNA residues) when a 3'-terminal RNA residue is present in the primer, compared to wild-type Taq DNA polymerase. This example demonstrates that the novel mutant Taq DNA polymerase of the present invention improves the specificity of rhPCR and SNP discrimination.

[0176] Quantitative real-time rhPCR was performed to compare the performance of wild-type OptiTaq DNA polymerase with that of mutant Taq DNA polymerase mutants ID2, 3, 10, and 18. The design of two different block-type cleavage primers was tested, including Generation 1 (Gen1) "RDDDDx" primers and Generation 2 (Gen2) "RDxxD" primers (see U.S. Patent Application Publication No. 2012 / 0258455 by Behlke et al. entitled "RNASE H-BASED ASSAYS UTILIZING MODIFIED RNA MONOMERS"). The amplification reaction was performed using the same synthetic oligonucleotide template employed in Example 5, where a single base was varied (SNP site) such that RNA residues were positioned in both Gen1 and Gen2 block-type cleavage (rhPCR) primers. Synthetic templates with each of the four possible bases at this position were employed. Reverse primers with each of the four possible complementary bases at this position were employed (RNA bases). The same forward primer was used in all reactions. Relative amplification efficiency was evaluated using real-time PCR.

[0177] Quantitative rhPCR was performed in a 10 μL reaction volume in a 384-well format using 2 × 10 6 copies of a 103 bp synthetic template (SEQ ID NOs: 51-54). The final reaction conditions used were 20 mM Tris-HCL (pH 8.4 at 25 °C), 50 mM KCL, 3 mM MgCl 2, 0.01% Triton X-100, 800 μM total dNTP, 200 nM universal forward primer (SEQ ID NO: 60), 200 nM reverse primer, and 200 nM 5'-nuclease detection probe (SEQ ID NO: 61). The reverse primer included allele-specific rhPCR primers of the Gen1 RDDDDx configuration (SEQ ID NOs: 66-69), allele-specific rhPCR primers of the Gen2 RDxxD configuration (SEQ ID NOs: 70-73), and a control universal reverse primer (SEQ ID NO: 59). Each of the rhPCR block-type cleavage reverse primers was tested for each of the 4 SNP templates. The reaction utilized either 0.5 U (10.8 ng / 11.1 nM / 111 fmol) of wild-type OptiTaq DNA polymerase or 0.5 U of one of 4 Taq DNA polymerase mutants (MUT ID2 V783F; MUT ID3 H784Q; MUT ID10, A661E I665W F667L; or MUT ID18, V783L H784Q). P. abyssi RNase H2 was added to each reaction in a 1 μL volume. Reactions using the control and Gen1 block-type cleavage RDDDDx rhPCR primers employed 2.6 mU RNase H2 / 10 μL reaction (5 fmole, 0.5 nM enzyme). Reactions using the Gen2 block-type cleavage RDxxD rhPCR primers employed 25 mM RNase H2 10 μL reaction (48 fmole, 4.8 nM enzyme) for the rC and rA primers (SEQ ID NOs: 71 and 72), and 200 mU RNase H2 / 10 μL reaction (384 fmole, 38 nM enzyme) for the rG and rU primers (SEQ ID NOs: 70 and 73). Cycling was performed on a Roche LightCycler® 480 (Roche Applied Science, Indianapolis, IN, USA) as follows: 95°C for 3 minutes, followed by 75 cycles of 95°C for 10 seconds and 60°C for 30 seconds. All reactions were performed in triplicate. The oligonucleotide reagents used in this example are shown in Table 14.

[0178]

Table 14

[0179] MUT ID10 (A661E, I665W, F667L) unexpectedly showed a large amplification delay when the primer matched the SNP site in the target in the rhPCR reaction using this synthetic amplicon system. However, this polymerase did not show any delay when using the human genomic DNA system for rhPCR (see Examples 8 and 9). Therefore, MUT ID10 was excluded from the analysis in the synthetic system experiment. Data generated using the other three mutant polymerases were analyzed, and the ΔCq values were calculated by comparing the match vs. mismatch primer / template pairs. In this case, ΔCq = Cq mismatch - Cq match. The results are shown in Table 15 for the Gen1 RDDDDx block-type cleavage primers rhPCR primers and in Table 16 for the Gen2 RDxxD block-type cleavage primers rhPCR primers.

[0180]

Table 15

[0181]

Table 16

[0182] In almost all cases, mismatch discrimination was excellent for rhPCR reactions performed using mutant Taq DNA polymerases compared to wild-type OptiTaq. The magnitude of the improvement was best seen by examining the ΔΔCq values, which is the difference in discrimination seen using wild-type OptiTaq and the mutant (ΔΔCq = ΔCq mutant - ΔCq wild-type). These results are shown in Table 17 for the Gen1 RDDDDx primers and in Table 18 for the Gen2 RDxxD primers. When using the Gen1 RDDDDx primers, the overall maximum benefit was seen when the mismatched base was "C" in the target nucleic acid, and the minimum benefit was seen when the block-type cleavage primer paired with an rG containing a mismatched T in the target. The greatest improvement was obtained using the mutant Taq DNA polymerase MUT ID18 (V783L H784Q). The average ΔΔCq for MUT ID2 (V783F) was 1.0. The average ΔΔCq for MUT ID3 (H784Q) was 2.0. The average ΔΔCq for MUT ID18 (V783L, H784Q) was 3.6. The benefits obtained using mutant Taq DNA polymerases were lower for the Gen2 RDxxD primers that already showed high ΔCq values using wild-type OptiTaq. The average ΔΔCq for the three mutant polymerases studied in the examples were 0.6, 2.1, and 1.2. Thus, the maximum benefit when using the Gen2 RDxxD primers was seen with MUT ID3 (H784Q).

[0183]

Table 17

[0184]

Table 18

[0185] [Example 8] Improved Mismatch Discrimination in rhPCR Using Mutant Taq DNA Polymerase in a Human Genome DNA SNP Assay Example 7 demonstrated the utility of the novel mutant Taq DNA polymerase of the present invention in a synthetic amplicon rhPCR SNP discrimination assay system. This example demonstrated the utility of the novel mutant Taq DNA polymerase in a human genome DNA rhPCR SNP discrimination assay system and examined the SNP site in the SMAD7 gene (NM_005904, C / T SNP, rs4939827). The assay employed target DNAs GM18562 (homozygous C / C) and GM18537 (homozygous T / T) from the Coriell Institute for Medical Research (Camden, NJ, USA). Two different block-type cleavage primer designs were tested and included Generation 1 (Gen1) "RDDDDx" primers and Generation 2 (Gen2) "RDxxD" primers (see U.S. Patent Application No. 2012 / 0258455 by Behlke et al. entitled "RNASE H-BASED ASSAYS UTILIZING MODIFIED RNA MONOMERS").

[0186] Quantitative real-time rhPCR was performed in a 10 μL reaction volume in a 384-well format containing 20 ng (equivalent to 6600 copies of the target) of human genomic DNA (GM18562 or GM18537). The reaction utilized either 0.5 U (10.8 ng / 11.1 nM / 111 fmol) of wild-type OptiTaq DNA polymerase or 0.5 U of any one of four Taq DNA polymerase mutants (MUT ID2, V783F; MUT ID3, H784Q; MUT ID10, A661E I665W F667L; or MUT ID18, V783L H784Q). The final reaction conditions used were 20 mM Tris-HCL (pH 8.4 at 25°C), 50 mM KCL, 3 mM MgCl 2, 0.01% Triton X-100, 800 μM total dNTP, 200 nM forward primer (SEQ ID NOs: 75-79), 200 nM universal reverse primer (SEQ ID NO: 74), and 200 nM SMAD7 probe (SEQ ID NO: 80). The sequence of the 85 bp SMAD7 amplicon is shown as SEQ ID NO: 81. The forward primers included the RDDDDx-constructed Gen1 allele-specific rhPCR primers (SEQ ID NOs: 76 and 77), the RDxxD-constructed Gen2 allele-specific rhPCR primers (SEQ ID NOs: 78 and 79), and the non-allele-specific control universal forward primer (SEQ ID NO: 75). The oligonucleotide reagents employed in this example are shown in Table 19. The reactions contained 1 μL of P.a.RNase H2 at a concentration of 2.6 mU per 10 μL reaction (5 fmole, 0.5 nM) for the Gen1 RDDDDx primers and the control primers (SEQ ID NOs: 75-77), or at a concentration of 200 mU per 10 μL reaction (384 fmole, 38.4 nM) for the Gen2 RDxxD primers (SEQ ID NOs: 78 and 79). Amplification was performed on a Roche LightCycler® 480 (Roche Applied Science, Indianapolis, IN, USA) as follows: 3 minutes at 95°C, followed by 75 cycles of 10 seconds at 95°C and 30 seconds at 60°C. All reactions were performed in triplicate.

[0187]

Table 19

[0188] The results using the Gen1 RDDDDx rhPCR primers are shown in Table 20, and the results using the Gen2 RDxxD rhPCR primers are shown in Table 21. Overall, the use of the mutant Taq DNA polymerase showed a small but real improvement in SNP discrimination in this human genomic DNA rhPCR assay using the Gen1 RDDDDx primers. However, a large improvement in discrimination was seen using the Gen2 RDxxD primers. The Gen2 RDxxD primers essentially showed greater SNP discrimination, and these levels increased such that the ΔCq values were greater than 40 amplification cycles between match and mismatch in some cases; since the qPCR reaction rarely went beyond 45 - 50 cycles and a positive signal was not detected in these cases until after 70 cycles, this level of discrimination is "greater than the assay" for most users (Table 21). Thus, the use of the novel mutant Taq DNA polymerase improves SNP discrimination in the rhPCR genotyping assay.

[0189]

Table 20

[0190]

Table 21

[0191] The ΔCq values for the SMAD7 SNP genotyping assay are graphically summarized in FIG. 5A for the Gen1 RDDDDx primers and in FIG. 6A for the Gen2 RDxxD primers. Regarding the rhPCR genotyping assay studied in Example 8, it is interesting to note that MUT ID10 (A661E I665W F667L) showed the greatest improvement compared to wild-type OptiTaq, especially when using the Gen2 RDxxD primers. Example 5 demonstrated the usefulness of mutant Taq DNA polymerases in AS-PCR, in which case the use of MUT ID18 (V783L H784Q) showed the greatest benefit and MUT ID3 (H784Q) showed the next greatest relative benefit. It is also clear that the various mutant Taq DNA polymerases of the present invention are useful not only in different amplification assays, but also that different mutants show various levels of benefit depending on the nature of the assay being used. Thus, it is beneficial to have a collection of mutant polymerases that can be matched to assays / applications with different characteristics to obtain the greatest benefit.

[0192] [Example 9] Improved discrimination of low-frequency alleles in genomic DNA using rhPCR with mutant Taq DNA polymerase The use of Gen2 RDxxD block-type cleavage primers in rhPCR can detect the presence of SNPs at levels from 1:1,000 to 1:10,000 in the background of wild-type genomic DNA using the native (wild-type) Taq DNA polymerase (see U.S. Patent Application No. 2012 / 0258455 by Behlke et al. entitled "RNASE H-BASED ASSAYS UTILIZING MODIFIED RNA MONOMERS"). This example demonstrates that the mutant Taq DNA polymerases of the present invention improve low-frequency allele discrimination in the rhPCR assay.

[0193] The low-frequency allele detection experiment was designed to detect the nucleotide identity at the SNP site in the SMAD7 gene (NM_005904, C / T SNP, rs4939827), and the target DNAs GM18562 (homozygous C / C) and GM18537 (homozygous T / T) (Coriell Institute for Medical Research, Camden, NJ, USA) were employed. Control reactions were set up using 2 ng (660 copies), 0.2 ng (66 copies) or 0.02 ng (6.6 copies) of the input, matched target DNA. The low-frequency allele detection reactions were set up using 2 ng (660 copies), 0.2 ng (66 copies) or 0.02 ng (6.6 copies) of the input, matched target DNA + 200 ng (66,000 copies) of the other (mismatched) gene for one allele. The background was established in reactions containing 0 copies of the matched target DNA + 200 ng (66,000 copies) of the mismatched target DNA. Both combinations were tested: GM18562 (C / C) as the low-frequency allele in the presence of excess GM18537 (T / T) and GM18537 (T / T) as the low-frequency allele in the presence of excess GM18562 (C / C).

[0194] Quantitative real-time rhPCR was performed in a 10 μL reaction volume in a 384-well format. The final reaction conditions used were 10 mM Tris-HCL (pH 8.4 at 25 °C), 50 mM KCL, 3.5 mM MgCl 2, 0.01% Triton X-100, 0.8 mM dNTP, one of 200 nM SMAD7 forward primers (SEQ ID NOs: 75, 78, and 79), 200 nM SMAD7 reverse primer (SEQ ID NO: 74), and 200 nM SMAD7 probe (SEQ ID NO: 80). The 85 bp SMAD7 amplicon defined by these primers is shown as SEQ ID NO: 81. Note that the forward primer was unmodified (control, SEQ ID NO: 75), or specific for the SMAD7 C-allele (SEQ ID NO: 78) or SMAD7 T-allele (SEQ ID NO: 79) using the block-type cleavage rhPCR Gen2 RDxxD design. The reaction used either 0.5 U of wild-type OptiTaq DNA polymerase or 0.5 U of one of the four Taq DNA polymerase mutants studied (MUT ID No.2, V783F; MUT ID NO.3, H784Q; MUT ID NO.10, A661E I665W F667L; or MUT ID NO.18, V783L H784Q). The reaction contained P. abyssi RNase H2 at a concentration of 200 mU (384 fmole) per 10 μL reaction when using the SMAD7 For rC DxxD (SEQ ID NO: 78) primer and P. abyssi RNase H2 at a concentration of 500 - 600 mU (960 - 1152 fmole) per 10 μL reaction for the control reaction or when using the SMAD7 For rU DxxD (SEQ ID NO: 79) primer. The oligonucleotide reagents used in this example are shown in Table 22. Cycling was performed on a Roche LightCycler® 480 (Roche Applied Science, Indianapolis, IN, USA) as follows: 3 minutes at 95°C, followed by 65 cycles of 10 seconds at 95°C and 30 seconds at 60°C. All reactions were performed in triplicate.

[0195]

Table 22

[0196] The results are analyzed and shown in Table 23. The control column shows the Cq values for the matched primer / target reactions, with no mismatched target present, to establish the quantitative standard curve. The low-frequency allele detection column shows the Cq values for the detection of 660, 66, 6, or 0 (background control) copies of the matched primer / target in the presence of 66,000 copies of the mismatched target. At least a 3-cycle difference (ΔCq = 3.0 or greater) between the background and the positive signal is required to call a "positive" reaction for low-frequency allele detection; a 5-cycle difference (ΔCq = 5.0 or greater) is generally assumed to be preferred. In this system, the background is the signal observed when amplification is performed using an input target that does not match the primer, and thus the signal only arises from amplification originating from the mismatched target.

[0197] Using wild-type OptiTaq DNA polymerase, the detection of the "C" allele in an excess "T" background and the detection of the "T" allele in an excess "C" background both met the ΔCq 3.0 and ΔCq 5.0 levels of stringency required to be called a 1:1000 low-frequency allele detection event (66 copies of the matched target in the presence of 66,000 copies of the mismatched target). The 1:10,000 reaction (6 copies of the matched target in the presence of 66,000 copies of the mismatched target) did not meet either of these criteria. Thus, rhPCR had a 1:1000 low-frequency allele detection limit using wild-type OptiTaq in this genomic DNA SNP system.

[0198] In contrast, rhPCR using each of the four mutants showed a low-frequency allele detection limit of 1:10,000 for both the "C" and "T" allele targets, and the ΔCq stringency cutoff was 3.0. MUT ID3 (H784Q) showed a low-frequency allele detection limit of 1:10,000 for both the "C" and "T" targets in this genomic SNP system for a higher ΔCq stringency cutoff of 5.0. The other three mutant Taq DNA polymerases (MUT ID No.2, V783F; MUT ID NO.10, A661E I665W F667L; and MUT ID NO.18, V783L H784Q) showed a low-frequency allele detection limit of 1:10,000 for the "C" allele target with a ΔCq stringency cutoff of 5.0 and a low-frequency allele detection limit of 1:10,000 for the "T" allele target with a ΔCq stringency cutoff of 3.0. Therefore, the inventors conclude that the novel mutant Taq DNA polymerase of the present invention provides an improved low-frequency allele detection reaction using block-type cleavage primers in rhPCR as compared to the use of wild-type DNA polymerase.

[0199]

Table 23

[0200] [Example 10] Sequence of Taq DNA polymerase mutants showing improved discrimination for mismatches or presence of RNA residues at the 3'-end of the primer The complete amino acid and nucleotide sequences of the codon-optimized mutant enzymes employed in Examples 5-9 are shown below. These sequences are readily derivable by those skilled in the art from the information provided in Tables 1, 3, 4, and 5, but for clarity, the finally assembled sequences are provided. Base changes are identified in bold underlined font for nucleic acid and amino acid substitutions.

[0201] Nucleotide sequence of SEQ ID NO: 82, Mutant ID2 (V783F).

[0202]

Chem.

[0203] Amino acid sequence of SEQ ID NO: 83, Mutant ID2 (V783F).

[0204]

Chem.

[0205] Nucleotide sequence of SEQ ID NO: 84, Mutant ID3 (H784Q).

[0206]

Chem.

[0207] Amino acid sequence of SEQ ID NO: 85, Mutant ID3 (H784Q).

[0208]

Chem.

[0209] Nucleotide sequence of SEQ ID NO: 86, Mutant ID10 (A661E, I665W, F667L).

[0210]

Chem.

[0211] Amino acid sequence of SEQ ID NO: 87, Mutant ID10 (A661E, I665W, F667L).

[0212]

Chem.

[0213] Nucleotide sequence of SEQ ID NO: 88, Mutant ID 18 (V783L, H784Q).

[0214] [Chemical formula]

[0215] Amino acid sequence of SEQ ID NO: 89, Mutant ID 18 (V783L, H784Q).

[0216] [Chemical formula]

[0217] [Example 11] BLAST search for additional wild-type VH-related DNA polymerases A BLAST search using the entire Taq DNA polymerase sequence G755 as a comparison window against P812 (SEQ ID NO: 90) was performed using the online databases available through the National Center for Biotechnology Information within the National Library of Medicine of the National Institutes of Health (http: / / www.ncbi.nlm.nih.gov / ). The BLAST search revealed numerous wild-type DNA polymerases from other species that share extensive sequence identity with Taq DNA polymerase, including identity at positions V783 and H784 of Taq DNA polymerase (the "VH-related DNA polymerases"). An exemplary list of these thermostable polymerases is shown in Table 24, and a similar list of putative thermosensitive polymerases is shown in Table 25. In all identified wild-type polymerase genes except one polymerase gene (Phaclamydia hominis), the amino acids corresponding to V783 and H784 of Taq DNA polymerase were conserved. However, in the exceptional case, i.e., in the case of Phaclamydia hominis, Ile is naturally present at the residue position of Taq DNA polymerase corresponding to V783. However, the Taq DNA polymerase mutant corresponding to mutant ID1 containing this specific substitution behaves like the wild-type Taq DNA polymerase. In this way, the DNA polymerase of Phaclamydia hominis appears to result from a strong selection for Val at this position and is assumed to maintain wild-type activity when either a Val or Ile residue is present. These BLAST results confirm a natural counter-selection against DNA polymerases with increased template discrimination activity and provide strong evidence that the disclosed, engineered Taq DNA polymerase mutants with these properties are novel and non-obvious.

[0218] These identified DNA polymerases share extensive sequence homology with Taq DNA polymerase in the region containing residues V783 and V784 of Taq DNA polymerase. As observed in engineered Taq DNA polymerase mutants, each of the identified non-Taq DNA polymerases represents a sequence space in which engineered mutant enzymes with increased template discrimination activity can arise, compared to their respective unmodified counterparts. The magnitude of the increased template discrimination activity obtained for the same amino acid substitution in non-Taq DNA polymerases need not be the same when compared to their respective unmodified non-Taq DNA polymerases or even when compared to the magnitude of the increased template discrimination activity observed for the corresponding Taq DNA polymerase mutants. Nevertheless, a powerful prediction of this disclosure is that at least some amino acid substitutions in non-Taq DNA polymerases having homology to residues V783 and / or H784 of Taq DNA polymerase will exhibit increased template discrimination activity compared to their respective unmodified counterparts.

[0219]

Table 24

[0220]

Table 25

[0221] [Example 12] Generation of Additional Codon-Optimized Taq DNA Polymerase Mutants at Position H784 After determining the properties of the first 18 mutant versions of Taq polymerase (Table 3, Mut ID1-18), an additional 18 mutant versions of Taq DNA polymerase (Table 3, Mut ID19-30) were generated by site-directed mutagenesis of the cloned OptiTaq codon-optimized WT Taq DNA polymerase. The complete set represents all possible amino acid changes at position 784 in Taq polymerase. Specific mutations were introduced into the OptiTaq sequence using the PCR site-directed mutagenesis method (Weiner MP et al., Gene., 151(1-2):119-23 (1994)). Each mutagenesis reaction was carried out in 1× KOD PCR buffer with 10 pmol of two complementary oligonucleotides (Table 26) containing the desired base change, annealed to double-stranded OptiTaq plasmid (20 ng), 5 U KOD DNA polymerase (Novagen-EMD Chemicals, San Diego, CA), 1.5 mM MgSO 4 was employed. The thermal cycling parameters were 16 cycles of 95°C for 3 minutes (95°C for 20 seconds - 55°C for 20 seconds - 70°C for 2.5 minutes), followed by an incubation at 70°C for 4 minutes. After PCR site-directed mutagenesis, the amplification products were treated with 10 U of DpnI (NEB, Ipswish, MA) at 37°C for 1 hour and then inactivated at 80°C for 20 minutes. One-tenth of the digested material was transformed into XL-1 Blue competent bacteria. Bacterial clones were isolated, plasmid DNA was prepared, and individual mutations were confirmed by Sanger DNA sequencing. All mutants were retained in the pET-27b(+) expression vector suitable for expressing recombinant proteins in E. coli. As described in Example 3, the recombinant mutants of Taq polymerase were expressed and purified.

[0222]

Table 26

[0223] [Example 13] Characterization of the properties of 18 mutant Taq DNA polymerases modified at position H784 in PCR The 18 mutant Taq DNA polymerase enzymes described in Example 12 were characterized for polymerase activity and the ability to discriminate 3'-RNA residues in primer oligonucleotides.

[0224] The unit activity of the purified wild-type protein was determined by comparing the performance of known amounts of OptiTaq and each mutant in qPCR with that of commercially available native non-hot-start Taq DNA polymerase, Taq-B DNA polymerase (Enzymatics, Beverly, MA). The quantification cycle value (Cq, the number of amplification cycles at which a positive signal is first detected) and the amplification curve shape were analyzed to determine the nanogram amounts at which both enzymes perform similarly within the sub-optimal range for each. Using these nanogram amounts and the known unit value of Taq-B DNA polymerase, relative activity unit values could be extrapolated for all of the mutant DNA polymerase enzymes that had sufficient activity to support PCR.

[0225] The following reaction conditions were employed: 1×qPCR buffer (20 mM Tris pH 8.4, 50 mM KCl, 3 mM MgCl 2 , 0.01% Triton-X100) in a final volume of 10 μL, 800 μM dNTP (200 μM each), 500 nM For primer (Hs HPRT F517, SEQ ID NO: 43), 500 nM Rev primer (Hs HPRT R591, SEQ ID NO: 44), 250 nM probe (Hs HPRT P554, SEQ ID NO: 45), 2×10 3Linear, cloned plasmid template of the copy (HPRT-targ, SEQ ID NO: 46). The amount of DNA polymerase added to each reaction was varied as follows: for wild-type (OptiTaq), the reactions were set up using 10, 1, 0.1, 0.01, 0.001 U / μL (220, 22, 2.2, 0.22 or 0.022 ng of protein per 10 μL reaction). The mutant polymerases were driven at similar concentrations. Also, those mutant enzymes showing polymerase activity were titrated more finely, and 220, 22, 10.6, 4.8, 2.2, 1.1, 0.48 and 0.22 ng of protein per 10 μL reaction were tested. Enzyme dilutions were made in enzyme dilution buffer (20 mM Tris pH 7.5, 100 mM NaCl, 1 mM DTT, 0.1% Triton-X100, 1 mg / mL BSA, 10% glycerol). The reactions were carried out in 384-well format on a BIO-RAD CFX384™ real-time system (BIO-RAD, Hercules, CA) using cycling parameters of 30 seconds at 95°C, followed by 60 cycles of [15 seconds at 95°C, followed by 1 minute at 60°C]. Detection was achieved using a fluorescence-quenched probe (5'-nuclease assay format. Note that the mutations introduced in this series of Taq mutants do not occur in the 5'-nuclease domain). The sequences of the primers, probe and template (plasmid insert) are shown in Table 27.

[0226]

Table 27

[0227] These 18 Taq DNA polymerase mutants were characterized as outlined above. The results are summarized in Table 28. Ten mutants, including mutants ID19, 23, 25, 28 and 31 to 36, showed no detectable DNA polymerase activity and were not further studied. Four mutants, mutants ID20, 21, 27 and 29, had DNA polymerase activity; however, the processing ability was reduced 4 - 6-fold compared to the wild-type enzyme. Three mutants, mutants ID24, 26 and 30, showed DNA polymerase activity similar to wild-type OptiTaq.

[0228]

Table 28

[0229] A subset of these mutant Taq DNA polymerases that showed appropriate levels of DNA polymerase activity was studied for their ability to discriminate between primers with 3'-DNA as opposed to 3'-RNA residues compared to the wild-type OptiTaq enzyme. Real-time PCR was performed as described above, employing in the reaction an amount of each mutant DNA polymerase equal to 0.5 units of wild-type OptiTaq per 10 μL reaction. The following reaction conditions were employed: 1×qPCR buffer (20 mM Tris pH 8.4, 50 mM KCl, 3 mM MgCl 2 , 0.01% Triton-X100) in a final volume of 10 μL, 800 μM dNTP (200 μM each), 500 nM For primer (Hs SFRS9 F569rU, SEQ ID NO: 47), 500 nM Rev primer (Hs SFRS9 R712 rA, SEQ ID NO: 48), 250 nM probe (Hs SFRS9 P644, SEQ ID NO: 49), 2×10 3Linear, cloned plasmid template for the copy (SFRS9-targ, SEQ ID NO: 50). The reaction was carried out in a 384-well format on a BIO-RAD CFX384™ real-time system (BIO-RAD, Hercules, CA) using cycling parameters of 30 seconds at 95°C, followed by 60 cycles of [15 seconds at 95°C, followed by 1 minute at 60°C]. Detection was achieved using a fluorescence-quenched probe (5'-nuclease assay format). The sequences of the primers, probe, and template (plasmid insert) are shown in Table 29.

[0230] [Table 29]

[0231] Eight Taq DNA polymerase mutants that support PCR were tested for their ability to use 3'-RNA-modified primers as outlined above. The results are summarized in Table 28. Mutants ID20 and 22 showed no difference between primers with 3'-DNA compared to 3'-RNA residues. Mutants ID21, 24, 26, 27, 29, and 30 showed amplification delays using 3'-RNA primers. Thus, additional Taq DNA polymerase mutants were identified and discriminated against priming from 3'-RNA residues. Those mutants that showed some delay with RNA priming and high throughput were studied for improvement in primer 3'-residue mismatch discrimination.

[0232] [Example 14] Improved Mismatch Discrimination in Allele-Specific PCR Using Mutant Taq DNA Polymerase Modified at Position H784 Of the 18 mutant enzymes studied in Examples 12 and 13, mutants ID21, 24, 26, 27, 29 and 30 showed the ability to discriminate 3'-RNA residues in the primer and retained high enzyme activity / processing ability. These six mutants and additionally mutants ID20 and 22 were studied for their ability to discriminate 3'-terminal DNA mismatches compared to wild-type OptiTaq DNA polymerase using an allele-specific qPCR assay. The amplification reaction was performed on a synthetic oligonucleotide template, in which a single base located at the 3'-end of the reverse primer was varied (SNP). Synthetic templates with each of the four possible bases at this position were employed. Reverse primers with each of the four possible bases at the 3'-end were employed. The relative amplification efficiency of all primer / template combinations was evaluated using qPCR.

[0233] Quantitative allele-specific real-time PCR (AS-qPCR) was performed in a 10 μL reaction volume in a 384-well format using 2×10 5 copies of a 103 bp synthetic template (SEQ ID NOs: 51-54). The final reaction conditions used were 20 mM Tris-HCL (pH 8.4 at 25°C), 50 mM KCL, and 3 mM MgCl 2, 0.01% Triton X-100, 800 μM total dNTP, and 200 nM universal forward primer (SEQ ID NO: 60), 200 nM reverse primer (separate reactions were set up for each of the allele-specific primer SEQ ID NOs: 55 - 58 or the control universal primer SEQ ID NO: 59), and 200 nM 5' nuclease detection probe (SEQ ID NO: 61). Each allele-specific primer was tested based on its respective SNP template. The reactions were carried out with 0.5 U (10.8 ng / 11.1 nM / 111 fmol) of wild-type OptiTaq DNA polymerase or 0.5 U of one of the nine Taq DNA polymerase mutants studied (mutant ID3 (H784Q) (10.8 ng / 11.1 nM / 111 fmol); mutant ID20 H784A (54 ng / 55.5 nM / 555 fmol); mutant ID22 H784T (10.8 ng / 11.1 nM / 111 fmol); mutant ID24 H784V (24 ng / 24.7 nM / 246.7 fmol); mutant ID26 H784I (21.6 ng / 22.2 nM / 222 fmol); mutant ID27 H784M (10.8 ng / 11.1 nM / 111 fmol); mutant ID29 H784F (49.1 ng / 49.4 nM / 494.5 fmol); mutant ID30 H784Y) (24 ng / 24.7 nM / 246.7 fmol)). Amplification was performed on a CFX384™ C1000™ thermocycler system (Bio-Rad, Hercules, CA) using the following cycling parameters: an initial denaturation at 95 °C for 30 seconds, followed by 60 cycles of 10 seconds at 95 °C and then 30 seconds at 60 °C. The oligonucleotide reagents used in this example are shown in Table 30.

[0234]

Table 30

[0235] First, all reactions were performed in triplicate. Similar results were obtained for all replicates when wild-type OptiTaq was used. However, the results showed greater variability for the mutant polymerases. Therefore, to obtain statistically significant results, each reaction was performed 24 times for the mutant polymerases and 21 times for the wild-type enzyme. The ΔCq value was calculated as the Cq value obtained for each mismatched base pair - the Cq value obtained for the matched base pair (ΔCq = Cq mismatch - Cq match). The ΔCq values for all 24 replicates were averaged and the standard deviation was calculated. The results are shown in Table 31 and summarized graphically in Figures 3C, 3D, 3D, 3F, and 3G. Note that the reverse primer is an allele-specific primer. Thus, the "Syn Rev T" primer (SEQ ID NO: 55) is a perfect match to template A (SEQ ID NO: 51), etc.

[0236]

Table 31

[0237] Wild-type OptiTaq showed an average ΔCq for AS-qPCR in this synthetic amplicon system of 4.1 in the range from -0.8 to 8.5. Mutant ID3 (H784Q) showed an average ΔCq of 9.9 in the range from 4.6 to 21.2. Mutant ID20 (H784A) showed an average ΔCq of 11.2 in the range from 6.3 to 14.9. Mutant ID21 (H784S) showed an average ΔCq of 15.3 in the range from 6.6 to 25.8. Mutant ID22 (H784T) showed an average ΔCq of 6.6 in the range from 1.5 to 13.3. Mutant ID24 (H784V) showed an average ΔCq of 4.1 in the range from -0.3 to 10.2. Mutant ID26 (H784I) showed an average ΔCq of 5.0 in the range from 0.3 to 11.3. Mutant ID27 (H784M) showed an average ΔCq of 9.8 in the range from 4.5 to 16.7. Mutant ID29 (H784F) showed an average ΔCq of 7.8 in the range from 3.5 to 13.3. Mutant ID30 (H784Y) showed an average ΔCq of 8.3 in the range from 5.3 to 15.7. Thus, in almost all pairs of combinations of the four template bases and the four 3'-terminal primer bases, the mutant Taq DNA polymerase of the present invention showed greater discrimination against mismatches than wild-type OptiTaq DNA polymerase. The magnitude of the improvement for each mismatch pair is defined by ΔΔCq, which is the difference in discrimination between the mutant and wild-type enzymes (ΔΔCq = ΔCq mutant - ΔCq wild-type). The ΔΔCq values were calculated and are shown in Table 32.

[0238]

Table 32

[0239] The mutant ID3 (H784Q) showed an average ΔΔCq of 4.9 compared to the wild-type OptiTaq. The mutant ID20 (H784A) showed an average ΔΔCq of 7.1 compared to the wild-type OptiTaq. The mutant ID21 (H784S) showed an average ΔΔCq of 11.2 compared to the wild-type OptiTaq. The mutant ID22 (H784T) showed an average ΔΔCq of 2.5 compared to the wild-type OptiTaq. The mutant ID24 (H784V) showed an average ΔΔCq of -0.2 compared to the wild-type OptiTaq. The mutant ID26 (H784I) showed an average ΔΔCq of 1.0 compared to the wild-type OptiTaq. The mutant ID27 (H784M) showed an average ΔΔCq of 5.7 compared to the wild-type OptiTaq. The mutant ID29 (H784F) showed an average ΔΔCq of 3.6 compared to the wild-type OptiTaq. The mutant ID30 (H784Y) showed an average ΔΔCq of 4.2 compared to the wild-type OptiTaq. Thus, except for the mutant ID24 (H784V), each of the mutant Taq DNA polymerases of the present invention showed a significant improvement in mismatch discrimination compared to the wild-type OptiTaq. Overall, the mutant ID21 (H784S) showed the best SNP discrimination within the set of nine mutant enzymes studied in this example using the AS-PCR assay.

[0240] [Example 15] Improved Mismatch Discrimination in rhPCR Using Mutant Taq DNA Polymerases in a Human Genome DNA SNP Assay Example 14 demonstrated the utility of the novel mutant Taq DNA polymerase of the present invention in a synthetic amplicon rhPCR SNP discrimination assay system. This example demonstrated the utility of the novel mutant Taq DNA polymerase in a human genomic DNA rhPCR SNP discrimination assay system and examined the SNP site in the SMAD7 gene (NM_005904, C / T SNP, rs4939827). The assay employed target DNAs GM18562 (homozygous C / C) and GM18537 (homozygous T / T) from the Coriell Institute for Medical Research (Camden, NJ, USA). Two different block-type cleavage primer designs were tested, including Generation 1 (Gen1) "RDDDDx" primers and Generation 2 (Gen2) "RDxxD" primers (see U.S. Patent Application No. 2012 / 0258455 by Behlke et al. entitled RNASE H-BASED ASSAYS UTILIZING MODIFIED RNA MONOMERS).

[0241] Quantitative real-time rhPCR was performed in a 10 μL reaction volume in a 384-well format containing 20 ng (equivalent to 6600 copies of the target) of human genomic DNA (GM18562 or GM18537). The reaction utilized either 0.5 U (10.8 ng / 11.1 nM / 111 fmol) of wild-type OptiTaq DNA polymerase or 0.5 U of any one of nine Taq DNA polymerase mutants (MUT ID3, H784Q; MUT ID20, H784A; MUT ID21, H784S; MUT ID22, H784T; MUT ID24, H784V; MUT ID26, H784I; MUT ID27 H784M; MUT ID29, H784F; MUT ID30, H784Y). The final reaction conditions used were 20 mM Tris-HCL (pH 8.4 at 25°C), 50 mM KCL, 3 mM MgCl 2, 0.01% Triton X-100, 800 μM total dNTP, 200 nM forward primer (SEQ ID NOs: 75-79), 200 nM universal reverse primer (SEQ ID NO: 74), and 200 nM SMAD7 probe (SEQ ID NO: 80). The sequence of the 85 bp SMAD7 amplicon is shown as SEQ ID NO: 81. The forward primer included the RDDDDx-constructed Gen1 allele-specific rhPCR primer (SEQ ID NOs: 76 and 77), the RDxxD-constructed Gen2 allele-specific rhPCR primer (SEQ ID NOs: 78 and 79), and a non-allele-specific control universal forward primer (SEQ ID NO: 75). The oligonucleotide reagents employed in this example are shown in Table 33. The reaction was carried out at a concentration of 2.6 mU per 10 μL reaction (5 fmole, 0.5 nM), except for MUT ID21 (H784S) where 200 mU per 10 μL (384 fmole, 38.4 nM) was used for the Gen1 RDDDDx primer and the control primer (SEQ ID NOs: 75-77), or at a concentration of 200 mU per 10 μL reaction (384 fmole, 38.4 nM) for the Gen2 RDxxD primer (SEQ ID NOs: 78 and 79), and contained 1 μL of P.a.RNase H2. Amplification was performed on a Roche LightCycler® 480 (Roche Applied Science, Indianapolis, IN, USA) as follows: 3 minutes at 95°C, followed by 95 cycles of 10 seconds at 95°C and 30 seconds at 60°C. All reactions were performed in triplicate.

[0242]

Table 33

[0243] The results using the Gen1 RDDDDx rhPCR primers are shown in Table 34, and the results using the Gen2 RDxxD rhPCR primers are shown in Table 35. The use of the mutant Taq DNA polymerase showed a significant improvement in SNP discrimination in this human genomic DNA rhPCR assay using the Gen1 RDDDDx primers, but the amplification efficiency often decreased, as indicated by the increase in the matched Cq. A large improvement in discrimination was seen using the Gen2 RDxxD primers, but the amplification efficiency was often lost here as well. The Gen2 RDxxD primers essentially showed greater SNP discrimination, and these levels increased such that the ΔCq value was more than 40 amplification cycles between match and mismatch in some cases; since the qPCR reaction was rarely carried out beyond 45 - 50 cycles and a positive signal was not detected in these cases until after 70 cycles, this level of discrimination is "greater than the assay" for most users (Table 35). Thus, the use of the novel mutant Taq DNA polymerase improves SNP discrimination in the rhPCR genotyping assay.

[0244] [Table 34] TIFF2025081508000069.tif226166TIFF2025081508000070.tif16163

[0245] [Table 35] TIFF2025081508000072.tif226167TIFF2025081508000073.tif16163

[0246] The ΔCq values for the SMAD7 SNP genotyping assay are graphically summarized in FIGS. 5B and 5C for the Gen1 RDDDDx primers and in FIGS. 6B and 6C for the Gen2 RDxxD primers. It is also clear that the various mutant Taq DNA polymerases of the present invention are not only useful in different amplification assays, but also that different mutants exhibit various levels of benefit depending on the nature of the assay being used. Thus, it is beneficial to have a collection of mutant polymerases that can be matched to assays / applications with different characteristics to obtain maximum benefit.

[0247] [Example 16] Improved discrimination of low-frequency alleles in genomic DNA using rhPCR with mutant Taq DNA polymerase The use of Gen2 RDxxD block-type cleavage primers in rhPCR can detect the presence of SNPs at levels from 1:1,000 to 1:10,000 in the background of wild-type genomic DNA using native (wild-type) Taq DNA polymerase (see U.S. Patent Application No. 2012 / 0258455 by Behlke et al., entitled RNASE H-BASED ASSAYS UTILIZING MODIFIED RNA MONOMERS). This example demonstrates that the mutant Taq DNA polymerases of the present invention improve low-frequency allele discrimination in rhPCR assays.

[0248] The low-frequency allele detection experiment was designed to detect the nucleotide identity of the SNP site in the SMAD7 gene (NM_005904, C / T SNP, rs4939827), and the target DNAs GM18562 (homozygous C / C) and GM18537 (homozygous T / T) (Coriell Institute for Medical Research, Camden, NJ, USA) were employed. The control reactions were set up using 2 ng (660 copies), 0.2 ng (66 copies) or 0.02 ng (6.6 copies) of the input, matched target DNA. The low-frequency allele detection reactions were set up using 2 ng (660 copies), 0.2 ng (66 copies) or 0.02 ng (6.6 copies) of the input, matched target DNA + 200 ng (66,000 copies) of the other (mismatched) allele for one allele. The background was established in reactions containing 0 copies of the matched target DNA + 200 ng (66,000 copies) of the mismatched target DNA. Both combinations were tested: GM18562 (C / C) as the low-frequency allele in the presence of excess GM18537 (T / T) and GM18537 (T / T) as the low-frequency allele in the presence of excess GM18562 (C / C).

[0249] Quantitative real-time rhPCR was performed in a 10 μL reaction volume in a 384-well format. The final reaction conditions used were 10 mM Tris-HCL (pH 8.4 at 25°C), 50 mM KCL, 3.5 mM MgCl 2, 0.01% Triton X-100, 0.8 mM dNTP, one of 200 nM SMAD7 forward primers (SEQ ID NOs: 75, 78, and 79), 200 nM SMAD7 reverse primer (SEQ ID NO: 74), and 200 nM SMAD7 probe (SEQ ID NO: 80). The 85 bp SMAD7 amplicon defined by these primers is shown as SEQ ID NO: 81. Note that the forward primer was unmodified (control, SEQ ID NO: 75), or specific for the SMAD7 C-allele (SEQ ID NO: 78) or SMAD7 T-allele (SEQ ID NO: 79) using the block-type cleavage rhPCR Gen2 RDxxD design. The reaction utilized either 0.5 U of wild-type OptiTaq DNA polymerase or 0.5 U of one of the three Taq DNA polymerase mutants studied (MUT ID20 (H784A); MUT ID27 (H784M); MUT ID30 (H784Y)). The reaction contained P. abyssi RNase H2 at a concentration of 200 mU (384 fmole) per 10 μL reaction when using the SMAD7 For rC DxxD (SEQ ID NO: 78) primer and P. abyssi RNase H2 at a concentration of 500 - 600 mU (960 - 1152 fmole) per 10 μL reaction for the control reaction or when using the SMAD7 For rU DxxD (SEQ ID NO: 79) primer. The oligonucleotide reagents used in this example are shown in Table 36. Cycling was performed on a Roche LightCycler® 480 (Roche Applied Science, Indianapolis, IN, USA) as follows: 3 minutes at 95°C, followed by 65 cycles of 10 seconds at 95°C and 30 seconds at 60°C. All reactions were performed in triplicate.

[0250]

Table 36

[0251] The results are analyzed and shown in Table 37. The control column shows the Cq values for the matched primer / target reactions, with no mismatched targets present, to establish the quantitative standard curve. MUT ID NO.3, H784Q, is included in the data analysis for comparison. The low-frequency allele detection column shows the Cq values for the detection of 660, 66, 6, or 0 (background control) copies of the matched primer / target in the presence of 66,000 copies of the mismatched target. At least a 3-cycle difference (ΔCq = 3.0 or greater) between the background and the positive signal is required to call a "positive" reaction for low-frequency allele detection; a 5-cycle difference (ΔCq = 5.0 or greater) is generally assumed to be preferred. In this system, the background is the signal observed when amplification is performed using an input non-target that matches the primer, and thus, the signal only results from amplification originating from the mismatched target.

[0252] Using wild-type OptiTaq DNA polymerase, the detection of the "C" allele in an excess "T" background and the detection of the "T" allele in an excess "C" background both met the ΔCq 3.0 and ΔCq 5.0 levels of stringency required to be called a 1:1000 low-frequency allele detection event (66 copies of the matched target in the presence of 66,000 copies of the mismatched target). The 1:10,000 reaction (6 copies of the matched target in the presence of 66,000 copies of the mismatched target) did not meet either of these criteria. Thus, rhPCR had a 1:1000 low-frequency allele detection limit using wild-type OptiTaq in this genomic DNA SNP system.

[0253] In contrast, rhPCR using each of the four mutants showed a low-frequency allele detection limit of 1:10,000 for both the "C" and "T" allele targets, and the ΔCq stringency cutoff was 3.0. MUT ID3 (H784Q) showed a low-frequency allele detection limit of 1:10,000 for both the "C" and "T" targets in this genomic SNP system for a higher ΔCq stringency cutoff of 5.0. The other three mutant Taq DNA polymerases (MUT ID20 (H784A); MUT ID27 (H784M); MUT ID30 (H784Y)) showed a low-frequency allele detection limit of 1:10,000 for the "C" allele target with a ΔCq stringency cutoff of 5.0 and a low-frequency allele detection limit of 1:10,000 for the "T" allele target with a ΔCq stringency cutoff of 3.0. Therefore, the inventors conclude that the novel mutant Taq DNA polymerase of the present invention provides an improved low-frequency allele detection reaction using block-type cleavage primers in rhPCR as compared to the use of wild-type DNA polymerase.

[0254]

Table 37

[0255] [Example 17] Sequences of Taq DNA polymerase mutants showing improved discrimination for mismatches or the presence of RNA residues at the 3' end of primers The complete amino acid and nucleotide sequences of the codon-optimized mutant enzymes employed in Examples 11-15 are shown below. These sequences are readily derivable by those skilled in the art from the information provided in Tables 1, 3, 4, and 26, but for clarity, the finally assembled sequences are provided. Base changes are identified in bold underlined font for nucleic acid and amino acid substitutions.

[0256] Nucleotide sequence of SEQ ID NO: 146, Mutant ID 20 (H784A).

[0257] [Chemical formula]

[0258] Amino acid sequence of SEQ ID NO: 147, Mutant ID 20 (H784A).

[0259] [Chemical formula]

[0260] Nucleotide sequence of SEQ ID NO: 148, Mutant ID 21 (H784S).

[0261] [Chemical formula]

[0262] Amino acid sequence of SEQ ID NO: 149, Mutant ID 21 (H784S).

[0263] [Chemical formula]

[0264] Nucleotide sequence of SEQ ID NO: 150, Mutant ID 22 (H784T).

[0265] [Chemical formula]

[0266] Amino acid sequence of SEQ ID NO: 151, Mutant ID 22 (H784T).

[0267] [Chemical formula]

[0268] Nucleotide sequence of SEQ ID NO: 152, Mutant ID 24 (H784V).

[0269]

Chem.

[0270] Amino acid sequence of SEQ ID NO: 153, Mutant ID 24 (H784V).

[0271]

Chem.

[0272] Nucleotide sequence of SEQ ID NO: 154, Mutant ID 26 (H784I).

[0273]

Chem.

[0274] Amino acid sequence of SEQ ID NO: 155, Mutant ID 26 (H784I).

[0275]

Chem.

[0276] Nucleotide sequence of SEQ ID NO: 156, Mutant ID 27 (H784M).

[0277]

Chem.

[0278] Amino acid sequence of SEQ ID NO: 157, Mutant ID 27 (H784M).

[0279]

Chem.

[0280] Nucleotide sequence of SEQ ID NO: 158, Mutant ID 29 (H784F).

[0281] [Chem.]

[0282] Amino acid sequence of SEQ ID NO: 159, Mutant ID 29 (H784F).

[0283] [Chem.]

[0284] Nucleotide sequence of SEQ ID NO: 160, Mutant ID 30 (H784Y).

[0285] [Chem.]

[0286] Amino acid sequence of SEQ ID NO: 161, Mutant ID 30 (H784Y).

[0287] [Chem.]

[0288] [Example 18] Generation of a codon-optimized Taq DNA polymerase mutant modified to eliminate 5'exonuclease activity Additional Taq DNA polymerase mutants were generated that eliminated the 5'exonuclease activity of several mutants in Table 3. Taq DNA polymerase lacking 5'-exonuclease activity was previously named "KlenTaq" (Barnes, W.M., Gene 112: 29-35, 1992). Deletion of the N-terminal 5'exonuclease domain of Taq polymerase improves the enzyme's mismatch discrimination (Barnes, W.M., Gene 112: 29-35, 1992). This study characterized whether the specificity improvement seen in the Taq DNA polymerase mutants of the present invention was combined with mutations that eliminated 5'-exonuclease activity. The examples presented herein are intended to be illustrative and in no way limit the scope of the claims. Specific mutations were introduced into the OptiTaq sequence using the PCR site-directed mutagenesis method (Weiner MP et al., Gene., 151(1-2): 119-23 (1994)). Each mutagenesis reaction employed 10 pmol of two oligonucleotides (Table 38) to amplify around the plasmid containing the DNA polymerase, except for the 5'exonuclease domain. These primers were manufactured to contain 5'phosphates that would allow religation after amplification. Briefly, these primers were in 1×KOD PCR buffer with the previously characterized mutant DNA polymerases (MUT ID2, 3, 10, 18, 21, and 30) (20 ng each), 5 U KOD DNA polymerase (Novagen-EMD Chemicals, San Diego, CA), 1.5 mM MgSO 4Annealed to a double-stranded plasmid containing. The thermal cycle parameters were 25 cycles of 3 minutes at 95 °C (20 seconds at 95 °C - 20 seconds at 55 °C - 2 minutes at 70 °C), followed by an immersion at 70 °C for 4 minutes. After PCR site-directed mutagenesis, the amplification product was treated with 10 U of DpnI (NEB, Ipswish, MA) at 37 °C for 1 hour, and then inactivated at 80 °C for 20 minutes. One-sixth of the digested material was ligated with T4 DNA ligase (NEB, Ipswich, MA) at 16 °C for 20 minutes, followed by inactivation at 65 °C for 10 minutes. One-fifteenth of the ligated digested material was transformed into XL-1 Blue competent bacteria. Bacterial clones were isolated, plasmid DNA was prepared, and the deletion of the 5' exonuclease domain was confirmed by Sanger DNA sequencing. All mutants were retained in the pET-27b(+) expression vector suitable for expressing recombinant proteins in E. coli. As described in Example 3, the recombinant mutants of Taq polymerase were expressed and purified.

[0289]

Table 38

[0290] [Example 19] Characterization of the properties of seven 5'-exonuclease deletion mutants of Taq DNA polymerase in PCR The seven mutant Taq DNA polymerase enzymes described in Example 18 were characterized for polymerase activity.

[0291] The unit activity of the purified wild-type protein was determined by comparing the performance of known amounts of OptiTaq and each mutant in qPCR with that of commercially available non-hot start Taq DNA polymerase, Taq-B DNA polymerase (Enzymatics, Beverly, MA). The quantification cycle value (Cq, the number of amplification cycles at which a positive signal is first detected) and the amplification curve shape were analyzed to determine the nanogram amounts at which both enzymes perform similarly within the sub-optimal range for each. Using these nanogram amounts and the known unit value of Taq-B DNA polymerase, relative activity unit values could be extrapolated for all mutant DNA polymerase enzymes having sufficient activity to support PCR. Also, tests were conducted to determine the MgCl 2 concentration at which the polymerase exhibits optimal activity.

[0292] The following reaction conditions were employed: 1×qPCR buffer (20 mM Tris pH 8.4, 50 mM KCl, 0.01% Triton-X100) in a final volume of 10 μL, 800 μM dNTP (200 μM each), 500 nM For primer (Hs HPRT F517, SEQ ID NO: 43), 500 nM Rev primer (Hs HPRT R591, SEQ ID NO: 44), 250 nM RNase H2-cleavable probe (Hs HPRT RN2 probe, SEQ ID NO: 164), 20 mU Pyrococcus abyssi RNase H2, 2×10 3 copies of linearized cloned plasmid template (HPRT-targ, SEQ ID NO: 46). MgCl 2It was tested at 3, 4 or 5 mM in each case. The amount of DNA polymerase added to each reaction was varied as follows: for wild type (OptiTaq), the reactions were set up using 10, 1, 0.1, 0.01, 0.001 U / μL (220, 22, 2.2, 0.22 or 0.022 ng of protein per 10 μL reaction). The mutant polymerases were driven at similar concentrations. Also, those mutant enzymes showing polymerase activity were titrated more finely and tested with 220, 22, 10.6, 4.8, 2.2, 1.1, 0.48 and 0.22 ng of protein per 10 μL reaction. The polymerase dilutions were made in enzyme dilution buffer (20 mM Tris pH 7.5, 100 mM NaCl, 1 mM DTT, 0.1% Triton-X100, 1 mg / mL BSA, 10% glycerol). The reactions were carried out in 384-well format on a BIO-RAD CFX384™ real-time system (BIO-RAD, Hercules, CA) using cycling parameters of 30 seconds at 95°C followed by 60 cycles of [15 seconds at 95°C followed by 1 minute at 60°C]. Detection was achieved using a fluorescence-quenched probe (cleaved by the action of P.a.RNase H2 enzyme). The sequences of the primers, probes and templates (plasmid inserts) are shown in Table 39.

[0293]

Table 39

[0294] These seven Taq DNA polymerase 5'-exonuclease deletion mutants were characterized as outlined above. The results are summarized in Table 40. All seven mutants had DNA polymerase activity; however, the processing ability in mutants ID38, 39, 40, 41, 42, and 43 was reduced 10 - 50-fold compared to the wild-type enzyme. One mutant, ID37 (OptiTaq KlenTaq), showed DNA polymerase activity that was nearly identical to wild-type OptiTaq. Thus, in combination with point mutations that improve polymerase specificity, complete deletion of the 5'-exonuclease domain of Taq DNA polymerase all significantly impaired enzyme activity and processing ability.

[0295] [Table 40]

[0296] [Example 20] Improved Mismatch Discrimination in Allele-Specific PCR Using Mutant Taq DNA Polymerases That Also Have Deletions in the 5'-Exonuclease Domain Of the seven mutant enzymes studied in Examples 18 and 19, mutants ID37, 38, 39, 40, 41, 42, and 43 retained sufficient enzyme activity / processing ability to be characterized. These seven mutants were studied for their ability to discriminate 3'-terminal DNA mismatches compared to wild-type OptiTaq DNA polymerase using an allele-specific qPCR assay. The amplification reaction was performed on a synthetic oligonucleotide template in which a single base (SNP) located at the 3'-end of the reverse primer was varied. Synthetic templates with each of the four possible bases at this position were employed. Reverse primers with each of the four possible bases at the 3'-end were employed. Relative amplification efficiency was evaluated using qPCR.

[0297] Quantitative allele-specific real-time PCR (AS-qPCR) was performed with 2×10 5Using a 103 bp synthetic template for the copy (SEQ ID NOs: 51 - 54), it was performed in a 10 μL reaction volume in a 384 - well format. The final reaction conditions used were 20 mM Tris - HCL (pH 8.4 at 25°C), 50 mM KCL, an amount of MgCl 2 determined to be optimal for each polymerase in Example 19, 0.01% Triton X - 100, 800 μM total dNTP, as well as 200 nM universal forward primer (SEQ ID NO: 60), 200 nM reverse primer (separate reactions were set up for each of the allele - specific primer SEQ ID NOs: 55 - 58 or the control universal primer SEQ ID NO: 59), and 200 nM RNase H2 - cleavable probe (SEQ ID NO: 165). Also, 20 mU Pyrococcus abyssi RNase H2 was included in each reaction. Each allele - specific primer was tested based on its respective SNP template. The reaction was carried out using either 0.5 U (10.8 ng / 11.1 nM / 111 fmol) of OptiTaq KlenTaq DNA polymerase (mutant ID37) or 0.5 U of one of the six Taq DNA polymerase mutants studied (mutant ID38 (108 ng / 111 nM / 1110 fmol); mutant ID39 (216 ng / 222 nM / 2220 fmol); mutant ID40 (360 ng / 370 nM / 3700 fmol); mutant ID41 (1060 ng / 555 nM / 5550 fmol); mutant ID42 (1060 ng / 555 nM / 5550 fmol); mutant ID43 (216 ng / 222 nM / 2220 fmol)). Amplification was carried out on a CFX384™ C1000™ thermocycler system (Bio - Rad, Hercules, CA) using the following cycling parameters: an initial denaturation at 95°C for 30 seconds, followed by 60 cycles of 10 seconds at 95°C and then 30 seconds at 60°C. The oligonucleotide reagents used in this example are shown in Table 41.

[0298]

Table 41

[0299] First, all reactions were performed in triplicate. When wild-type OptiTaq was used, similar results were obtained for all replicates. However, the results showed greater variability for the mutant polymerases. Therefore, to obtain statistically significant results, each reaction was performed 24 times for the mutant polymerases and 21 times for the wild-type enzyme. The ΔCq value was calculated as the Cq value obtained for each mismatched base pair - the Cq value obtained for the matched base pair (ΔCq = Cq mismatch - Cq match). The ΔCq values for all 24 replicates were averaged and the standard deviation was calculated. The results are shown in Table 42 and summarized graphically in Figures 7A, 7B, and 7C. Note that the reverse primer is an allele-specific primer. Thus, the "Syn Rev T" primer (SEQ ID NO: 55) is a perfect match to template A (SEQ ID NO: 51), etc.

[0300]

Table 42

[0301] OptiTaq KlenTaq mutant ID37 showed an average ΔCq for AS-qPCR in this synthetic amplicon system of 9.8 in the range from 3.9 to 14.7. Mutant ID38 (A661E, I665W, F667L KlenTaq) showed an average ΔCq of 11.9 in the range from 7.9 to 17.4. Mutant ID39 (V783F KlenTaq) showed an average ΔCq of 11.3 in the range from 6.9 to 17.1. Mutant ID40 (H784Q KlenTaq) showed an average ΔCq of 11.9 in the range from 7.9 to 18.2. Mutant ID41 (V783L H784Q KlenTaq) showed an average ΔCq of 10.5 in the range from 5.8 to 15.8. Mutant ID42 (H784S KlenTaq) showed an average ΔCq of 11.9 in the range from 8.3 to 15.8. Mutant ID43 (H784Y KlenTaq) showed an average ΔCq of 11.2 in the range from 6.5 to 15.5. Thus, in all combinations of pairs of all four template bases and all four 3'-terminal primer bases, the mutant Taq DNA polymerase of the present invention showed greater discrimination against mismatches than OptiTaq or OptiTaq KlenTaq DNA polymerase. The magnitude of the improvement for each mismatch pair is defined by ΔΔCq, which is the difference in discrimination between the mutant and wild-type KlenTaq enzymes (ΔΔCq = ΔCq mutant KlenTaq - ΔCq OptiTaq KlenTaq). The ΔΔCq values were calculated and are shown in Table 43.

[0302]

Table 43

[0303] The mutant ID38 (A661E, I665W, F667L KlenTaq) showed an average ΔΔCq of 1.7 compared to OptiTaq KlenTaq. The mutant ID39 (V783F KlenTaq) showed an average ΔΔCq of 2.0 compared to OptiTaq KlenTaq. The mutant ID40 (H784Q KlenTaq) showed an average ΔΔCq of 2.1 compared to OptiTaq KlenTaq. The mutant ID41 (V783L H784Q KlenTaq) showed an average ΔΔCq of 0.7 compared to OptiTaq KlenTaq. The mutant ID42 (V784S KlenTaq) showed an average ΔΔCq of 2.1 compared to OptiTaq KlenTaq. The mutant ID43 (H784Y KlenTaq) showed an average ΔΔCq of 2.0 compared to OptiTaq KlenTaq. Thus, each of the mutant Taq DNA polymerases of the present invention has a complete deletion of the 5'-exonuclease domain, but showed a significant improvement in mismatch discrimination compared to OptiTaq KlenTaq which contains no other second mutations. Overall, mutants ID40 and 42 (H784Q KlenTaq and H784S KlenTaq) showed the best SNP discrimination within the set of mutant enzymes studied in this example using the AS-PCR assay.

[0304] [Example 21] Improved Mismatch Discrimination in rhPCR Using Mutant KlenTaq DNA Polymerase in a Human Genome DNA SNP Assay Example 20 demonstrated the usefulness of the novel mutant Taq DNA polymerase of the present invention in a synthetic amplicon rhPCR SNP discrimination assay system. This example demonstrated the usefulness of the novel mutant Taq DNA polymerase in a human genomic DNA rhPCR SNP discrimination assay system and examined the SNP site in the SMAD7 gene (NM_005904, C / T SNP, rs4939827). The assay employed target DNAs GM18562 (homozygous C / C) and GM18537 (homozygous T / T) from the Coriell Institute for Medical Research (Camden, NJ, USA). One block-type cleavage primer design, Generation 1 (Gen1) "RDDDDx" primer (see U.S. Patent Application No. 2012 / 0258455 by Behlke et al. entitled RNASE H-BASED ASSAYS UTILIZING MODIFIED RNA MONOMERS) was tested.

[0305] Quantitative real-time rhPCR was performed in a 10 μL reaction volume in a 384-well format containing 20 ng (equivalent to 6600 copies of the target) of human genomic DNA (GM18562 or GM18537). The reaction utilized either 0.5 U (10.8 ng / 11.1 nM / 111 fmol) of OptiTaq KlenTaq DNA polymerase or 0.5 U of one of three Taq DNA polymerase mutants (mutant ID40 (360 ng / 370 nM / 3700 fmol); mutant ID41 (1060 ng / 555 nM / 5550 fmol); mutant ID43 (216 ng / 222 nM / 2220 fmol)). The final reaction conditions used were 20 mM Tris-HCL (pH 8.4 at 25 °C), 50 mM KCL, 3 mM MgCl 2, 0.01% Triton X-100, 800 μM total dNTP, 200 nM forward primer (SEQ ID NOs: 75-79), 200 nM universal reverse primer (SEQ ID NO: 74), and 200 nM RNase H2-cleavable SMAD7 probe (SEQ ID NO: 166). The 85 bp SMAD7 amplicon is shown as SEQ ID NO: 81. The forward primers included the RDDDDx-constructed Gen1 allele-specific rhPCR primers (SEQ ID NOs: 76 and 77), and a non-allele-specific control universal forward primer (SEQ ID NO: 75). The oligonucleotide reagents employed in this example are shown in Table 44. The reaction contained 1 μL of P.a.RNase H2 at a concentration of 2.6 mU (5 fmole, 0.5 nM) per 10 μL reaction. Amplification was performed on a Roche LightCycler® 480 (Roche Applied Science, Indianapolis, IN, USA) as follows: 3 minutes at 95°C, followed by 95 cycles of 10 seconds at 95°C and 30 seconds at 60°C. All reactions were performed in triplicate.

[0306] [Table 44]

[0307] The results using the Gen1 RDDDDx rhPCR primers are shown in Table 45. The use of mutant Taq DNA polymerase showed a significant improvement in SNP discrimination in this human genomic DNA rhPCR assay using the Gen1 RDDDDx primers, but the amplification efficiency often decreased as indicated by an increase in the match Cq. Thus, the use of the novel mutant KlenTaq DNA polymerase improves SNP discrimination in the rhPCR genotyping assay.

[0308] [Table 45]

[0309] [Example 22] Sequence of Taq DNA polymerase mutants showing improved discrimination regarding mismatches or presence of RNA residues at the 3'-end of primers The complete amino acid and nucleotide sequences of the codon-optimized mutant enzymes employed in Examples 18-21 are shown below. These sequences can be readily derived by those skilled in the art from the information provided in Tables 1, 3, 4, 26 and 38, but for clarity, the finally assembled sequences are provided. Base changes are identified in a font with bold underlining for nucleic acid substitutions and amino acid substitutions.

[0310] SEQ ID NO: 167, nucleotide sequence of mutant ID37 (OptiTaq KlenTaq).

[0311]

Chem.

[0312] SEQ ID NO: 168, amino acid sequence of mutant ID37 (OptiTaq KlenTaq).

[0313]

Chem.

[0314] SEQ ID NO: 169, nucleotide sequence of mutant ID38 (A661E, I665W, F667L KlenTaq).

[0315]

Chem.

[0316] SEQ ID NO: 170, amino acid sequence of mutant ID38 (A661E, I665W, F667L KlenTaq).

[0317]

Chem.

[0318] Nucleotide sequence of Accession No. 171, Mutant ID 39 (V783F KlenTaq).

[0319]

Chem.

[0320] Amino acid sequence of Accession No. 172, Mutant ID 39 (V783F KlenTaq).

[0321]

Chem.

[0322] Nucleotide sequence of Accession No. 173, Mutant ID 40 (H784Q KlenTaq).

[0323]

Chem.

[0324] Amino acid sequence of Accession No. 174, Mutant ID 40 (H784Q KlenTaq).

[0325]

Chem.

[0326] Nucleotide sequence of Accession No. 175, Mutant ID 41 (V783L H784Q KlenTaq).

[0327]

Chem.

[0328] Amino acid sequence of Accession No. 176, Mutant ID 41 (V783L H784Q KlenTaq).

[0329] [Chemical]

[0330] Nucleotide sequence of SEQ ID NO: 177, Mutant ID42 (H784S KlenTaq).

[0331] [Chemical]

[0332] Amino acid sequence of SEQ ID NO: 178, Mutant ID42 (H784S KlenTaq).

[0333] [Chemical]

[0334] Nucleotide sequence of SEQ ID NO: 179, Mutant ID43 (H784Y KlenTaq).

[0335] [Chemical] TIFF2025081508000120.tif53155

[0336] Amino acid sequence of SEQ ID NO: 180, Mutant ID43 (H784Y KlenTaq).

[0337] [Chemical]

[0338] Incorporation by reference All publications, patents, patent applications, and deposit number data described herein are hereby incorporated by reference in their entirety, as if each individual publication, patent, patent application, or deposit number data were specifically and individually indicated to be incorporated by reference. In the case of citation and reference to deposit number data, the corresponding DNA polymerase amino acid and nucleotide sequences are hereby incorporated by reference as if such sequences were disclosed by SEQ ID NO. In case of conflict, this application, including any definitions herein, will control.

[0339] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. In regard to substantially any plural and / or singular terms used herein, those skilled in the art can translate from the plural to the singular as appropriate for the context and / or application. Various plural / singular permutations may be explicitly set forth herein for clarity.

[0340] Although the invention has been described with reference to particular embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention. Furthermore, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Accordingly, the invention is not intended to be limited to the particular embodiments or examples disclosed, but the invention is intended to cover all embodiments included within the scope of the appended claims.

Claims

1. A mutant Taq DNA polymerase having increased template discrimination activity compared to an unmodified Taq DNA polymerase of SEQ ID NO: 1, wherein the mutant Taq DNA polymerase consists of SEQ ID NO: 85 or 174, and the increased template discrimination activity comprises at least one property selected from the group consisting of increased 3'-mismatch discrimination and increased 3'-nucleotide discrimination.

2. 2. The mutant Taq DNA polymerase of claim 1, wherein the level of polymerase activity is at least about 0.01-fold the polymerase activity of the unmodified Taq DNA polymerase of SEQ ID NO:

1.

3. 2. The mutant Taq DNA polymerase of claim 1, wherein the increased template discrimination activity comprises increased 3'-mismatch discrimination, and wherein the mutant Taq DNA polymerase has an average ΔΔCq of at least about 1.0 when assessed for increased 3'-mismatch discrimination by an allele-specific PCR assay.

4. 2. The mutant Taq DNA polymerase of claim 1, wherein the increased template discrimination activity comprises increased 3'-nucleotide discrimination, and when the mutant Taq DNA polymerase is assessed for increased 3'-nucleotide discrimination by quantitative PCR, the average ΔΔCq is at least about 1.

0.

5. 2. The mutant Taq DNA polymerase of claim 1, wherein the increased template discrimination activity comprises increased 3'-mismatch discrimination by rhPCR, and wherein the mutant Taq DNA polymerase has an average ΔΔCq of at least about 1.0 when assessed for increased 3'-mismatch discrimination by rhPCR using RDDDDx blocked-cleavable rhPCR primers.

6. 2. The mutant Taq DNA polymerase of claim 1, wherein the increased template discrimination activity comprises increased 3'-mismatch discrimination by rhPCR, and wherein the mutant Taq DNA polymerase has an average ΔΔCq of at least about 0.50 when assessed for increased 3'-mismatch discrimination by rhPCR using RDxxD blocked-cleavable rhPCR primers.

7. 2. The mutant Taq DNA polymerase of claim 1, wherein the increased template discrimination activity comprises increased 3'-mismatch discrimination by rhPCR SNP discrimination assay, and wherein the mutant Taq DNA polymerase has an average ΔΔCq of at least about 1.0 when assessed for increased 3'-mismatch discrimination by rhPCR SNP discrimination assay of SMAD7 gene (NM_005904, C / T SNP, rs4939827) using RDDDDx blocked-cleavable rhPCR primers consisting of SEQ ID NOs:76 and 77.

8. 2. The mutant Taq DNA polymerase of claim 1, wherein the increased template discrimination activity comprises increased 3'-mismatch discrimination by rhPCR SNP discrimination assay, and wherein the mutant Taq DNA polymerase has an average ΔΔCq of at least about 1.0 when assessed for increased 3'-mismatch discrimination by rhPCR SNP discrimination assay of SMAD7 gene (NM_005904, C / T SNP, rs4939827) using RDxxD blocked-cleavable rhPCR primers consisting of SEQ ID NOs:78 and 79.

9. 2. The mutant Taq DNA polymerase of claim 1, wherein the increased template discrimination activity comprises increased 3'-mismatch discrimination by rhPCR SNP discrimination assay, and wherein the mutant Taq DNA polymerase has an average ΔΔCq of at least about 5.0 when assessed for increased 3'-mismatch discrimination by rhPCR SNP discrimination assay of SMAD7 gene (NM_005904, C / T SNP, rs4939827) using RDxxD blocked-cleavable rhPCR primers consisting of SEQ ID NOs:78 and 79.

10. 2. The mutant Taq DNA polymerase of claim 1, wherein the increased template discrimination activity comprises increased low frequency allele discrimination, and wherein the mutant Taq DNA polymerase has an increased low frequency allele discrimination of at least 1:10,000 when assessed by an rhPCR SNP discrimination assay of the SMAD7 gene (NM_005904, C / T SNP, rs4939827) with a ΔCq of at least 3.0 using RDxxD blocked-cleavable rhPCR primers consisting of SEQ ID NOs:78 and 79.

11. 10. A kit for generating extended primers comprising at least one container providing the mutant DNA polymerase of claim 1.

12. 12. The kit of claim 11, further comprising one or more additional containers selected from the group consisting of: (a) a container providing a primer capable of hybridizing to a predetermined polynucleotide template under primer extension conditions; (b) a container providing a nucleoside triphosphate; and (c) a container providing a buffer suitable for primer extension.

13. 12. The kit of claim 11, further comprising one or more additional containers selected from the group consisting of: (a) a container containing a blocked-cleavable primer, and (b) a container containing RNase H2.

14. A reaction mixture comprising the mutant DNA polymerase of claim 1, at least one primer, a polynucleotide template, and nucleoside triphosphates.

15. 15. The reaction mixture of claim 14, wherein at least one primer comprises a blocked-cleavable primer.

16. 15. The reaction mixture of claim 14, further comprising RNase H2.

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