KR101958659B1 - Dna polymerases with increased mutation specific amplification - Google Patents

Abstract

The present invention relates to a DNA polymerase having an increased specific gene amplification efficiency and a use thereof. More specifically, the present invention relates to a DNA polymerase which is mutated at a specific amino acid position to increase gene-specific amplification efficiency, A vector comprising the nucleic acid sequence, and a host cell transformed with the vector. The present invention also provides a method for detecting at least one gene mutation or SNP in vitro in at least one template using a DNA polymerase having increased gene mutation-specific amplification efficiency, a method for detecting a gene containing the DNA polymerase A variant or a composition for detecting SNP, and a PCR kit comprising the composition.

Description

DNA POLYMERASES WITH INCREASED MUTATION SPECIFIC AMPLIFICATION [0002]

The present invention relates to a DNA polymerase having an increased specific gene amplification efficiency and a use thereof. More specifically, the present invention relates to a DNA polymerase which is mutated at a specific amino acid position to increase gene-specific amplification efficiency, A vector comprising the nucleic acid sequence, and a host cell transformed with the vector. The present invention also provides a method for detecting at least one gene mutation or SNP in vitro in at least one template using a DNA polymerase having increased gene mutation-specific amplification efficiency, a method for detecting a gene containing the DNA polymerase A variant or a composition for detecting SNP, and a PCR kit comprising the composition.

Since the first human genome sequence has been discovered, researchers have focused on finding genetic differences between individuals, such as single base mutations (single nucleotide polymorphisms, SNPs). It is of interest because it is becoming increasingly clear that single base mutations in the genome are associated with different drug resistance or disease predisensors for a wide variety of diseases. Knowledge of future, clinically relevant nucleotide variations can apply treatments to the individual's genetic supply and prevent drug treatment that can be ineffective or cause side effects (Shi, Expert Rev. Mol. Diagn 1, 363-365 (2001)). The development of techniques that enable time and cost-effective identification of nucleotide variations will lead to further advances in pharmacogenetics.

 SNPs occupy a major genetic variation in the human genome and cause over 90% of differences among individuals (Kwok, Annu. Rev. Genomics Hum, Genet. 2, 235-258 (2001); Kwok and Chen, Curr. Mol. Biol., 5, 43-60 (2003); Twyman and Primrose, Pharmacogenomics 4, 67-79 (2003)). In order to detect other nucleic acid variations, such as genetic mutations and mutations, a variety of methods can be used. For example, identifying a variant of a target nucleic acid can be accomplished by hybridizing a nucleic acid sample to be analyzed with a hybridizing primer specific for the sequence variant under appropriate hybridization conditions (Guo et al., Nat. Biotechnol. 15, 331- 335 (1997)).

However, it has been found that such hybridization methods do not, in particular, satisfy clinical requirements in terms of the essential sensitivity of the assay. Thus, PCR has been used extensively in molecular biology and diagnostic testing methods for mutation detection such as SNPs and other allelic sequence variants (Saiki et al., Science 239, 487-490 (1988)), where the presence of variants The target nucleic acid to be examined was amplified by polymerase chain reaction (PCR) before hybridization. As hybridization probes for such assays, single stranded oligonucleotides are generally used. Modified embodiments of the assay include using fluorescent hybridization probes (Livak, Genet. Anal. 14, 143-149 (1999)). In general, efforts have been made to automate methods of measuring SNPs and other sequence variations (Gut, Hum. Mutat. 17, 475-492 (2001)).

An alternative to the sequence mutation-specific hybridization known in the art is provided by so-called gene mutation-specific amplification. In this detection method, during amplification, a mutation-specific amplification primer is used, which usually has a so-called differential terminal nucleotide residue at the 3'-end of the primer, and the residue is only one specific variation of the target nucleic acid to be detected Only complementary. In this method, the nucleotide variants are measured by the presence or absence of the DNA product after PCR amplification. The principle of gene mutation-specific amplification is based on the formation of a canonical or non-standard primer-template complex at the end of a gene mutation-specific amplification primer. At precisely paired 3 'primer ends, amplification by DNA polymerase occurs while extension at the mismatched primer ends is suppressed.

For example, U.S. Pat. Pat. No. 5,595,890 discloses methods for gene mutation-specific amplification and its application for detection of point mutations that are clinically associated with, for example, the k-ras oncogene. Also, U.S. Pat. Pat. No. 5,521,301 discloses an allele-specific amplification method for genotype analysis of the ABO blood group system. In contrast, U.S.A. Pat. No. 5,639,611 discloses the use of allele-specific amplification associated with the detection of point mutations that cause sickle cell anemia. However, gene mutation-specific amplification or allele-specific amplification is problematic in that it has low selectivity, which requires more complex and time- and cost-intensive optimization steps.

Such a method for detecting sequence mutations, polymorphisms and mainly point mutations is particularly useful when allelic-specific amplification (or deletion) is required when the sequence variant to be detected is deficient compared to the dominant variant of the same nucleic acid segment Gene mutation-specific amplification).

This situation arises, for example, when the sporadic tumor cells are detected in body fluids such as blood, serum or plasma by gene mutation-specific amplification (U.S. Pat. No. 5,496,699). To this end, DNA is first isolated from body fluids such as blood, serum or plasma, and DNA consists of deficient, sporadic tumor cells and excess non-proliferative cells. Thus, mutations that are important in tumor DNA in the k-ras gene should be detected from several replicates in the presence of excess wild-type DNA.

All methods for gene-specific amplification disclosed in the prior art have the disadvantage that 3 ' -terminal oligonucleotide residues must be used. Also, despite the use of 3 ' -different nucleotide residues, they have the disadvantage that primer extension occurs at low levels in the presence of the appropriate DNA polymerase even if the target nucleic acid does not exactly match the sequence variant that is to be detected. In particular, when a particular sequence variant is detected with an excess of background nucleic acid comprising another sequence variant, the false positive result will result. The main reason for the disadvantage of this PCR-based method is the incompatibility of the polymerase used in the method for sufficiently discriminating mismatched bases. Therefore, it is not yet possible to directly obtain clear information on the presence or absence of mutation in PCR. To date, additional time and cost intensive purification and assay methods have been required for a clear diagnosis of mutations. Therefore, a novel method for improving the selectivity of gene mutation-specific or allele-specific PCR amplification will have a significant impact on the reliability and robustness of direct gene mutation or SNP analysis by PCR.

The present inventors have made efforts to develop a novel DNA polymerase capable of enhancing the selectivity of gene mutation-specific PCR amplification. As a result, when a mutation is caused in an amino acid residue at a specific position of Taq polymerase, The efficiency was remarkably increased, and the present invention was completed.

Disclosure of the Invention The present invention provides a DNA polymerase for detecting one or more gene mutations or SNPs in a target sequence containing a gene mutation or SNP.

Another object of the present invention is to provide a nucleic acid sequence encoding a DNA polymerase according to the present invention, a vector containing the nucleic acid sequence, and a host cell transformed with the vector.

Still another object of the present invention is to provide a method for producing a DNA polymerase according to the present invention.

It is still another object of the present invention to provide a method for in vitro detection of one or more gene mutations or SNPs in one or more templates using the DNA polymerase of the present invention.

Still another object of the present invention is to provide a composition for detecting a gene mutation or SNP comprising a DNA polymerase according to the present invention.

It is still another object of the present invention to provide a kit for detecting a gene mutation or SNP comprising a composition for detecting a gene mutation or a SNP according to the present invention.

In order to achieve the above object, the present invention provides a DNA polymerase having a Taq polymerase comprising the amino acid sequence of SEQ ID NO: 1,

(a) substitution of the 507th amino acid residue in the amino acid sequence of SEQ ID NO: 1; And

(b) a substitution of a 536th amino acid residue, a substitution of a 660th amino acid residue, or a substitution of both 536th and 660th amino acid residues in the amino acid sequence of SEQ ID NO: 1.

According to a preferred embodiment of the present invention, substitution of the 507th amino acid residue is replaced by lysine (K) in glutamic acid (E), and substitution of arginine (R) to lysine (K) , And the substitution of the 660th amino acid residue may be substituted with valine (V) in arginine (R).

According to another preferred embodiment of the present invention, the DNA polymerase distinguishes between a mismatched primer and a mismatched primer, wherein the mismatched primer and the mismatched primer are hybridized with the target sequence, May include non-standard nucleotides at the 3 ' end thereof for the target sequence to be hybridized.

According to another preferred embodiment of the present invention, the DNA polymerase has an improved Ct value (higher amplification efficiency) than the amplification of the target sequence containing the mismatched primer in amplification of the target sequence containing the matched primer Lt; / RTI >

The present invention also provides a nucleic acid sequence encoding a DNA polymerase according to the present invention, a vector comprising the nucleic acid sequence, and a host cell transformed with the vector.

The present invention also relates to a method for producing a host cell comprising the steps of: culturing the host cell; And separating the DNA polymerase from the culture and the culture supernatant thereof.

The present invention also relates to a DNA polymerase according to the present invention

a) one or more molds;

b) one or more matched primers, one or more mismatched primers or one or more matched primers and one or more mismatched primers; And

c) contacting with a nucleoside triphosphate,

Wherein the one or more matched primers and the mismatched primers are hybridized to the target sequence and the mismatched primers hybridize to non-canonical nucleotides at up to seven base positions from its 3 ' A method for in vitro detection of one or more gene mutations or SNPs in one or more templates.

According to one preferred embodiment of the present invention, the method may comprise melting point analysis using a double-strand specific dye.

According to another preferred embodiment of the present invention, the method comprises real-time PCR, analysis on agarose gel after standard PCR, gene-specific amplification or allele-specific amplification through real-time PCR, tetra-primer amplification- Lt; RTI ID = 0.0 > mutation < / RTI > system PCR or isothermal amplification.

The present invention also provides a composition for detecting a gene mutation or SNP comprising a DNA polymerase according to the present invention.

The present invention also provides a PCR kit comprising a composition for detecting said gene mutation or SNP.

According to a preferred embodiment of the present invention, the PCR kit further comprises one or more matched primers, one or more mismatched primers or one or more matched primers and one or more mismatched primers, Primer and one or more mismatched primers are hybridized to the target sequence and the mismatched primer comprises non-canonical nucleotides at positions 3 to 7 bases from its 3 ' end to the hybridizing target sequence .

According to another preferred embodiment of the present invention, the PCR kit may further comprise nucleoside triphosphate.

According to another preferred embodiment of the present invention, the PCR kit comprises

a) one or more buffers;

b) a reagent for quantification binding to double stranded DNA;

c) a polymerase blocking antibody;

d) one or more control values or control sequences; And

e) one or more molds; May be further included.

Since the DNA polymerase of the present invention having an increased gene amplification-specific amplification efficiency has a higher mismatch-to-matched kidney selectivity than the conventional Taq polymerase, a reliable gene mutation-specific amplification is possible without any substrate modification do. In addition, the DNA polymerase of the present invention can be usefully used for medical diagnosis of diseases and research on recombinant DNA.

FIG. 1 shows a preparation process of a Taq DNA polymerase containing R536K, R660V and R536K / R660V mutations, wherein (a) is a schematic representation of fragment PCR and overlap PCR, and (b) (C) shows the results obtained by amplifying the total length by overlap PCR and confirming the amplified products by electrophoresis. FIG.
FIG. 2 shows the results of electrophoresis of the overlap PCR product of purified pUC19 vector treated with SAP and digested with restriction enzyme EcoRI / XbaI and purified (FIG. 1 (c)) for gel extraction.
FIG. 3 is a schematic representation of fragment PCR and overlap PCR during the preparation of Taq DNA polymerase comprising E507K, E507K / R536K, E507K / R660V and E507K / R536K / R660V mutations, respectively.
FIG. 4 shows the results of electrophoresis of the overlap PCR product of FIG. 3 purified with pUC19 vector treated with SAP after digestion with restriction enzyme EcoRI / XbaI for gel extraction.
FIG. 5 is a diagram illustrating a process of preparing a PCR template by collecting oral epithelial cells.
Fig. 6 shows the results of AS-qPCR performed on rs1408799 using the E507K / R536K, E507K / R660V and E507K / R536K / R660V Taq polymerase of the present invention. As a control group, Taq polymerizing Enzyme was used.
FIG. 7 shows the results of AS-qPCR performed on rs1015362 using Taq polymerase having the E507K / R536K, E507K / R660V and E507K / R536K / R660V mutations of the present invention, and the control group includes the E507K mutation Taq polymerase was used.
FIG. 8 shows the results of performing AS-qPCR on rs4911414 using Taq polymerase having the E507K / R536K, E507K / R660V and E507K / R536K / R660V mutations of the present invention, and the control group includes the E507K mutation Taq polymerase was used.

Hereinafter, the present invention will be described in more detail.

As described above, in order to improve the drawbacks of the gene mutation-specific amplification method disclosed in the prior art, there is a continuing need to develop a method for improving the selectivity of gene mutation-specific amplification efficiency. Will have a significant impact on the reliability and robustness of direct SNP analysis by PCR. As a result of efforts to develop a method for improving the selectivity of gene mutation-specific PCR amplification, the present inventors have found that when a mutation is caused in an amino acid residue at a specific position of Taq polymerase, the gene mutation-specific amplification efficiency is remarkably increased And completed the present invention.

Since the DNA polymerase of the present invention having an increased gene amplification-specific amplification efficiency has a higher mismatch-to-matched kidney selectivity than the conventional Taq polymerase, a reliable gene mutation-specific amplification is possible without any substrate modification do. In addition, the DNA polymerase of the present invention can be usefully used for medical diagnosis of diseases and research on recombinant DNA.

Hereinafter, terms used in the present application will be described.

&Quot; Amino acid " refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. As used herein, the term "amino acid" includes the following 20 natural or genetically encoded alpha-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N) (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Gln or G) (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).

Amino acids are typically organic acids, which include substituted or unsubstituted amino groups, substituted or unsubstituted carboxy groups, and one or more side chains or groups, or any analog of these groups. Exemplary side chains include, for example, thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynyl, Boronate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thio acid, hydroxylamine, or any combination of these groups.

Other exemplary amino acids include, but are not limited to, amino acids, including photoactivatable crosslinking agents, metal-bonded amino acids, spin-labeled amino acids, fluorescent amino acids, metal-containing amino acids, amino acids with novel functionalities, Amino acids that are covalently or non-covalently interacting, light caged and / or photoisomerizable amino acids, radioactive amino acids, biotin or biotin analogs, glycosylated amino acids, other carbohydrate modified amino acids, Carbon-linked sugar-containing amino acids, redox-active amino acids, aminothioic acid-containing amino acids, and one or more toxic < RTI ID = 0.0 > Amino acids.

In the DNA polymerase of the present invention, the term " mutant " means a recombinant polypeptide comprising at least one amino acid substitution relative to the corresponding naturally occurring or unmodified DNA polymerase.

The term " thermostable polymerase " (which refers to a thermostable enzyme) is thermally resistant and has sufficient activity to achieve subsequent polynucleotide elongation reactions and is subjected to elevated temperatures for the time required to achieve denaturation of the double- It is not irreversibly denatured (inactivated). As used herein, it is suitable for use at temperatures that cyclize reactions such as PCR. The irreversible denaturation here is permanent and refers to a complete loss of enzyme activity. For thermostable polymerases, the enzyme activity refers to catalyzing the combination of nucleotides in a suitable manner to form a polynucleotide elongation product complementary to the template nucleic acid strand. Thermostable bacterial-derived thermostable DNA polymerases include, for example, the following: Thermotoga maritima, Thermus aquaticus, Thermus thermophilus, Thermus flabus, Thermodipyliporphis, Thermus species DNA polymerase derived from Sps17, Thermus species Z05, Thermus caldophilus, Bacillus caldotenax, Thermotoga neopolitanica, and Thermosipo africanus.

The term " thermoactive " refers to an enzyme that retains its catalytic properties at the temperatures typically used in reverse transcription or annealing / extension steps in RT-PCR and / or PCR reactions (i.e., 45-80 ° C). Thermostable enzymes are those that are irreversibly inactivated or denatured when treated with elevated temperatures required for nucleic acid denaturation. The thermoactive enzyme may or may not be thermostable. The thermoactive DNA polymerase may be DNA or RNA that is dependent on thermophilic species or mesophilic species, including, but not limited to,

The term " host cell " refers to both single-cellular prokaryotic and eukaryotic organisms (e. G., Bacteria, yeast, and actinomycetes) and single cells from higher plants or animals when cultured in cell culture media.

The term " vector " is a DNA molecule capable of replicating foreign DNA such as a gene to a recipient cell, such as plasmid, phage, and artificial chromosome. &Quot; Plasmid ", " vector " or " plasmid vector " may be used interchangeably herein.

The term " nucleotide " is a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) present in the form of a single strand or a double strand, Lt; RTI ID = 0.0 > nucleotides < / RTI >

The term " nucleic acid " or " polynucleotide " refers to a polymer that can correspond to DNA or RNA polymers, or analogs thereof. The nucleic acid can be, for example, a chromosome or a chromosome segment, a vector (e.g., an expression vector), an expression cassette, a naked DNA or RNA polymer, a product of a polymerase chain reaction (PCR), an oligonucleotide, Or may include the same. The nucleic acid may be, for example, single-stranded, double-stranded, or tri-stranded, but is not limited to any particular length. Unless otherwise stated, a particular nucleic acid sequence includes or encodes a complementary sequence in addition to any sequence specified.

The term " primer " refers to a polynucleotide that can act as a starting point for nucleic acid synthesis in the template-orientation when the polynucleotide stretch is placed under the conditions of initiation. Primers can also be used in a variety of other oligonucleotide-mediated synthesis processes, including as de novo RNA synthesis and as initiators of in vitro transcription-related processes. The primer is typically a single-stranded oligonucleotide (e.g., oligodeoxyribonucleotide). The appropriate length of primer will typically depend on the intended use in the range of 6 to 40 nucleotides, more typically in the range of 15 to 35 nucleotides. Short primer molecules generally require a lower temperature to form a sufficiently stable hybridization complex with the template. The primer is not required to reflect the correct sequence of the template, but the primer must be sufficiently complementary to hybridize with the template to be elongated. In certain embodiments, the term "primer pair" includes a 5'-sense primer that is complementarily hybridized to the 5'-end of the nucleic acid sequence to be amplified and a 3'-antisense primer that hybridizes to the 3'end of the amplified sequence ≪ / RTI > The primer can be labeled, if necessary, by incorporating a label that can be detected by spectroscopic, photochemical, biochemical, immunochemical or chemical means. For example, useful labels include: ³² P, fluorescent dyes, electron-dense reagents, enzymes (commonly used in ELISA assays), biotin, or hapten, and proteins that can be used with antisera or monoclonal antibodies .

The term " 5'-nucleic acid hydrolase (nuclease) probe " refers to an oligonucleotide comprising at least one luminescent marker moiety used in a 5'-nucleic acid hydrolase reaction to target nucleic acid detection. In some embodiments, for example, the 5'-nucleic acid hydrolase probe comprises only a single light emitting moiety (e.g., a fluorescent dye, etc.). In certain embodiments, the 5 ' -nucleic acid hydrolase probe comprises a self-complementary region such that the probe can form a hairpin structure under selected conditions. In some embodiments, the 5 ' -nucleic acid hydrolase probe comprises two or more label portions, one of the two labels is separated or degraded from the oligonucleotide and then released with increased radiant intensity. In certain embodiments, the 5'-nucleic acid hydrolase probe is labeled with two different fluorescent dyes, for example a 5'-terminal reporter dye and a 3'-terminal light quencher dye or moiety. In some embodiments, the 5'-nuclease probe is labeled at one or more positions in addition to, or in addition to, the terminus. When the probe is intact, energy transfer typically occurs between the two fluorescent materials so that fluorescence emission from the reporter dye is partially or more extinguished. During the elongation step of the polymerase chain reaction, for example, the 5'-nucleic acid hydrolase probe bound to the template nucleic acid has an activity such that the fluorescence emission of the reporter dye is no longer quenched, for example, Taq polymerase or other Is degraded by the 5 ' to 3 ' -nucleic acid hydrolase activity of the polymerase. In some embodiments, a 5'-nucleic acid hydrolase probe can be labeled with two or more different reporter dyes and a 3'-terminal quencher dye or moiety.

The term "FRET" or "fluorescence resonance energy transfer" or "forester resonance energy transfer" refers to the transfer of energy between two or more chromophores, a donor chromophore and a receptor chromophore (referred to as a quencher). The donor typically transfers energy to the acceptor when the donor is excited by emitting light of the appropriate wavelength. The receptors typically re-emit energy transferred in the form of light that is emitted at different wavelengths. When the receptor is a "dark" quencher, it disperses the energy transferred in a form other than light. Whether a particular fluorescent substance acts as a donor or acceptor depends on the properties of other members of the FRET pair. Commonly used donor-acceptor pairs include FAM-TAMRA pairs. The commonly used quenchers are DABCYL and TAMRA. Commercially available dark quenching agents include: BlackHole Quenchers (BHQ), (Biosearch Technologies, Inc., Novato, Cal.), Iowa Black (Integrated DNA Tech., Inc., Coralville, Iowa) And BlackBerry 占 Quencher 650 (BBQ-650) (Berry & Assoc., Dexter, Mich.).

The term " conventional " or " native " when referring to a nucleic acid base, nucleoside triphosphate, or nucleotide refers to naturally occurring in the polynucleotides described (i.e., for DNA they are dATP, dGTP, dCTP, dTTP). In addition, dITP and 7-deaza-dGTP are frequently used instead of dGTP and can be used in place of dATP in in vitro DNA synthesis reactions such as sequencing.

The term " unusual " or " modified " when referring to a nucleic acid base, nucleoside, or nucleotide refers to a variation, derivative, or modification of a naturally occurring conventional base, nucleoside, or nucleotide in a particular polynucleotide ≪ / RTI > Certain unusual nucleotides are modified at the 2 ' position of the ribose sugar compared to conventional dNTPs. Thus, even though the naturally occurring nucleotides for RNA are ribonucleotides (i.e., ATP, GTP, CTP, UTP, aggregated rNTP), because the nucleotides have hydroxyl groups at the 2 ' As used herein, a ribonucleotide is a nucleotide that is not conventional as a substrate for a DNA polymerase. As used herein, unconventional nucleotides include, but are not limited to, compounds that are used as terminators for nucleic acid sequencing. Exemplary terminator compounds include, but are not limited to, compounds having a 2 ', 3 ' -dideoxy structure, which is referred to as dideoxynucleoside triphosphate. Dideoxy nucleoside triphosphate ddATP, ddTTP, ddCTP and ddGTP are collectively referred to as ddNTP. A further example of a terminator compound comprises a 2'-PO ₄ analogs of ribonucleotides. Other non-conventional nucleotides include, but are not limited to, phosphorothioate dNTPs ([[?] -S] dNTPs, 5 '- [?] -Borano- dNTPs, [?] - methylphosphonate dNTPs, and ribonucleosides Triphosphate (rNTP). Unusual bases include radioactive isotopes such as ³² P, ³³ P, or ³⁵ S; Fluorescent labeling; Labeling of chemiluminescence; Labeling of bioluminescence; Hapten labels such as biotin; Or enzymatic labels such as streptavidin or avidin. Fluorescent labels may include negatively charged dyes, such as dyes of the fluoxane family, or neutrally charged dyes such as the rhodamine family, or positively charged dyes such as the cyanine family. Dyes of the fluoxane family include, for example, FAM, HEX, TET, JOE, NAN and ZOE. The dyes of the Rhodamine family include Texas Red, ROX, R110, R6G, and TAMRA. Various dyes or nucleotides labeled with FAM, HEX, TET, JOE, NAN, ZOE, ROX, R110, R6G, Texas Red and TAMRA are available from Perkin-Elmer (Boston, Mass.), Applied Biosystems / Molecular Probes (Eugene, OR). The dyes of the cyanine family include Cy2, Cy3, Cy5, and Cy7 and are marketed by GE Healthcare UK Limited (Amersham Place, Little Chalfont, Buckinghamshire, England).

The term " mismatch discrimination " is intended to encompass the term " mismatch discrimination " when a nucleic acid (e.g., a primer or other oligonucleotide) is extended in a template-dependent manner by affixing one or more nucleotides Refers to the ability of a biocatalyst (e. G., An enzyme, e. G., A polymerase, ligase, etc.) to distinguish a fully complementary sequence from a match-containing sequence. The term " mismatch discrimination " refers to a mismatch-containing (substantially complementary) nucleic acid in which an extended nucleic acid (e.g., a primer or other oligonucleotide) has a mismatch in the 3'-terminal nucleic acid relative to a template in which the nucleic acid is hybridized Quot; refers to the ability of the biocatalyst to distinguish completely complementary sequences from the sequence. In some embodiments, the extended nucleic acid comprises a mismatch at the 3 ' -terminus to a fully complementary sequence. In some embodiments, the extended nucleic acid comprises a mismatch at the second (N-1) 3'-position and / or N-2 position at the terminus to a fully complementary sequence.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

The present invention relates to a DNA polymerase having a Taq polymerase comprising the amino acid sequence of SEQ ID NO: 1, which comprises (a) substitution of the 507th amino acid residue in the amino acid sequence of SEQ ID NO: 1; And (b) a substitution of a 536th amino acid residue in the amino acid sequence of SEQ ID NO: 1, a substitution of the 660th amino acid residue, or both of the 536th and 660th amino acid residues.

The " Taq polymerase " was first isolated from the bacterium by a heat-resistant DNA polymerase named Thermus aquaticus , a thermophilic bacterium. Thermos Aqua ticus is a bacterium inhabited by hot springs and hydrothermal vesicles. Taq polymerase has been identified as an enzyme capable of withstanding the protein denaturation conditions (high temperature) required in the PCR process. The optimal activity temperature of Taq polymerase is 75-80 ° C. It has a half-life of 92 hours at 92.5 ° C, 40 minutes at 95 ° C, and 9 minutes at 97.5 ° C. It replicates 1000 base pairs of DNA within 10 seconds at 72 ° C . This lacks the 3 'to 5' exonuclease correcting activity, and the error rate is measured in about one out of 9,000 nucleotides. For example, when heat resistant Taq is used, PCR can be performed at a high temperature (60 ° C or higher). For the Taq polymerase, the amino acid sequence shown in SEQ ID NO: 1 is used as the reference sequence.

According to a preferred embodiment of the present invention, substitution of the 507th amino acid residue is replaced by lysine (K) in glutamic acid (E), and substitution of arginine (R) to lysine (K) , And the substitution of the 660th amino acid residue may be substituted with valine (V) in arginine (R).

In the present invention, the Taq polymerase in which the 507th amino acid residue in the amino acid sequence of SEQ ID NO: 1 was substituted with lysine (K) in glutamic acid (E) was named "E507K" (SEQ ID NO: 2); The Taq polymerase in which the 507th amino acid residue in the amino acid sequence of SEQ ID NO: 1 is substituted with lysine (K) in glutamic acid (E) and the lysine (K) in arginine (R) is substituted with the 536th amino acid residue is referred to as "E507K / R536K Quot; (SEQ ID NO: 6); The Taq polymerase in which the 507th amino acid residue in the amino acid sequence of SEQ ID NO: 1 is substituted with lysine (K) in glutamic acid (E) and the 660th amino acid residue is substituted with valine (V) in arginine (R) is "E507K / R660V Quot; (SEQ ID NO: 7); Finally, the 507th amino acid residue in the amino acid sequence of SEQ ID NO: 1 is substituted with lysine (K) in glutamic acid (E), the 536th amino acid residue is substituted with lysine (K) in arginine (R) The Taq polymerase substituted with valine (V) in arginine (R) was named "E507K / R536K / R660V" (SEQ ID NO: 8).

According to a preferred embodiment of the present invention, the DNA polymerase distinguishes between a matched primer and a mismatched primer, wherein the mismatched primer and the mismatched primer are hybridized with the target sequence, and the mismatched primer is hybridized Lt; RTI ID = 0.0 > 3 ' end < / RTI >

The mismatched primer is a hybrid oligonucleotide that must be sufficiently complementary to hybridize with the target sequence but does not reflect the exact sequence of the target sequence.

The "canonical nucleotide" or "complementary nucleotide" refers to the standard Watson-Crick base pair, A-U, A-T, and G-C.

The term "non-canonical nucleotide" or "non-complementary nucleotide" refers to A-C, A-G, G-U, G-T, T-C, T-U, A-A, G-G, T-T, U-U, C-C and C-U in addition to the Watson-

According to a preferred embodiment of the present invention, the DNA polymerase may exhibit a lower Ct value than the amplification of the target sequence comprising the mismatched primer in amplification of the target sequence comprising the matched primer.

For example, a DNA polymerase can elongate primers matched with greater efficiency than mismatched primers in a target sequence-dependent manner by covalently linking one or more nucleotides to the primer. Where greater efficiency can be observed, for example, lower Ct values for primers matched than mismatched primers in RT-PCR. The Ct value difference between the matched primers and the mismatched primers is at least 10, preferably between 10 and 20, or there may be no synthesis of the amplicon by mismatched primers.

For example, a product formed by standard PCR using the forward primer matched in the first reaction and the reverse primer, and the reverse primer matched with the mismatched forward primer in the second reaction in the same experimental setup, Which means that it is bigger.

The Ct (Threshold Crossing Cycle) value represents the DNA quantification method by quantitative PCR, which relies on plotting fluorescence over log count cycles. The threshold for DNA-based fluorescence detection is set at least slightly higher than the background. The number of cycles for which the fluorescence exceeds the threshold value is called Ct, or Cq (quantification cycle) according to the MIQE guideline. The Ct value for a given reaction is defined as the number of cycles in which the fluorescence emission crosses a fixed limit value. For example, SYBR Green I and fluorescent probes can be used for real-time PCR for template DNA quantification. Fluorescence from the sample is collected every cycle during the PCR and plotted against the number of cycles. The starting template concentration is inversely proportional to the time the fluorescence signal first appears. The higher the concentration of the template, the earlier the signal appears (at low cycle counts).

The present invention also relates to a nucleic acid sequence encoding the aforementioned DNA polymerase and a vector and a host cell comprising the nucleic acid sequence. Various vectors can be prepared using the nucleic acid encoding the DNA polymerase of the present invention. Any vector may be used, including replicons and control sequences derived from species compatible with the host cell. The vector of the present invention may be an expression vector and includes a transcriptional and translational control nucleic acid region operably linked to a nucleic acid sequence encoding a DNA polymerase of the present invention. Regulatory sequences refer to DNA sequences required for the expression of coding sequences operatively linked in a particular host organism. For example, control sequences suitable for prokaryotes include promoters, any operative sequences and ribosome binding sites. In addition, the vector may contain a " Positive Retroregulatory Element (PRE) " to enhance the half-life of the transcribed mRNA. The transcriptional and translational regulatory nucleic acid domains will generally be appropriate for host cells used to express the polymerase. Various types of appropriate expression vectors, and suitable regulatory sequences, are known in the art for a variety of host cells. Generally, transcriptional and translational control sequences may include, for example, promoter sequences, ribosomal binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancers or activation sequences. In a typical embodiment, regulatory sequences include promoters and transcription initiation and termination sequences. The vector also typically comprises a polylinker region containing several restriction sites for insertion of foreign DNA. In certain embodiments, a " fusion flag " is used to facilitate purification and, if necessary, a tag / flag is subsequently removed (e.g., " His-Tag "). However, these are generally unnecessary when purifying thermostable and / or thermostable proteins from a mesophilic host (e. G., E. coli) in which the "heat-step" is used. Construction of suitable vectors containing DNA coding replication sequences, regulatory sequences, phenotypic selection genes, and the mutant polymer of interest are prepared using standard recombinant DNA techniques. Isolated plasmids, viral vectors, and DNA fragments are cleaved and truncated and ligated together in a particular order to produce the desired vectors as is known in the art.

In one preferred embodiment of the invention, the expression vector contains a selectable marker gene for selection of the transformed host cell. Selection genes are known in the art and will vary for the host cells used. Suitable selection genes may include gene coding for resistance to ampicillin and / or tetracycline, which can transform cells that cultivate these vectors in the presence of these antibiotics.

In a preferred embodiment of the present invention, the nucleic acid sequence encoding the DNA polymerase of the present invention is introduced into cells either alone or in combination with a vector. The introduction or equivalent expression thereof means that the nucleic acid enters the cell in a manner suitable for subsequent integration, amplification and / or expression. Methods of introduction include, for example, CaPO ₄ precipitation, liposome fusion, LIPOFECTIN®, electrophoresis, viral infection, and the like.

Prokaryotes are used as host cells for the initial cloning step of the present invention. In order to produce large quantities of DNA rapidly, to prepare single-stranded DNA templates used for site-directed mutagenesis, to screen many mutants simultaneously, for DNA sequencing of the resulting mutants, Do. Suitable host cells of prokaryotic cells are E. coli K12 strain 94 (ATCC No. 31,446), E. coli strain W3110 (ATCC No. 27,325), E. coli K12 strain DG116 (ATCC No. 53,606), E. coli X1776 ATCC No. 31,537), and E. coli B; It is well known in the art that many different strains of E. coli such as HB101, JM101, NM522, NM538, NM539 and other species, including Vassilis such as Bacillus subtilis, other Enterobacteriaceae such as Salmonella typhimurium) or Serratia marcesans, and various Pseudomonas species (Pseudomonas sp.) can be used as hosts. Plasmids typically used for transformation of E. coli include pBR322, pUCI8, pUCI9, pUCIl8, pUC119 and Bluescript M13. However, many other suitable vectors may also be used.

The present invention also relates to a method for producing a host cell comprising the steps of: culturing the host cell; And separating the DNA polymerase from the culture and the culture supernatant thereof.

The DNA polymerase of the present invention is prepared by culturing a host cell transformed with an expression vector containing a nucleic acid sequence encoding a DNA polymerase under appropriate conditions that induce or cause expression of the DNA polymerase. Methods for culturing transformed host cells under conditions suitable for protein expression are known in the art. Suitable host cells for the production of the polymerase from lambda pL promoter-containing plasmid vectors include the E. coli strain DG116 (ATCC No. 53606). Depending on the expression, the polymerase can be harvested and separated.

Once purified, the mismatch discrimination of the DNA polymerase of the present invention can be assayed. For example, mismatch distinguishing activity is measured by comparing the amplification of a target sequence perfectly matched to a primer for amplification of a target with a single base mismatch at the 3-terminus of the primer. Amplification can be detected in real time, for example, by the use of a TaqMan ^TM probe. The ability of the polymerase to distinguish between two target sequences can be estimated by comparing the Ct of the two reactions.

Therefore, the present invention provides DNA polymerase according to the present invention

a) one or more molds;

b) nucleoside triphosphate; And

c) contacting one or more matched primers, one or more mismatched primers, or one or more matched primers with one or more mismatched primers, wherein the one or more matched primers and the mismatched primers comprise a target sequence Wherein the mismatched primer comprises one or more mutations or SNPs in one or more templates, including non-canonical nucleotides at up to seven base positions from its 3 'end to the hybridizing target sequence A method for detection in vitro is provided.

The term " SNP (Single Nucleotide Polymorphisms) " refers to a genetic change or mutation showing a difference in one base sequence (A, T, G, or C) in a DNA base sequence.

In the in vitro gene mutation or SNP detection method of the present invention, the target sequence may be present in the test sample and include DNA, cDNA or RNA, preferably genomic DNA. The test sample may be a bacterium, a bacterial culture, or a cell lysate prepared from a cell culture. In addition, the test sample may be contained in an animal, preferably a vertebrate animal, more preferably a human subject. The target sequence may be comprised in a genomic DNA, preferably a genomic DNA of a subject, more preferably a bacterial or vertebrate animal, most preferably a human subject.

The SNP detection method of the present invention can include melting temperature analysis using a double-strand specific dye such as SYBR Green I.

Melting temperature curve analysis can be performed in a real-time PCR device such as ABI 5700/7000 (96 well format) or ABI 7900 (384 well format) device containing onboard software (SDS 2.1). Alternatively, the melting temperature curve analysis can be performed as a termination analysis.

&Quot; Dyes that bind to double-stranded DNA " or " double-stranded specific dyes " can be used when they have high fluorescence when bound to double-stranded DNA rather than unbound. Examples of such dyes include SOYTO-9, SOYTO-13, SOYTO-16, SOYTO-60, SOYTO-64, SYTO-82, EtBr, SYTOX Orange, TO- TO-PRO-3 or EvaGreen. Except for EtBr and EvaGreen (Quiagen), these dyes have been tested in real-time applications.

The in vitro gene mutation or SNP detection method of the present invention can be used for real-time PCR, analysis on agarose gel after standard PCR, gene-specific amplification or allele-specific amplification through real-time PCR, tetra-primer amplification - can be carried out by a refractory mutation system PCR or isothermal amplification.

The " standard PCR " is a technique for amplifying a single or several copies of DNA or cDNA known to the ordinary skilled artisan. Most PCR uses a Taq polymerase or a thermostable DNA polymerase such as Klen Taq. DNA polymerase enzymatically assemble new DNA strands from nucleotides by using single stranded DNA as template and using oligonucleotides (primers). Amplicons generated by PCR can be analyzed, for example, on agarose gels.

The " real time PCR " can monitor the process in real time when performing PCR. Thus, the data are collected throughout the PCR process, not at the end of the PCR. In real-time PCR, the reaction is characterized by the time point during the cycle when the amplification is first detected rather than the amount of target accumulated after a fixed number of cycles. Two methods, mainly dye-based detection and probe-based detection, are used to perform quantitative PCR.

The " Allele Specific Amplification (ASA) " is an amplification technique designed so that a single nucleotide residue can distinguish PCR primers from other templates.

The above-mentioned " allele-specific amplification or gene mutation-specific amplification by real-time PCR " detects a gene mutation or a SNP in a highly efficient manner. Unlike most other methods for detecting gene mutations or SNPs, preliminary amplification of the target gene material is not required. ASA combines amplification and detection in a single reaction based on the distinction between matched and mismatched primer / target sequence complexes. The increase in amplified DNA during the reaction can be monitored in real time by an increase in the fluorescence signal caused by a dye such as SYBR Green I that emits light as it binds to double stranded DNA. Allele-specific amplification or gene mutation-specific amplification by real-time PCR appears to delay or absent the fluorescence signal for mismatches. In gene mutation or SNP detection, this provides information on the presence or absence of a gene mutation or SNP.

The " tetra-primer amplification-refractory mutation system PCR " amplifies both wild-type and mutant alleles together with the control fragment in a single-tube PCR reaction. Non-allele-specific control amplicons are amplified by two common (outer) primers on the side of the mutation region. The two allele-specific (inner) primers are designed in the opposite direction to the common primer, and both wild-type and mutant amplicons can be amplified simultaneously with a common primer. As a result, the two allele-specific amplicons have different lengths and can be easily separated by standard gel electrophoresis because the mutations are asymmetrically located relative to the common (outer) primer. The control amplicon provides internal control for false negatives as well as amplification failure, and at least one of the two allele-specific amplicons is always present in the tetra-primer amplification-refractory mutant system PCR.

This " isothermal amplification " means that the amplification of the nucleic acid is performed at a lower temperature without depending on the thermocycler, preferably without requiring the temperature to change during amplification. The temperature used in isothermal amplification may be between room temperature (22-24 ° C.) and about 65 ° C., or about 60-65 ° C., 45-50 ° C., 37 ° C. -42 ° C., or 22-24 ° C. The products of isothermal amplification are bioanalyzer, which is a chip-based capillary electrophoresis device for analyzing gel electrophoresis, ELISA, enzyme linked oligosorbent assay (ELOSA), real time PCR, ECL (improved chemiluminescence), RNA, DNA and protein or turbidity As shown in FIG.

In one embodiment of the present invention, the use of E507K / R536K, E507K / R660V, or E507K / R536K / R660V Taq polymerase to extend mismatched primers for templates containing SNPs (rs1408799, rs1015362 and / or rs4911414) And whether the ability to do so was reduced.

As a result, it can be confirmed that the amplification by the mismatch primer is delayed in E507K / R536K, E507K / R660V, or E507K / R536K / R660V Taq polymerase as compared to E507K Taq polymerase as shown in FIGS. 6 to 8 , The effect was most pronounced in E507K / R536K / R660V Taq polymerase.

As a result, it was confirmed that the DNA polymerase of the present invention had a higher mismatch elongation selectivity as compared with the conventional Taq polymerase (E507K). Therefore, the DNA polymerase of the present invention is expected to be useful for medical diagnosis of disease and research of recombinant DNA.

The present invention also relates to a composition for detecting a gene mutation or SNP comprising a DNA polymerase according to the present invention and a PCR kit containing the same.

The PCR kit of the present invention may include any reagent or other element that is known to the skilled artisan for use in the primer extension process.

According to a preferred embodiment of the present invention, the PCR kit further comprises one or more matched primers, one or more mismatched primers or one or more matched primers and one or more mismatched primers, Primer and one or more mismatched primers are hybridized to the target sequence and the mismatched primer comprises non-canonical nucleotides at positions 3 to 7 bases from its 3 ' end to the hybridizing target sequence .

The PCR kit of the present invention may further comprise a nucleoside triphosphate.

The PCR kit of the present invention also includes a) one or more buffers; b) a reagent for quantification binding to double stranded DNA; c) a polymerase blocking antibody; d) one or more control values or control sequences; And e) one or more molds; May be further included.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Taq  Mutagenesis of polymerase

1-1. snippet PCR

In this embodiment, the 536th amino acid residue in the amino acid sequence of SEQ ID NO: 1 is replaced with Taq DNA polymerase (hereinafter referred to as "R536K") substituted with lysine at arginine, the Taq DNA polymerase substituted with valine at arginine (Hereinafter referred to as "R660V") and a 536th amino acid residue were substituted with lysine in arginine and the 660th amino acid residue was substituted with valine in arginine (hereinafter referred to as "R536K / R660V") was prepared as follows Respectively.

First, Taq DNA polymerase fragments (F1 to F5) were amplified by PCR using the mutation-specific primers shown in Table 1 as shown in Fig. 1 (a). The reaction conditions are shown in Table 2.

As a result of confirming the PCR products on electrophoresis, the band for each fragment was confirmed as shown in FIG. 1 (b), and it was confirmed that the desired fragment was amplified.

1-2. Overlap PCR

Each fragment amplified in 1-1 above was used as a template and the entire length was amplified using primers (Eco-F and Xba-R primers) at both ends. The reaction conditions are shown in Tables 3 and 4.

As a result, it was confirmed that Taq polymerase of "R536K", "R660V" and "R536K / R660V" was amplified as shown in FIG. 1 (c).

1-3. Ligation

pUC19 was digested with restriction enzymes EcoRI / XbaI at 37 DEG C for 4 hours under the conditions shown in Table 5, and DNA was purified. The purified DNA was treated with SAP at 37 DEG C for 1 hour under the conditions of Table 6 to prepare a vector .

For the insert, the overlap PCR product of Example 1-2 was purified and digested with restriction enzyme EcoRI / XbaI for 3 hours at 37 ° C under the conditions of Table 7, and then gel-extracted with the prepared vector (FIG. 2 ).

After ligation at room temperature (RT) for 2 hours under the conditions of Table 8, E. coli DH5α was transformed and selected on a medium containing ampicillin. The prepared plasmids were sequenced from the obtained colonies to obtain Taq DNA polymerase mutants (" R536K "," R660V ", and " R536K / R660V ") into which desired mutations were introduced.

Introduction of E507K mutation

2-1. Short PCR

R536K, R660V, and R536K / R660V produced in Example 1 were tested for activity of Taq polymerase activity (data not shown) as a result of testing the Taq polymerase activity of R536K, R660V, and R536K / R660V , The E507K mutation (substitution of the 507th amino acid residue in the amino acid sequence of SEQ ID NO: 1 with lysine in glutamic acid) was introduced, and the E507K mutation was also introduced into the wild type Taq DNA polymerase (WT) for use as a control. The production method of the Taq DNA polymerase into which the E507K mutation was introduced is the same as in Example 1. [

Using the mutation-specific primers shown in Table 9, the Taq DNA polymerase fragments (F6 to F7) were amplified by PCR as shown in Fig. The reaction conditions are shown in Table 10.

2-2. Overlap PCR

Each fragment amplified in the above 2-1 was used as a template to amplify the total length using primers (Eco-F and Xba-R primers) at both ends. The reaction conditions are shown in Table 11.

2-3. Ligation

pUC19 was digested with restriction enzyme EcoRI / XbaI at 37 ° C for 4 hours under the conditions shown in Table 5, and DNA was purified. The purified DNA was treated with SAP at 37 ° C for 1 hour under the conditions shown in Table 6 to prepare a vector .

For the insert, the overlap PCR product of Example 2-2 was purified and digested with restriction enzyme EcoRI / XbaI for 3 hours at 37 ° C under the conditions of Table 7, followed by gel extraction with the prepared vector (FIG. 4 ).

After ligation at room temperature (RT) for 2 hours under the conditions of Table 8, E. coli was transformed into DH5? Or DH10? And selected on a medium containing ampicillin. The plasmids prepared from the obtained colonies were sequenced to obtain Taq DNA polymerase mutants ("E507K / R536K", "E507K / R660V" and "E507K / R536K / R660V") into which the E507K mutation was introduced.

Using the DNA polymerase of the present invention qPCR  Perform

A Taq polymerase containing the mutations "E507K / R536K", "E507K / R660V" and "E507K / R536K / R660V" obtained in Example 2 above was used to extend the mismatched primers for the template containing the SNP The ability to do so was reduced. As a control group, " E507K " Taq polymerase containing the E507K mutation was used.

The templates containing the SNP used in this Example are rs1408799, rs1015362 and rs4911414, and the genotype of each template and the sequence information of the specific primer (IDT, USA) for each of them are as shown in Tables 12 and 13 below.

The qPCR conditions (Applied Biosystems 7500 Fast) are shown in Table 14 below.

The probes were double-labeled as shown in Table 15 below.

Oral epithelial cells were collected using an oral epithelial cell collection kit purchased from Noble Bio, dissolved in 500 μl of lysis solution, and centrifuged at 12,000 × g for 3 minutes. The supernatant was transferred to a new tube and used in an amount of 1 쨉 l per experiment (Fig. 5).

Table 16 shows the reaction conditions, and Table 17 shows the composition of the reaction buffer.

Except for the specific primers shown in Table 13, were prepared in two tubes in the same manner and qPCR was performed by adding each allele-specific primer. At this time, fluorescence signals detected in each tube were merged to analyze the difference in the cycle (Ct) values reaching the threshold fluorescence value calculated and calculated on the AB 7500 software (v2.0.6). It is judged that the longer the Ct value in the amplification by the mismatched primer is, the better the gene mutation specificity or the allele specificity.

The results of AS-qPCR for rs1408799, rs1015362 and rs4911414 show that for the Tap polymerase comprising E507K / R536K, E507K / R660V, or E507K / R536K / R660V mutations compared to the control group E507K as shown in FIGS. 6 to 8, Amplification of the primer was delayed, and the effect was most remarkable in the mutation of E507K / R536K / R660V.

It was confirmed that the Tap DNA polymerase including the E507K / R536K, E507K / R660V, or E507K / R536K / R660V mutants of the present invention had excellent mismatch renal selectivity as compared with the Taq polymerase including the E507K mutation. Therefore, the Taq DNA polymerase of the present invention is expected to be useful for medical diagnosis of diseases and recombinant DNA studies.

<110> GeneCast Co., Ltd. <120> DNA POLYMERASES WITH INCREASED MUTATION SPECIFIC AMPLIFICATION <130> 1062317 <160> 36 <170> Kopatentin 2.0 <210> 1 <211> 832 <212> PRT <213> Artificial Sequence <220> <223> Thermus aquaticus DNA polymerase (Taq) <400> 1 Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu   1 5 10 15 Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly              20 25 30 Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala          35 40 45 Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val      50 55 60 Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly  65 70 75 80 Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu                  85 90 95 Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu             100 105 110 Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys         115 120 125 Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp     130 135 140 Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly 145 150 155 160 Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro                 165 170 175 Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn             180 185 190 Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu         195 200 205 Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu     210 215 220 Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys 225 230 235 240 Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val                 245 250 255 Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe             260 265 270 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu         275 280 285 Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly     290 295 300 Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp 305 310 315 320 Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val Val His Arg Ala Pro                 325 330 335 Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu             340 345 350 Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro         355 360 365 Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn     370 375 380 Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 385 390 395 400 Glu Ala Gly Glu Arg Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu                 405 410 415 Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu             420 425 430 Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly         435 440 445 Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala     450 455 460 Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His 465 470 475 480 Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp                 485 490 495 Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg             500 505 510 Ser Thr Ser Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile         515 520 525 Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr     530 535 540 Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu 545 550 555 560 His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser                 565 570 575 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln             580 585 590 Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala         595 600 605 Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly     610 615 620 Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr 625 630 635 640 Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro                 645 650 655 Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly             660 665 670 Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu         675 680 685 Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg     690 695 700 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val 705 710 715 720 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg                 725 730 735 Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro             740 745 750 Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu         755 760 765 Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His     770 775 780 Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala 785 790 795 800 Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro                 805 810 815 Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu             820 825 830 <210> 2 <211> 832 <212> PRT <213> Artificial Sequence <220> <223> Taq E507K <400> 2 Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu   1 5 10 15 Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly              20 25 30 Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala          35 40 45 Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val      50 55 60 Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly  65 70 75 80 Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu                  85 90 95 Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu             100 105 110 Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys         115 120 125 Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp     130 135 140 Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly 145 150 155 160 Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro                 165 170 175 Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn             180 185 190 Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu         195 200 205 Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu     210 215 220 Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys 225 230 235 240 Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val                 245 250 255 Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe             260 265 270 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu         275 280 285 Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly     290 295 300 Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp 305 310 315 320 Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val Val His Arg Ala Pro                 325 330 335 Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu             340 345 350 Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro         355 360 365 Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn     370 375 380 Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 385 390 395 400 Glu Ala Gly Glu Arg Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu                 405 410 415 Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu             420 425 430 Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly         435 440 445 Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala     450 455 460 Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His 465 470 475 480 Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp                 485 490 495 Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Lys Lys Thr Gly Lys Arg             500 505 510 Ser Thr Ser Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile         515 520 525 Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr     530 535 540 Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu 545 550 555 560 His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser                 565 570 575 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln             580 585 590 Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala         595 600 605 Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly     610 615 620 Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr 625 630 635 640 Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro                 645 650 655 Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly             660 665 670 Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu         675 680 685 Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg     690 695 700 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val 705 710 715 720 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg                 725 730 735 Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro             740 745 750 Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu         755 760 765 Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His     770 775 780 Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala 785 790 795 800 Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro                 805 810 815 Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu             820 825 830 <210> 3 <211> 832 <212> PRT <213> Artificial Sequence <220> <223> Taq R536K <400> 3 Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu   1 5 10 15 Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly              20 25 30 Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala          35 40 45 Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val      50 55 60 Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly  65 70 75 80 Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu                  85 90 95 Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu             100 105 110 Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys         115 120 125 Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp     130 135 140 Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly 145 150 155 160 Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro                 165 170 175 Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn             180 185 190 Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu         195 200 205 Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu     210 215 220 Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys 225 230 235 240 Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val                 245 250 255 Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe             260 265 270 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu         275 280 285 Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly     290 295 300 Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp 305 310 315 320 Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val Val His Arg Ala Pro                 325 330 335 Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu             340 345 350 Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro         355 360 365 Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn     370 375 380 Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 385 390 395 400 Glu Ala Gly Glu Arg Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu                 405 410 415 Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu             420 425 430 Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly         435 440 445 Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala     450 455 460 Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His 465 470 475 480 Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp                 485 490 495 Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg             500 505 510 Ser Thr Ser Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile         515 520 525 Val Glu Lys Ile Leu Gln Tyr Lys Glu Leu Thr Lys Leu Lys Ser Thr     530 535 540 Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu 545 550 555 560 His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser                 565 570 575 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln             580 585 590 Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala         595 600 605 Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly     610 615 620 Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr 625 630 635 640 Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro                 645 650 655 Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly             660 665 670 Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu         675 680 685 Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg     690 695 700 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val 705 710 715 720 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg                 725 730 735 Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro             740 745 750 Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu         755 760 765 Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His     770 775 780 Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala 785 790 795 800 Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro                 805 810 815 Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu             820 825 830 <210> 4 <211> 832 <212> PRT <213> Artificial Sequence <220> <223> Taq R660V <400> 4 Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu   1 5 10 15 Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly              20 25 30 Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala          35 40 45 Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val      50 55 60 Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly  65 70 75 80 Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu                  85 90 95 Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu             100 105 110 Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys         115 120 125 Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp     130 135 140 Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly 145 150 155 160 Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro                 165 170 175 Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn             180 185 190 Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu         195 200 205 Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu     210 215 220 Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys 225 230 235 240 Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val                 245 250 255 Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe             260 265 270 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu         275 280 285 Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly     290 295 300 Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp 305 310 315 320 Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val Val His Arg Ala Pro                 325 330 335 Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu             340 345 350 Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro         355 360 365 Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn     370 375 380 Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 385 390 395 400 Glu Ala Gly Glu Arg Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu                 405 410 415 Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu             420 425 430 Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly         435 440 445 Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala     450 455 460 Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His 465 470 475 480 Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp                 485 490 495 Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg             500 505 510 Ser Thr Ser Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile         515 520 525 Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr     530 535 540 Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu 545 550 555 560 His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser                 565 570 575 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln             580 585 590 Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala         595 600 605 Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly     610 615 620 Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr 625 630 635 640 Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro                 645 650 655 Leu Met Arg Val Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly             660 665 670 Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu         675 680 685 Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg     690 695 700 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val 705 710 715 720 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg                 725 730 735 Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro             740 745 750 Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu         755 760 765 Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His     770 775 780 Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala 785 790 795 800 Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro                 805 810 815 Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu             820 825 830 <210> 5 <211> 832 <212> PRT <213> Artificial Sequence <220> <223> Taq R536K / R660V <400> 5 Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu   1 5 10 15 Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly              20 25 30 Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala          35 40 45 Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val      50 55 60 Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly  65 70 75 80 Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu                  85 90 95 Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu             100 105 110 Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys         115 120 125 Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp     130 135 140 Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly 145 150 155 160 Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro                 165 170 175 Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn             180 185 190 Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu         195 200 205 Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu     210 215 220 Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys 225 230 235 240 Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val                 245 250 255 Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe             260 265 270 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu         275 280 285 Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly     290 295 300 Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp 305 310 315 320 Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val Val His Arg Ala Pro                 325 330 335 Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu             340 345 350 Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro         355 360 365 Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn     370 375 380 Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 385 390 395 400 Glu Ala Gly Glu Arg Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu                 405 410 415 Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu             420 425 430 Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly         435 440 445 Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala     450 455 460 Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His 465 470 475 480 Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp                 485 490 495 Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg             500 505 510 Ser Thr Ser Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile         515 520 525 Val Glu Lys Ile Leu Gln Tyr Lys Glu Leu Thr Lys Leu Lys Ser Thr     530 535 540 Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu 545 550 555 560 His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser                 565 570 575 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln             580 585 590 Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala         595 600 605 Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly     610 615 620 Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr 625 630 635 640 Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro                 645 650 655 Leu Met Arg Val Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly             660 665 670 Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu         675 680 685 Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg     690 695 700 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val 705 710 715 720 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg                 725 730 735 Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro             740 745 750 Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu         755 760 765 Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His     770 775 780 Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala 785 790 795 800 Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro                 805 810 815 Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu             820 825 830 <210> 6 <211> 832 <212> PRT <213> Artificial Sequence <220> <223> Taq E507K / R536K <400> 6 Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu   1 5 10 15 Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly              20 25 30 Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala          35 40 45 Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val      50 55 60 Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly  65 70 75 80 Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu                  85 90 95 Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu             100 105 110 Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys         115 120 125 Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp     130 135 140 Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly 145 150 155 160 Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro                 165 170 175 Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn             180 185 190 Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu         195 200 205 Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu     210 215 220 Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys 225 230 235 240 Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val                 245 250 255 Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe             260 265 270 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu         275 280 285 Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly     290 295 300 Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp 305 310 315 320 Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val Val His Arg Ala Pro                 325 330 335 Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu             340 345 350 Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro         355 360 365 Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn     370 375 380 Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 385 390 395 400 Glu Ala Gly Glu Arg Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu                 405 410 415 Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu             420 425 430 Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly         435 440 445 Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala     450 455 460 Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His 465 470 475 480 Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp                 485 490 495 Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Lys Lys Thr Gly Lys Arg             500 505 510 Ser Thr Ser Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile         515 520 525 Val Glu Lys Ile Leu Gln Tyr Lys Glu Leu Thr Lys Leu Lys Ser Thr     530 535 540 Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu 545 550 555 560 His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser                 565 570 575 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln             580 585 590 Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala         595 600 605 Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly     610 615 620 Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr 625 630 635 640 Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro                 645 650 655 Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly             660 665 670 Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu         675 680 685 Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg     690 695 700 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val 705 710 715 720 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg                 725 730 735 Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro             740 745 750 Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu         755 760 765 Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His     770 775 780 Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala 785 790 795 800 Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro                 805 810 815 Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu             820 825 830 <210> 7 <211> 832 <212> PRT <213> Artificial Sequence <220> <223> Taq E507K / R660V <400> 7 Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu   1 5 10 15 Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly              20 25 30 Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala          35 40 45 Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val      50 55 60 Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly  65 70 75 80 Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu                  85 90 95 Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu             100 105 110 Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys         115 120 125 Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp     130 135 140 Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly 145 150 155 160 Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro                 165 170 175 Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn             180 185 190 Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu         195 200 205 Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu     210 215 220 Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys 225 230 235 240 Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val                 245 250 255 Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe             260 265 270 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu         275 280 285 Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly     290 295 300 Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp 305 310 315 320 Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val Val His Arg Ala Pro                 325 330 335 Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu             340 345 350 Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro         355 360 365 Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn     370 375 380 Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 385 390 395 400 Glu Ala Gly Glu Arg Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu                 405 410 415 Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu             420 425 430 Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly         435 440 445 Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala     450 455 460 Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His 465 470 475 480 Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp                 485 490 495 Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Lys Lys Thr Gly Lys Arg             500 505 510 Ser Thr Ser Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile         515 520 525 Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr     530 535 540 Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu 545 550 555 560 His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser                 565 570 575 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln             580 585 590 Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala         595 600 605 Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly     610 615 620 Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr 625 630 635 640 Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro                 645 650 655 Leu Met Arg Val Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly             660 665 670 Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu         675 680 685 Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg     690 695 700 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val 705 710 715 720 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg                 725 730 735 Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro             740 745 750 Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu         755 760 765 Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His     770 775 780 Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala 785 790 795 800 Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro                 805 810 815 Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu             820 825 830 <210> 8 <211> 832 <212> PRT <213> Artificial Sequence <220> <223> Taq E507K / R536K / R660V <400> 8 Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu   1 5 10 15 Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly              20 25 30 Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala          35 40 45 Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val      50 55 60 Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly  65 70 75 80 Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu                  85 90 95 Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu             100 105 110 Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys         115 120 125 Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp     130 135 140 Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly 145 150 155 160 Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro                 165 170 175 Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn             180 185 190 Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu         195 200 205 Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu     210 215 220 Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys 225 230 235 240 Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val                 245 250 255 Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe             260 265 270 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu         275 280 285 Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly     290 295 300 Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp 305 310 315 320 Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val Val His Arg Ala Pro                 325 330 335 Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu             340 345 350 Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro         355 360 365 Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn     370 375 380 Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 385 390 395 400 Glu Ala Gly Glu Arg Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu                 405 410 415 Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu             420 425 430 Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly         435 440 445 Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala     450 455 460 Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His 465 470 475 480 Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp                 485 490 495 Glu Leu Gly Leu Pro Ala Ile Gly Lys Glu Lys Lys Thr Gly Lys Arg             500 505 510 Ser Thr Ser Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile         515 520 525 Val Glu Lys Ile Leu Gln Tyr Lys Glu Leu Thr Lys Leu Lys Ser Thr     530 535 540 Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu 545 550 555 560 His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser                 565 570 575 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln             580 585 590 Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala         595 600 605 Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly     610 615 620 Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr 625 630 635 640 Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro                 645 650 655 Leu Met Arg Val Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly             660 665 670 Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu         675 680 685 Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg     690 695 700 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val 705 710 715 720 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg                 725 730 735 Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro             740 745 750 Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu         755 760 765 Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His     770 775 780 Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala 785 790 795 800 Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro                 805 810 815 Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu             820 825 830 <210> 9 <211> 2499 <212> DNA <213> Artificial Sequence <220> <223> Thermus aquaticus DNA polymerase (Taq) <400> 9 atgaggggga tgctgcccct ctttgagccc aagggccggg tcctcctggt ggacggccac 60 cacctggcct accgcacctt ccacgccctg aagggcctca ccaccagccg gggggagccg 120 gtgcaggcgg tctacggctt cgccaagagc ctcctcaagg ccctcaagga ggacggggac 180 gcggtgatcg tggtctttga cgccaaggcc ccctccttcc gccacgaggc ctacgggggg 240 tacaaggcgg gccgggcccc cacgccggag gactttcccc ggcaactcgc cctcatcaag 300 gagctggtgg acctcctggg gctggcgcgc ctcgaggtcc cgggctacga ggcggacgac 360 gtcctggcca gcctggccaa gaaggcggaa aaggagggct acgaggtccg catcctcacc 420 gccgacaaag acctttacca gctcctttcc gaccgcatcc acgtcctcca ccccgagggg 480 tacctcatca ccccggcctg gctttgggaa aagtacggcc tgaggcccga ccagtgggcc 540 gactaccggg ccctgaccgg ggacgagtcc gacaaccttc ccggggtcaa gggcatcggg 600 gagaagacgg cgaggaagct tctggaggag tgggggagcc tggaagccct cctcaagaac 660 ctggaccggc tgaagcccgc catccgggag aagatcctgg cccacatgga cgatctgaag 720 ctctcctggg acctggccaa ggtgcgcacc gacctgcccc tggaggtgga cttcgccaaa 780 aggcgggagc ccgaccggga gaggcttagg gcctttctgg agaggcttga gtttggcagc 840 ctcctccacg agttcggcct tctggaaagc cccaaggccc tggaggaggc cccctggccc 900 ccgccggaag gggccttcgt gggctttgtg ctttcccgca aggagcccat gtgggccgat 960 cttctggccc tggccgccgc cagggggggc cgggtccacc gggcccccga gccttataaa 1020 gccctcaggg acctgaagga ggcgcggggg cttctcgcca aagacctgag cgttctggcc 1080 ctgagggaag gccttggcct cccgcccggc gacgacccca tgctcctcgc ctacctcctg 1140 gacccttcca acaccacccc cgagggggtg gcccggcgct acggcgggga gtggacggag 1200 gaggcggggg agcgggccgc cctttccgag aggctcttcg ccaacctgtg ggggaggctt 1260 gagggggagg agaggctcct ttggctttac cgggaggtgg agaggcccct ttccgctgtc 1320 ctggcccaca tggaggccac gggggtgcgc ctggacgtgg cctatctcag ggccttgtcc 1380 ctggaggtgg ccgaggagat cgcccgcctc gaggccgagg tcttccgcct ggccggccac 1440 cccttcaacc tcaactcccg ggaccagctg gaaagggtcc tctttgacga gctagggctt 1500 cccgccatcg gcaagacgga gaagaccggc aagcgctcca ccagcgccgc cgtcctggag 1560 gccctccgcg aggcccaccc catcgtggag aagatcctgc agtaccggga gctcaccaag 1620 ctgaagagca cctacattga ccccttgccg gacctcatcc accccaggac gggccgcctc 1680 cacacccgct tcaaccagac ggccacggcc acgggcaggc taagtagctc cgatcccaac 1740 ctccagaaca tccccgtccg caccccgctt gggcagagga tccgccgggc cttcatcgcc 1800 gaggaggggt ggctattggt ggccctggac tatagccaga tagagctcag ggtgctggcc 1860 cacctctccg gcgacgagaa cctgatccgg gtcttccagg aggggcggga catccacacg 1920 gagaccgcca gctggatgtt cggcgtcccc cgggaggccg tggaccccct gatgcgccgg 1980 gcggccaaga ccatcaactt cggggtcctc tacggcatgt cggcccaccg cctctcccag 2040 gagctagcca tcccttacga ggaggcccag gccttcattg agcgctactt tcagagcttc 2100 cccaaggtgc gggcctggat tgagaagacc ctggaggagg gcaggaggcg ggggtacgtg 2160 gagaccctct tcggccgccg ccgctacgtg ccagacctag aggcccgggt gaagagcgtg 2220 cgggaggcgg ccgagcgcat ggccttcaac atgcccgtcc agggcaccgc cgccgacctc 2280 atgaagctgg ctatggtgaa gctcttcccc aggctggagg aaatgggggc caggatgctc 2340 cttcaggtcc acgacgagct ggtcctcgag gccccaaaag agagggcgga ggccgtggcc 2400 cggctggcca aggaggtcat ggagggggtg tatcccctgg ccgtgcccct ggaggtggag 2460 gtggggatag gggaggactg gctctccgcc aaggagtga 2499 <210> 10 <211> 2499 <212> DNA <213> Artificial Sequence <220> <223> Taq E507K <400> 10 atgaggggga tgctgcccct ctttgagccc aagggccggg tcctcctggt ggacggccac 60 cacctggcct accgcacctt ccacgccctg aagggcctca ccaccagccg gggggagccg 120 gtgcaggcgg tctacggctt cgccaagagc ctcctcaagg ccctcaagga ggacggggac 180 gcggtgatcg tggtctttga cgccaaggcc ccctccttcc gccacgaggc ctacgggggg 240 tacaaggcgg gccgggcccc cacgccggag gactttcccc ggcaactcgc cctcatcaag 300 gagctggtgg acctcctggg gctggcgcgc ctcgaggtcc cgggctacga ggcggacgac 360 gtcctggcca gcctggccaa gaaggcggaa aaggagggct acgaggtccg catcctcacc 420 gccgacaaag acctttacca gctcctttcc gaccgcatcc acgtcctcca ccccgagggg 480 tacctcatca ccccggcctg gctttgggaa aagtacggcc tgaggcccga ccagtgggcc 540 gactaccggg ccctgaccgg ggacgagtcc gacaaccttc ccggggtcaa gggcatcggg 600 gagaagacgg cgaggaagct tctggaggag tgggggagcc tggaagccct cctcaagaac 660 ctggaccggc tgaagcccgc catccgggag aagatcctgg cccacatgga cgatctgaag 720 ctctcctggg acctggccaa ggtgcgcacc gacctgcccc tggaggtgga cttcgccaaa 780 aggcgggagc ccgaccggga gaggcttagg gcctttctgg agaggcttga gtttggcagc 840 ctcctccacg agttcggcct tctggaaagc cccaaggccc tggaggaggc cccctggccc 900 ccgccggaag gggccttcgt gggctttgtg ctttcccgca aggagcccat gtgggccgat 960 cttctggccc tggccgccgc cagggggggc cgggtccacc gggcccccga gccttataaa 1020 gccctcaggg acctgaagga ggcgcggggg cttctcgcca aagacctgag cgttctggcc 1080 ctgagggaag gccttggcct cccgcccggc gacgacccca tgctcctcgc ctacctcctg 1140 gacccttcca acaccacccc cgagggggtg gcccggcgct acggcgggga gtggacggag 1200 gaggcggggg agcgggccgc cctttccgag aggctcttcg ccaacctgtg ggggaggctt 1260 gagggggagg agaggctcct ttggctttac cgggaggtgg agaggcccct ttccgctgtc 1320 ctggcccaca tggaggccac gggggtgcgc ctggacgtgg cctatctcag ggccttgtcc 1380 ctggaggtgg ccgaggagat cgcccgcctc gaggccgagg tcttccgcct ggccggccac 1440 cccttcaacc tcaactcccg ggaccagctg gaaagggtcc tctttgacga gctagggctt 1500 cccgccatcg gcaagacgaa aaagaccggc aagcgctcca ccagcgccgc cgtcctggag 1560 gccctccgcg aggcccaccc catcgtggag aagatcctgc agtaccggga gctcaccaag 1620 ctgaagagca cctacattga ccccttgccg gacctcatcc accccaggac gggccgcctc 1680 cacacccgct tcaaccagac ggccacggcc acgggcaggc taagtagctc cgatcccaac 1740 ctccagaaca tccccgtccg caccccgctt gggcagagga tccgccgggc cttcatcgcc 1800 gaggaggggt ggctattggt ggccctggac tatagccaga tagagctcag ggtgctggcc 1860 cacctctccg gcgacgagaa cctgatccgg gtcttccagg aggggcggga catccacacg 1920 gagaccgcca gctggatgtt cggcgtcccc cgggaggccg tggaccccct gatgcgccgg 1980 gcggccaaga ccatcaactt cggggtcctc tacggcatgt cggcccaccg cctctcccag 2040 gagctagcca tcccttacga ggaggcccag gccttcattg agcgctactt tcagagcttc 2100 cccaaggtgc gggcctggat tgagaagacc ctggaggagg gcaggaggcg ggggtacgtg 2160 gagaccctct tcggccgccg ccgctacgtg ccagacctag aggcccgggt gaagagcgtg 2220 cgggaggcgg ccgagcgcat ggccttcaac atgcccgtcc agggcaccgc cgccgacctc 2280 atgaagctgg ctatggtgaa gctcttcccc aggctggagg aaatgggggc caggatgctc 2340 cttcaggtcc acgacgagct ggtcctcgag gccccaaaag agagggcgga ggccgtggcc 2400 cggctggcca aggaggtcat ggagggggtg tatcccctgg ccgtgcccct ggaggtggag 2460 gtggggatag gggaggactg gctctccgcc aaggagtga 2499 <210> 11 <211> 2499 <212> DNA <213> Artificial Sequence <220> <223> Taq R536K <400> 11 atgaggggga tgctgcccct ctttgagccc aagggccggg tcctcctggt ggacggccac 60 cacctggcct accgcacctt ccacgccctg aagggcctca ccaccagccg gggggagccg 120 gtgcaggcgg tctacggctt cgccaagagc ctcctcaagg ccctcaagga ggacggggac 180 gcggtgatcg tggtctttga cgccaaggcc ccctccttcc gccacgaggc ctacgggggg 240 tacaaggcgg gccgggcccc cacgccggag gactttcccc ggcaactcgc cctcatcaag 300 gagctggtgg acctcctggg gctggcgcgc ctcgaggtcc cgggctacga ggcggacgac 360 gtcctggcca gcctggccaa gaaggcggaa aaggagggct acgaggtccg catcctcacc 420 gccgacaaag acctttacca gctcctttcc gaccgcatcc acgtcctcca ccccgagggg 480 tacctcatca ccccggcctg gctttgggaa aagtacggcc tgaggcccga ccagtgggcc 540 gactaccggg ccctgaccgg ggacgagtcc gacaaccttc ccggggtcaa gggcatcggg 600 gagaagacgg cgaggaagct tctggaggag tgggggagcc tggaagccct cctcaagaac 660 ctggaccggc tgaagcccgc catccgggag aagatcctgg cccacatgga cgatctgaag 720 ctctcctggg acctggccaa ggtgcgcacc gacctgcccc tggaggtgga cttcgccaaa 780 aggcgggagc ccgaccggga gaggcttagg gcctttctgg agaggcttga gtttggcagc 840 ctcctccacg agttcggcct tctggaaagc cccaaggccc tggaggaggc cccctggccc 900 ccgccggaag gggccttcgt gggctttgtg ctttcccgca aggagcccat gtgggccgat 960 cttctggccc tggccgccgc cagggggggc cgggtccacc gggcccccga gccttataaa 1020 gccctcaggg acctgaagga ggcgcggggg cttctcgcca aagacctgag cgttctggcc 1080 ctgagggaag gccttggcct cccgcccggc gacgacccca tgctcctcgc ctacctcctg 1140 gacccttcca acaccacccc cgagggggtg gcccggcgct acggcgggga gtggacggag 1200 gaggcggggg agcgggccgc cctttccgag aggctcttcg ccaacctgtg ggggaggctt 1260 gagggggagg agaggctcct ttggctttac cgggaggtgg agaggcccct ttccgctgtc 1320 ctggcccaca tggaggccac gggggtgcgc ctggacgtgg cctatctcag ggccttgtcc 1380 ctggaggtgg ccgaggagat cgcccgcctc gaggccgagg tcttccgcct ggccggccac 1440 cccttcaacc tcaactcccg ggaccagctg gaaagggtcc tctttgacga gctagggctt 1500 cccgccatcg gcaagacgga gaagaccggc aagcgctcca ccagcgccgc cgtcctggag 1560 gccctccgcg aggcccaccc catcgtggag aagatcctgc agtacaagga gctcaccaag 1620 ctgaagagca cctacattga ccccttgccg gacctcatcc accccaggac gggccgcctc 1680 cacacccgct tcaaccagac ggccacggcc acgggcaggc taagtagctc cgatcccaac 1740 ctccagaaca tccccgtccg caccccgctt gggcagagga tccgccgggc cttcatcgcc 1800 gaggaggggt ggctattggt ggccctggac tatagccaga tagagctcag ggtgctggcc 1860 cacctctccg gcgacgagaa cctgatccgg gtcttccagg aggggcggga catccacacg 1920 gagaccgcca gctggatgtt cggcgtcccc cgggaggccg tggaccccct gatgcgccgg 1980 gcggccaaga ccatcaactt cggggtcctc tacggcatgt cggcccaccg cctctcccag 2040 gagctagcca tcccttacga ggaggcccag gccttcattg agcgctactt tcagagcttc 2100 cccaaggtgc gggcctggat tgagaagacc ctggaggagg gcaggaggcg ggggtacgtg 2160 gagaccctct tcggccgccg ccgctacgtg ccagacctag aggcccgggt gaagagcgtg 2220 cgggaggcgg ccgagcgcat ggccttcaac atgcccgtcc agggcaccgc cgccgacctc 2280 atgaagctgg ctatggtgaa gctcttcccc aggctggagg aaatgggggc caggatgctc 2340 cttcaggtcc acgacgagct ggtcctcgag gccccaaaag agagggcgga ggccgtggcc 2400 cggctggcca aggaggtcat ggagggggtg tatcccctgg ccgtgcccct ggaggtggag 2460 gtggggatag gggaggactg gctctccgcc aaggagtga 2499 <210> 12 <211> 2499 <212> DNA <213> Artificial Sequence <220> <223> Taq R660V <400> 12 atgaggggga tgctgcccct ctttgagccc aagggccggg tcctcctggt ggacggccac 60 cacctggcct accgcacctt ccacgccctg aagggcctca ccaccagccg gggggagccg 120 gtgcaggcgg tctacggctt cgccaagagc ctcctcaagg ccctcaagga ggacggggac 180 gcggtgatcg tggtctttga cgccaaggcc ccctccttcc gccacgaggc ctacgggggg 240 tacaaggcgg gccgggcccc cacgccggag gactttcccc ggcaactcgc cctcatcaag 300 gagctggtgg acctcctggg gctggcgcgc ctcgaggtcc cgggctacga ggcggacgac 360 gtcctggcca gcctggccaa gaaggcggaa aaggagggct acgaggtccg catcctcacc 420 gccgacaaag acctttacca gctcctttcc gaccgcatcc acgtcctcca ccccgagggg 480 tacctcatca ccccggcctg gctttgggaa aagtacggcc tgaggcccga ccagtgggcc 540 gactaccggg ccctgaccgg ggacgagtcc gacaaccttc ccggggtcaa gggcatcggg 600 gagaagacgg cgaggaagct tctggaggag tgggggagcc tggaagccct cctcaagaac 660 ctggaccggc tgaagcccgc catccgggag aagatcctgg cccacatgga cgatctgaag 720 ctctcctggg acctggccaa ggtgcgcacc gacctgcccc tggaggtgga cttcgccaaa 780 aggcgggagc ccgaccggga gaggcttagg gcctttctgg agaggcttga gtttggcagc 840 ctcctccacg agttcggcct tctggaaagc cccaaggccc tggaggaggc cccctggccc 900 ccgccggaag gggccttcgt gggctttgtg ctttcccgca aggagcccat gtgggccgat 960 cttctggccc tggccgccgc cagggggggc cgggtccacc gggcccccga gccttataaa 1020 gccctcaggg acctgaagga ggcgcggggg cttctcgcca aagacctgag cgttctggcc 1080 ctgagggaag gccttggcct cccgcccggc gacgacccca tgctcctcgc ctacctcctg 1140 gacccttcca acaccacccc cgagggggtg gcccggcgct acggcgggga gtggacggag 1200 gaggcggggg agcgggccgc cctttccgag aggctcttcg ccaacctgtg ggggaggctt 1260 gagggggagg agaggctcct ttggctttac cgggaggtgg agaggcccct ttccgctgtc 1320 ctggcccaca tggaggccac gggggtgcgc ctggacgtgg cctatctcag ggccttgtcc 1380 ctggaggtgg ccgaggagat cgcccgcctc gaggccgagg tcttccgcct ggccggccac 1440 cccttcaacc tcaactcccg ggaccagctg gaaagggtcc tctttgacga gctagggctt 1500 cccgccatcg gcaagacgga gaagaccggc aagcgctcca ccagcgccgc cgtcctggag 1560 gccctccgcg aggcccaccc catcgtggag aagatcctgc agtaccggga gctcaccaag 1620 ctgaagagca cctacattga ccccttgccg gacctcatcc accccaggac gggccgcctc 1680 cacacccgct tcaaccagac ggccacggcc acgggcaggc taagtagctc cgatcccaac 1740 ctccagaaca tccccgtccg caccccgctt gggcagagga tccgccgggc cttcatcgcc 1800 gaggaggggt ggctattggt ggccctggac tatagccaga tagagctcag ggtgctggcc 1860 cacctctccg gcgacgagaa cctgatccgg gtcttccagg aggggcggga catccacacg 1920 gagaccgcca gctggatgtt cggcgtcccc cgggaggccg tggaccccct gatgcgcgtg 1980 gcggccaaga ccatcaactt cggggtcctc tacggcatgt cggcccaccg cctctcccag 2040 gagctagcca tcccttacga ggaggcccag gccttcattg agcgctactt tcagagcttc 2100 cccaaggtgc gggcctggat tgagaagacc ctggaggagg gcaggaggcg ggggtacgtg 2160 gagaccctct tcggccgccg ccgctacgtg ccagacctag aggcccgggt gaagagcgtg 2220 cgggaggcgg ccgagcgcat ggccttcaac atgcccgtcc agggcaccgc cgccgacctc 2280 atgaagctgg ctatggtgaa gctcttcccc aggctggagg aaatgggggc caggatgctc 2340 cttcaggtcc acgacgagct ggtcctcgag gccccaaaag agagggcgga ggccgtggcc 2400 cggctggcca aggaggtcat ggagggggtg tatcccctgg ccgtgcccct ggaggtggag 2460 gtggggatag gggaggactg gctctccgcc aaggagtga 2499 <210> 13 <211> 2499 <212> DNA <213> Artificial Sequence <220> <223> Taq R536K / R660V <400> 13 atgaggggga tgctgcccct ctttgagccc aagggccggg tcctcctggt ggacggccac 60 cacctggcct accgcacctt ccacgccctg aagggcctca ccaccagccg gggggagccg 120 gtgcaggcgg tctacggctt cgccaagagc ctcctcaagg ccctcaagga ggacggggac 180 gcggtgatcg tggtctttga cgccaaggcc ccctccttcc gccacgaggc ctacgggggg 240 tacaaggcgg gccgggcccc cacgccggag gactttcccc ggcaactcgc cctcatcaag 300 gagctggtgg acctcctggg gctggcgcgc ctcgaggtcc cgggctacga ggcggacgac 360 gtcctggcca gcctggccaa gaaggcggaa aaggagggct acgaggtccg catcctcacc 420 gccgacaaag acctttacca gctcctttcc gaccgcatcc acgtcctcca ccccgagggg 480 tacctcatca ccccggcctg gctttgggaa aagtacggcc tgaggcccga ccagtgggcc 540 gactaccggg ccctgaccgg ggacgagtcc gacaaccttc ccggggtcaa gggcatcggg 600 gagaagacgg cgaggaagct tctggaggag tgggggagcc tggaagccct cctcaagaac 660 ctggaccggc tgaagcccgc catccgggag aagatcctgg cccacatgga cgatctgaag 720 ctctcctggg acctggccaa ggtgcgcacc gacctgcccc tggaggtgga cttcgccaaa 780 aggcgggagc ccgaccggga gaggcttagg gcctttctgg agaggcttga gtttggcagc 840 ctcctccacg agttcggcct tctggaaagc cccaaggccc tggaggaggc cccctggccc 900 ccgccggaag gggccttcgt gggctttgtg ctttcccgca aggagcccat gtgggccgat 960 cttctggccc tggccgccgc cagggggggc cgggtccacc gggcccccga gccttataaa 1020 gccctcaggg acctgaagga ggcgcggggg cttctcgcca aagacctgag cgttctggcc 1080 ctgagggaag gccttggcct cccgcccggc gacgacccca tgctcctcgc ctacctcctg 1140 gacccttcca acaccacccc cgagggggtg gcccggcgct acggcgggga gtggacggag 1200 gaggcggggg agcgggccgc cctttccgag aggctcttcg ccaacctgtg ggggaggctt 1260 gagggggagg agaggctcct ttggctttac cgggaggtgg agaggcccct ttccgctgtc 1320 ctggcccaca tggaggccac gggggtgcgc ctggacgtgg cctatctcag ggccttgtcc 1380 ctggaggtgg ccgaggagat cgcccgcctc gaggccgagg tcttccgcct ggccggccac 1440 cccttcaacc tcaactcccg ggaccagctg gaaagggtcc tctttgacga gctagggctt 1500 cccgccatcg gcaagacgga gaagaccggc aagcgctcca ccagcgccgc cgtcctggag 1560 gccctccgcg aggcccaccc catcgtggag aagatcctgc agtacaagga gctcaccaag 1620 ctgaagagca cctacattga ccccttgccg gacctcatcc accccaggac gggccgcctc 1680 cacacccgct tcaaccagac ggccacggcc acgggcaggc taagtagctc cgatcccaac 1740 ctccagaaca tccccgtccg caccccgctt gggcagagga tccgccgggc cttcatcgcc 1800 gaggaggggt ggctattggt ggccctggac tatagccaga tagagctcag ggtgctggcc 1860 cacctctccg gcgacgagaa cctgatccgg gtcttccagg aggggcggga catccacacg 1920 gagaccgcca gctggatgtt cggcgtcccc cgggaggccg tggaccccct gatgcgcgtg 1980 gcggccaaga ccatcaactt cggggtcctc tacggcatgt cggcccaccg cctctcccag 2040 gagctagcca tcccttacga ggaggcccag gccttcattg agcgctactt tcagagcttc 2100 cccaaggtgc gggcctggat tgagaagacc ctggaggagg gcaggaggcg ggggtacgtg 2160 gagaccctct tcggccgccg ccgctacgtg ccagacctag aggcccgggt gaagagcgtg 2220 cgggaggcgg ccgagcgcat ggccttcaac atgcccgtcc agggcaccgc cgccgacctc 2280 atgaagctgg ctatggtgaa gctcttcccc aggctggagg aaatgggggc caggatgctc 2340 cttcaggtcc acgacgagct ggtcctcgag gccccaaaag agagggcgga ggccgtggcc 2400 cggctggcca aggaggtcat ggagggggtg tatcccctgg ccgtgcccct ggaggtggag 2460 gtggggatag gggaggactg gctctccgcc aaggagtga 2499 <210> 14 <211> 2499 <212> DNA <213> Artificial Sequence <220> <223> Taq E507K / R536 <400> 14 atgaggggga tgctgcccct ctttgagccc aagggccggg tcctcctggt ggacggccac 60 cacctggcct accgcacctt ccacgccctg aagggcctca ccaccagccg gggggagccg 120 gtgcaggcgg tctacggctt cgccaagagc ctcctcaagg ccctcaagga ggacggggac 180 gcggtgatcg tggtctttga cgccaaggcc ccctccttcc gccacgaggc ctacgggggg 240 tacaaggcgg gccgggcccc cacgccggag gactttcccc ggcaactcgc cctcatcaag 300 gagctggtgg acctcctggg gctggcgcgc ctcgaggtcc cgggctacga ggcggacgac 360 gtcctggcca gcctggccaa gaaggcggaa aaggagggct acgaggtccg catcctcacc 420 gccgacaaag acctttacca gctcctttcc gaccgcatcc acgtcctcca ccccgagggg 480 tacctcatca ccccggcctg gctttgggaa aagtacggcc tgaggcccga ccagtgggcc 540 gactaccggg ccctgaccgg ggacgagtcc gacaaccttc ccggggtcaa gggcatcggg 600 gagaagacgg cgaggaagct tctggaggag tgggggagcc tggaagccct cctcaagaac 660 ctggaccggc tgaagcccgc catccgggag aagatcctgg cccacatgga cgatctgaag 720 ctctcctggg acctggccaa ggtgcgcacc gacctgcccc tggaggtgga cttcgccaaa 780 aggcgggagc ccgaccggga gaggcttagg gcctttctgg agaggcttga gtttggcagc 840 ctcctccacg agttcggcct tctggaaagc cccaaggccc tggaggaggc cccctggccc 900 ccgccggaag gggccttcgt gggctttgtg ctttcccgca aggagcccat gtgggccgat 960 cttctggccc tggccgccgc cagggggggc cgggtccacc gggcccccga gccttataaa 1020 gccctcaggg acctgaagga ggcgcggggg cttctcgcca aagacctgag cgttctggcc 1080 ctgagggaag gccttggcct cccgcccggc gacgacccca tgctcctcgc ctacctcctg 1140 gacccttcca acaccacccc cgagggggtg gcccggcgct acggcgggga gtggacggag 1200 gaggcggggg agcgggccgc cctttccgag aggctcttcg ccaacctgtg ggggaggctt 1260 gagggggagg agaggctcct ttggctttac cgggaggtgg agaggcccct ttccgctgtc 1320 ctggcccaca tggaggccac gggggtgcgc ctggacgtgg cctatctcag ggccttgtcc 1380 ctggaggtgg ccgaggagat cgcccgcctc gaggccgagg tcttccgcct ggccggccac 1440 cccttcaacc tcaactcccg ggaccagctg gaaagggtcc tctttgacga gctagggctt 1500 cccgccatcg gcaagacgaa aaagaccggc aagcgctcca ccagcgccgc cgtcctggag 1560 gccctccgcg aggcccaccc catcgtggag aagatcctgc agtacaagga gctcaccaag 1620 ctgaagagca cctacattga ccccttgccg gacctcatcc accccaggac gggccgcctc 1680 cacacccgct tcaaccagac ggccacggcc acgggcaggc taagtagctc cgatcccaac 1740 ctccagaaca tccccgtccg caccccgctt gggcagagga tccgccgggc cttcatcgcc 1800 gaggaggggt ggctattggt ggccctggac tatagccaga tagagctcag ggtgctggcc 1860 cacctctccg gcgacgagaa cctgatccgg gtcttccagg aggggcggga catccacacg 1920 gagaccgcca gctggatgtt cggcgtcccc cgggaggccg tggaccccct gatgcgccgg 1980 gcggccaaga ccatcaactt cggggtcctc tacggcatgt cggcccaccg cctctcccag 2040 gagctagcca tcccttacga ggaggcccag gccttcattg agcgctactt tcagagcttc 2100 cccaaggtgc gggcctggat tgagaagacc ctggaggagg gcaggaggcg ggggtacgtg 2160 gagaccctct tcggccgccg ccgctacgtg ccagacctag aggcccgggt gaagagcgtg 2220 cgggaggcgg ccgagcgcat ggccttcaac atgcccgtcc agggcaccgc cgccgacctc 2280 atgaagctgg ctatggtgaa gctcttcccc aggctggagg aaatgggggc caggatgctc 2340 cttcaggtcc acgacgagct ggtcctcgag gccccaaaag agagggcgga ggccgtggcc 2400 cggctggcca aggaggtcat ggagggggtg tatcccctgg ccgtgcccct ggaggtggag 2460 gtggggatag gggaggactg gctctccgcc aaggagtga 2499 <210> 15 <211> 2499 <212> DNA <213> Artificial Sequence <220> <223> Taq E507K / R660V <400> 15 atgaggggga tgctgcccct ctttgagccc aagggccggg tcctcctggt ggacggccac 60 cacctggcct accgcacctt ccacgccctg aagggcctca ccaccagccg gggggagccg 120 gtgcaggcgg tctacggctt cgccaagagc ctcctcaagg ccctcaagga ggacggggac 180 gcggtgatcg tggtctttga cgccaaggcc ccctccttcc gccacgaggc ctacgggggg 240 tacaaggcgg gccgggcccc cacgccggag gactttcccc ggcaactcgc cctcatcaag 300 gagctggtgg acctcctggg gctggcgcgc ctcgaggtcc cgggctacga ggcggacgac 360 gtcctggcca gcctggccaa gaaggcggaa aaggagggct acgaggtccg catcctcacc 420 gccgacaaag acctttacca gctcctttcc gaccgcatcc acgtcctcca ccccgagggg 480 tacctcatca ccccggcctg gctttgggaa aagtacggcc tgaggcccga ccagtgggcc 540 gactaccggg ccctgaccgg ggacgagtcc gacaaccttc ccggggtcaa gggcatcggg 600 gagaagacgg cgaggaagct tctggaggag tgggggagcc tggaagccct cctcaagaac 660 ctggaccggc tgaagcccgc catccgggag aagatcctgg cccacatgga cgatctgaag 720 ctctcctggg acctggccaa ggtgcgcacc gacctgcccc tggaggtgga cttcgccaaa 780 aggcgggagc ccgaccggga gaggcttagg gcctttctgg agaggcttga gtttggcagc 840 ctcctccacg agttcggcct tctggaaagc cccaaggccc tggaggaggc cccctggccc 900 ccgccggaag gggccttcgt gggctttgtg ctttcccgca aggagcccat gtgggccgat 960 cttctggccc tggccgccgc cagggggggc cgggtccacc gggcccccga gccttataaa 1020 gccctcaggg acctgaagga ggcgcggggg cttctcgcca aagacctgag cgttctggcc 1080 ctgagggaag gccttggcct cccgcccggc gacgacccca tgctcctcgc ctacctcctg 1140 gacccttcca acaccacccc cgagggggtg gcccggcgct acggcgggga gtggacggag 1200 gaggcggggg agcgggccgc cctttccgag aggctcttcg ccaacctgtg ggggaggctt 1260 gagggggagg agaggctcct ttggctttac cgggaggtgg agaggcccct ttccgctgtc 1320 ctggcccaca tggaggccac gggggtgcgc ctggacgtgg cctatctcag ggccttgtcc 1380 ctggaggtgg ccgaggagat cgcccgcctc gaggccgagg tcttccgcct ggccggccac 1440 cccttcaacc tcaactcccg ggaccagctg gaaagggtcc tctttgacga gctagggctt 1500 cccgccatcg gcaagacgaa aaagaccggc aagcgctcca ccagcgccgc cgtcctggag 1560 gccctccgcg aggcccaccc catcgtggag aagatcctgc agtaccggga gctcaccaag 1620 ctgaagagca cctacattga ccccttgccg gacctcatcc accccaggac gggccgcctc 1680 cacacccgct tcaaccagac ggccacggcc acgggcaggc taagtagctc cgatcccaac 1740 ctccagaaca tccccgtccg caccccgctt gggcagagga tccgccgggc cttcatcgcc 1800 gaggaggggt ggctattggt ggccctggac tatagccaga tagagctcag ggtgctggcc 1860 cacctctccg gcgacgagaa cctgatccgg gtcttccagg aggggcggga catccacacg 1920 gagaccgcca gctggatgtt cggcgtcccc cgggaggccg tggaccccct gatgcgcgtg 1980 gcggccaaga ccatcaactt cggggtcctc tacggcatgt cggcccaccg cctctcccag 2040 gagctagcca tcccttacga ggaggcccag gccttcattg agcgctactt tcagagcttc 2100 cccaaggtgc gggcctggat tgagaagacc ctggaggagg gcaggaggcg ggggtacgtg 2160 gagaccctct tcggccgccg ccgctacgtg ccagacctag aggcccgggt gaagagcgtg 2220 cgggaggcgg ccgagcgcat ggccttcaac atgcccgtcc agggcaccgc cgccgacctc 2280 atgaagctgg ctatggtgaa gctcttcccc aggctggagg aaatgggggc caggatgctc 2340 cttcaggtcc acgacgagct ggtcctcgag gccccaaaag agagggcgga ggccgtggcc 2400 cggctggcca aggaggtcat ggagggggtg tatcccctgg ccgtgcccct ggaggtggag 2460 gtggggatag gggaggactg gctctccgcc aaggagtga 2499 <210> 16 <211> 2499 <212> DNA <213> Artificial Sequence <220> <223> Taq E507K / R536K / R660V <400> 16 atgaggggga tgctgcccct ctttgagccc aagggccggg tcctcctggt ggacggccac 60 cacctggcct accgcacctt ccacgccctg aagggcctca ccaccagccg gggggagccg 120 gtgcaggcgg tctacggctt cgccaagagc ctcctcaagg ccctcaagga ggacggggac 180 gcggtgatcg tggtctttga cgccaaggcc ccctccttcc gccacgaggc ctacgggggg 240 tacaaggcgg gccgggcccc cacgccggag gactttcccc ggcaactcgc cctcatcaag 300 gagctggtgg acctcctggg gctggcgcgc ctcgaggtcc cgggctacga ggcggacgac 360 gtcctggcca gcctggccaa gaaggcggaa aaggagggct acgaggtccg catcctcacc 420 gccgacaaag acctttacca gctcctttcc gaccgcatcc acgtcctcca ccccgagggg 480 tacctcatca ccccggcctg gctttgggaa aagtacggcc tgaggcccga ccagtgggcc 540 gactaccggg ccctgaccgg ggacgagtcc gacaaccttc ccggggtcaa gggcatcggg 600 gagaagacgg cgaggaagct tctggaggag tgggggagcc tggaagccct cctcaagaac 660 ctggaccggc tgaagcccgc catccgggag aagatcctgg cccacatgga cgatctgaag 720 ctctcctggg acctggccaa ggtgcgcacc gacctgcccc tggaggtgga cttcgccaaa 780 aggcgggagc ccgaccggga gaggcttagg gcctttctgg agaggcttga gtttggcagc 840 ctcctccacg agttcggcct tctggaaagc cccaaggccc tggaggaggc cccctggccc 900 ccgccggaag gggccttcgt gggctttgtg ctttcccgca aggagcccat gtgggccgat 960 cttctggccc tggccgccgc cagggggggc cgggtccacc gggcccccga gccttataaa 1020 gccctcaggg acctgaagga ggcgcggggg cttctcgcca aagacctgag cgttctggcc 1080 ctgagggaag gccttggcct cccgcccggc gacgacccca tgctcctcgc ctacctcctg 1140 gacccttcca acaccacccc cgagggggtg gcccggcgct acggcgggga gtggacggag 1200 gaggcggggg agcgggccgc cctttccgag aggctcttcg ccaacctgtg ggggaggctt 1260 gagggggagg agaggctcct ttggctttac cgggaggtgg agaggcccct ttccgctgtc 1320 ctggcccaca tggaggccac gggggtgcgc ctggacgtgg cctatctcag ggccttgtcc 1380 ctggaggtgg ccgaggagat cgcccgcctc gaggccgagg tcttccgcct ggccggccac 1440 cccttcaacc tcaactcccg ggaccagctg gaaagggtcc tctttgacga gctagggctt 1500 cccgccatcg gcaagacgaa aaagaccggc aagcgctcca ccagcgccgc cgtcctggag 1560 gccctccgcg aggcccaccc catcgtggag aagatcctgc agtacaagga gctcaccaag 1620 ctgaagagca cctacattga ccccttgccg gacctcatcc accccaggac gggccgcctc 1680 cacacccgct tcaaccagac ggccacggcc acgggcaggc taagtagctc cgatcccaac 1740 ctccagaaca tccccgtccg caccccgctt gggcagagga tccgccgggc cttcatcgcc 1800 gaggaggggt ggctattggt ggccctggac tatagccaga tagagctcag ggtgctggcc 1860 cacctctccg gcgacgagaa cctgatccgg gtcttccagg aggggcggga catccacacg 1920 gagaccgcca gctggatgtt cggcgtcccc cgggaggccg tggaccccct gatgcgcgtg 1980 gcggccaaga ccatcaactt cggggtcctc tacggcatgt cggcccaccg cctctcccag 2040 gagctagcca tcccttacga ggaggcccag gccttcattg agcgctactt tcagagcttc 2100 cccaaggtgc gggcctggat tgagaagacc ctggaggagg gcaggaggcg ggggtacgtg 2160 gagaccctct tcggccgccg ccgctacgtg ccagacctag aggcccgggt gaagagcgtg 2220 cgggaggcgg ccgagcgcat ggccttcaac atgcccgtcc agggcaccgc cgccgacctc 2280 atgaagctgg ctatggtgaa gctcttcccc aggctggagg aaatgggggc caggatgctc 2340 cttcaggtcc acgacgagct ggtcctcgag gccccaaaag agagggcgga ggccgtggcc 2400 cggctggcca aggaggtcat ggagggggtg tatcccctgg ccgtgcccct ggaggtggag 2460 gtggggatag gggaggactg gctctccgcc aaggagtga 2499 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Eco-forward primer <400> 17 ggggtacctc atcaccccgg 20 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> R536K-reverse primer <400> 18 cttggtgagc tccttgtact gcaggat 27 <210> 19 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> R536K-forward primer <400> 19 atcctgcagt acaaggagct caccaag 27 <210> 20 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R660V-reverse primer <400> 20 gatggtcttg gccgccacgc gcatcagggg 30 <210> 21 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R660V-forward primer <400> 21 cccctgatgc gcgtggcggc caagaccatc 30 <210> 22 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Xba-reverse primer <400> 22 gctctagact atcactcctt ggcggagagc ca 32 <210> 23 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> E507K-reverse primer <400> 23 cttgccggtc tttttcgtct tgccgat 27 <210> 24 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> E507K-forward primer <400> 24 atcggcaaga cgaaaaagac cggcaag 27 <210> 25 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> rs1408799 forward primer <400> 25 ccagtgttag gttatttcta acttg 25 <210> 26 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> rs1408799 reverse_T primer <400> 26 gctcggagca catggtcaa 19 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> rs1408799 reverse_C primer <400> 27 gctcggagca catggtcag 19 <210> 28 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> rs1015362 forward primer <400> 28 tgaagagcag gaaagttctt ca 22 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs1015362 reverse_C primer <400> 29 actgtgtgtc tgaaacagtg 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs1015362 reverse_T primer <400> 30 actgtgtgtc tgaaacagta 20 <210> 31 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> rs4911414 forward_G primer <400> 31 gtaagtcttt gctgagaaat tcattg 26 <210> 32 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> rs4911414 forward_T primer <400> 32 gtaagtcttt gctgagaaat tcattt 26 <210> 33 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> rs4911414 reverse primer <400> 33 agtatccagg gttaatgtga aag 23 <210> 34 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> 1408799-FAM probe <400> 34 agatatttgt aaggtattct ggcct 25 <210> 35 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 1015362-HEX probe <400> 35 tgctgaacaa atagtcccga ccag 24 <210> 36 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 4911414-Texas Red probe <400> 36 tttctctagt tgcctttaag attt 24

Claims (15)

The glutamic acid (E), which is the 507th amino acid residue in the amino acid sequence of SEQ ID NO: 1, is substituted with lysine (K), arginine (R), which is the 536th amino acid residue, is substituted with lysine (K) R) is substituted with valine (V). delete 2. The method of claim 1, wherein the DNA polymerase distinguishes between a matched primer and a mismatched primer, wherein the mismatched primer and the mismatched primer hybridize to the target sequence, and the mismatched primer hybridizes to the hybridization target sequence Wherein the DNA polymerase comprises a non-standard nucleotide at the 3 ' end thereof. 2. The DNA polymerase according to claim 1, wherein the DNA polymerase exhibits a lower Ct value than the amplification of the target sequence comprising the mismatched primer, the amplification of the target sequence comprising the matched primer. A nucleic acid sequence encoding the DNA polymerase of claim 1. A vector comprising the nucleic acid sequence of claim 5. A host cell transformed with the vector of claim 6. The DNA polymerase of claim 1
a) one or more molds;
b) one or more matched primers, one or more mismatched primers or one or more matched primers and one or more mismatched primers; And
c) contacting with a nucleoside triphosphate,
Wherein the at least one matched primer and the mismatched primer hybridize to the target sequence, wherein at least one mutation or SNP in one or more templates is detected in vitro. 9. The method of claim 8, wherein the method comprises melting temperature analysis using a double-strand specific dye. 9. The method of claim 8, wherein the method is selected from the group consisting of real-time PCR, analysis on agarose gel after standard PCR, gene-specific amplification or allele-specific amplification through real-time PCR, tetra-primer amplification-refractory mutant system PCR, RTI ID = 0.0 &gt; isothermal &lt; / RTI &gt; amplification. A composition for detecting gene mutation or SNP comprising the DNA polymerase of claim 1. A PCR kit comprising the composition of claim 11. 13. The method of claim 12, further comprising one or more matched primers, one or more mismatched primers or one or more matched primers and one or more mismatched primers, wherein the one or more matched primers and one or more mismatched Wherein the primer is hybridized with the target sequence. 13. The PCR kit of claim 12, further comprising a nucleoside triphosphate. 13. The method of claim 12,
a) one or more buffers;
b) a reagent for quantification binding to double stranded DNA;
c) a polymerase blocking antibody;
d) one or more control values or control sequences; And
e) one or more molds; Wherein the PCR kit further comprises:

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