CN109251965B - PCR buffer composition for enhancing DNA polymerase activity with increased gene mutation specificity - Google Patents

PCR buffer composition for enhancing DNA polymerase activity with increased gene mutation specificity Download PDF

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CN109251965B
CN109251965B CN201810691172.6A CN201810691172A CN109251965B CN 109251965 B CN109251965 B CN 109251965B CN 201810691172 A CN201810691172 A CN 201810691172A CN 109251965 B CN109251965 B CN 109251965B
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李炳哲
朴日铉
李辉皓
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Genecast Co ltd
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Abstract

The present invention relates to a PCR buffer composition for enhancing DNA polymerase activity with increased gene mutation specificity and use thereof, and more particularly, to a PCR buffer composition for enhancing DNA polymerase activity with increased gene mutation specificity by inducing mutation at a specific amino acid position, a PCR kit for detecting gene mutation or SNP comprising the PCR buffer composition and/or DNA polymerase with increased gene mutation specificity, and a detection method for detecting at least one gene mutation or SNP in vitro (in vitro) from one or more templates using the kit.

Description

PCR buffer composition for enhancing DNA polymerase activity with increased gene mutation specificity
Technical Field
The present invention relates to a PCR buffer composition for enhancing DNA polymerase activity with increased gene mutation specificity and use thereof, and more particularly, to a PCR buffer composition for enhancing DNA polymerase activity with increased gene mutation specificity by inducing mutation at a specific amino acid position, a PCR kit for detecting gene mutation or SNP comprising the PCR buffer composition and/or DNA polymerase with increased gene mutation specificity, and a detection method for detecting at least one gene mutation or SNP in vitro (in vitro) from one or more templates using the kit.
Background
Since the first human genome sequence was discovered, researchers have focused on exploring genetic differences between individuals such as single nucleotide mutations (SNPs). It has become increasingly clear that single nucleotide mutations in the genome are associated with different resistance or predisposition to various diseases, and are therefore a major concern. Knowledge of future medically relevant nucleotide variations may be applied to treatment of the genetic supply of an individual and may prevent drug treatment that is ineffective or causes side effects (Shi, expert Rev. Mol. Diagn.1,363-365 (2001)). The development of time and cost efficient techniques for identifying nucleotide variations will lead to further advances in pharmacogenetics.
SNPs account for major genetic variation in the human genome, and can induce more than 90% of inter-individual variation. (Kwok, annu. Rev. Genomics Hum, genet.2,235-258 (2001); kwok and Chen, curr. Issues mol. Biol.5,43-60 (2003); twyman and Primrose, pharmacogenermics 4,67-79 (2003)). In order to detect other nucleic acid variations such as these genetic variations and mutations, various methods can be used. For example, identification of variants of a target nucleic acid can be achieved by hybridizing a nucleic acid sample to be analyzed with a hybridization 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 this hybridization method is not satisfactory in clinical practice particularly in terms of sensitivity required for measurement. Thus, PCR has been widely used in diagnostic detection methods for mutation detection in molecular biology, SNP and other allelic sequence variants (Saiki et al, science 239, 487-490 (1988)), in which a target nucleic acid to be detected is amplified by Polymerase Chain Reaction (PCR) before hybridization in consideration of the presence of the variant. For the hybridization primer used in such an assay, a single-stranded oligonucleotide is generally used. Specific examples of modifications of the assay include the use of fluorescent hybridization probes (Livak, genet.73-5 2017-07-12Anal.14,143-149 (1999)). In general, attempts have been made to automate the method of determining SNPs and other sequence variations (Gut, hum. Mutat.17,475-492 (2001)).
The sequence variation specific hybridization strategy known in the art is provided by so-called gene mutation specific amplification. In this detection method, variation-specific amplification primers are already used during amplification, usually primers having at their 3' -end so-called differential terminal nucleotide residues, which are complementary to only one specific variation of the target nucleic acid to be detected. In this method, nucleotide variants are measured as the presence or absence of DNA products after amplification by PCR. The principle of gene mutation specific amplification is based on the formation of canonical (canonical) or atypical primer-template complexes at the ends of gene mutation specific amplification primers. At the precisely paired 3' primer ends, amplification by DNA polymerase occurs, while extension is inhibited at the mismatched primer ends.
For example, U.S. Pat. No.5,595,890 discloses gene mutation-specific amplification methods and uses thereof, such as disclosing methods of use for detecting clinically relevant point mutations (point mutations) in k-ras tumor genes. In addition, U.S. Pat. No.5,521,301 discloses an allele-specific amplification method for genotyping of ABO blood group system. In contrast, U.S. Pat. No.5,639,611 discloses the use of allele-specific amplification in connection with detecting point mutations that cause sickle cell anemia. However, gene mutation-specific amplification or allele-specific amplification has the problem of low selectivity, thus requiring more complex, time and cost intensive optimization steps.
The methods for detecting sequence variations, polymorphisms and focusing on point mutations are particularly desirable for gene-specific amplification (or gene mutation-specific amplification) when the sequence variation to be detected is insufficient compared to the dominant variation of the same nucleic acid fragment (or the same gene).
This occurs, for example, when 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). For this purpose, DNA is first isolated from a body fluid such as blood, serum or plasma, and the DNA consists of insufficient sporadic tumor cells and an excess of non-proliferating cells. Thus, important mutations in tumor DNA in the k-ras gene should be detected from multiple replications in the presence of excess wild-type DNA.
All methods disclosed in the prior art for gene mutation specific amplification, despite the use of 3' -differential nucleotide residues, have the disadvantage that even if the target nucleic acid does not completely match the sequence variant to be detected, a lower level of primer extension takes place in the presence of a suitable DNA polymerase. In particular, when a particular sequence variant is detected by an excess of background nucleic acid comprising another sequence variant, a false positive result results. Another disadvantage of the known method is the need to use 3' -terminal differential oligonucleotide residues. The disadvantages of this PCR-based approach are mainly due to the inadequacy of the polymerase used in the method for sufficiently discriminating between mismatched bases. Therefore, it has not been possible to directly obtain clear information on the presence or absence of a mutation by PCR. To date, additional time and cost intensive purification and assay methods are required to definitively diagnose mutations. Therefore, new methods for improving the selectivity of gene mutation-specific or allele-specific PCR amplification will have a great impact on the reliability and robustness of direct gene mutation or SNP analysis by PCR.
Therefore, there is a continuing need to investigate DNA polymerases with increased gene mutation specificity and optimal reaction buffers for mixing various substances that allow the DNA polymerases to perform their functions.
The present inventors have made an effort to develop a novel DNA polymerase capable of improving gene mutation-specific PCR amplification selectivity and a reaction buffer for improving the activity thereof, and as a result, confirmed that gene mutation specificity is significantly increased when mutation is induced to amino acid residues at specific positions of Taq polymerase, and KCl, (NH) in a PCR buffer composition is controlled 4 ) 2 SO 4 And/or TMAC (tetramethylammonium chloride), the activity of the DNA polymerase having increased gene mutation specificity is enhanced, thereby completing the present invention.
Disclosure of Invention
The present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide a PCR buffer composition for enhancing DNA polymerase activity, which has increased specificity for gene mutation.
It is another object of the present invention to provide a PCR kit for detecting gene mutation or SNP, comprising the PCR buffer composition and/or a DNA polymerase having increased gene mutation specificity.
Another object of the present invention is to provide a method for in vitro (in vitro) detection of at least one gene mutation or SNP from more than one template using the PCR kit.
To achieve the object, the present invention provides a gene having an increased amountA mutation-specific DNA polymerase activity enhancing PCR buffer composition comprising 25 to 100mM KCl;1 to 15mM of (NH) 4 ) 2 SO 4 (ii) a The final pH was 8.0 to 9.0.
According to a preferred embodiment of the invention, the concentration of KCl is 60 to 90mM.
According to another preferred embodiment of the present invention, the (NH) is 4 ) 2 SO 4 Is 2 to 8mM.
According to yet another preferred embodiment of the invention, the concentration of KCl is 70 to 80mM, the (NH) 4 ) 2 SO 4 Is in a concentration of 4 to 6mM.
The present invention provides a PCR buffer composition for DNA polymerase activity enhancement with increased gene mutation specificity, further comprising 5 to 80mM TMAC (Tetra methyl ammonium chloride).
According to a preferred embodiment of the invention, the concentration of KCl is 40 to 90mM.
According to another preferred embodiment of the present invention, the (NH) 4 ) 2 SO 4 Is 1 to 7mM.
According to still another preferred embodiment of the present invention, the TMAC (tetramethylammonium chloride) is present in a concentration of 15 to 70mM, the KCl is present in a concentration of 50 to 80mM, and the (NH) is present 4 ) 2 SO 4 Is 1.5 to 6mM.
According to another preferred embodiment of the present invention, the PCR buffer composition may further comprise Tris-Cl and MgCl 2
The present invention provides a PCR kit for detecting gene mutation or SNP comprising the PCR buffer composition.
According to a preferred embodiment of the present invention, the PCR kit may further comprise a PCR primer consisting of SEQ ID NO:1 (base sequence of SEQ ID NO: 6), which may comprise (a) a DNA polymerase of Taq polymerase consisting of the amino acid sequence of SEQ ID NO:1, a substitution of the 507 amino acid residue in the amino acid sequence of 1; and (b) SEQ ID NO:1, the substitution of the 536 th amino acid residue, the substitution of the 660 th amino acid residue, or the substitution of the 536 th and 660 th, i.e., two, amino acid residues.
According to another preferred embodiment of the present invention, the substitution of the 507 th amino acid residue is a substitution of glutamic acid (E) with lysine (K), the substitution of the 536 th amino acid residue is a substitution of arginine (R) with lysine (K), and the substitution of the 660 th amino acid residue is a substitution of arginine (R) with valine (V).
According to another preferred embodiment of the present invention, the PCR kit further comprises: a) A nucleoside triphosphate; b) A quantification reagent that binds to double-stranded DNA; c) A polymerase blocking antibody; d) One or more control values or control sequences; e) One or more templates.
The invention also provides a detection method for detecting at least one gene mutation or SNP in vitro (in vitro) from more than one template by using the PCR kit.
Compared with the prior art, the invention has the following beneficial effects:
the present invention provides an optimal PCR buffer composition that enables a DNA polymerase having increased gene mutation specificity to perform its function, and is used together with a DNA polymerase having increased gene mutation specificity, thereby significantly improving the activity of the DNA polymerase and enabling reliable gene mutation-specific amplification. In addition, the kit comprising the PCR buffer composition of the present invention and/or the DNA polymerase having increased gene specificity can effectively detect gene mutation or SNP, and thus can be effectively used for medical diagnosis of diseases and research of recombinant DNA.
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Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 depicts the preparation of Taq DNA polymerase containing mutations R536K, R660V and R536K/R660V, respectively, wherein (a) is a schematic diagram of fragment PCR and overlap PCR, (B) shows the result of electrophoretic confirmation of the amplified product in the fragment PCR, and (c) shows the result of electrophoretic confirmation of the amplified product after amplification of the full length by overlap PCR;
FIG. 2 shows the result of electrophoretic confirmation of the SAP-treated pUC19 vector and the purified overlapping PCR product of FIG. 1 (c) after digestion with restriction enzymes EcoRI/XbaI for gel extraction;
FIG. 3 is a schematic of fragment PCR and overlap PCR in the preparation of Taq DNA polymerase containing mutations E507K, E507K/R536K, E507K/R660V and E507K/R536K/R660V, respectively;
FIG. 4 shows the results of electrophoretic confirmation of the SAP treated pUC19 vector and the purified PCR products of FIG. 3 after digestion with restriction enzymes EcoRI/XbaI in order to extract the gel;
FIG. 5 is a schematic view showing a process of preparing a PCR template by collecting oral epithelial cells;
FIG. 6 shows the results of AS-qPCR on rs1408799 using E507K/R536K, E507K/R660V and E507K/R536K/R660V Taq polymerase of the present invention; as a control group, taq polymerase including the E507K mutation was used;
FIG. 7 shows the results of AS-qPCR on rs1015362 using E507K/R536K, E507K/R660V and E507K/R536K/R660V Taq polymerase of the present invention; as a control group, taq polymerase including the E507K mutation was used;
FIG. 8 shows the results of AS-qPCR on rs4911414 using E507K/R536K, E507K/R660V and E507K/R536K/R660V Taq polymerase of the present invention; as a control group, taq polymerase including the E507K mutation was used;
FIG. 9 is a graph showing the confirmation of delayed effect of mismatch amplification according to the change of KCl concentration in a reaction buffer using E507K, E507K/R536K, E507K/R660V and E507K/R536K/R660V Taq polymerase of the present invention;
FIG. 10 shows that in order to determine the optimal KCl concentration in the reaction buffer, immobilization (NH) 4 ) 2 SO 4 And varying the concentration of KCl for amplification, and confirming the result of the PCR product by electrophoresis;
FIG. 11 shows that (NH) in the buffer was optimized for the determination of the reaction 4 ) 2 SO 4 Concentration, concentration of fixed KClDegree and change (NH) 4 ) 2 SO 4 After the concentration of (2) is amplified, the result of the PCR product is confirmed by electrophoresis;
FIG. 12 shows the results according to the (NH) in reaction buffer 4 ) 2 SO 4 Graph for confirming the effect of delay in mismatch amplification by change in concentration;
FIG. 13 shows the immobilization of KCl and (NH) in reaction buffer 4 ) 2 SO 4 A graph for confirming the effect of delaying mismatch amplification according to the change in the concentration of TMAC;
FIG. 14 shows the immobilization of TMAC and (NH) in reaction buffer 4 ) 2 SO 4 The concentration of (2) was determined from the change in KCl concentration, and a graph showing the effect of delaying mismatch amplification was obtained.
Detailed Description
The present invention will be described in detail below.
As described above, in order to overcome the drawbacks of the gene mutation-specific amplification methods disclosed in the prior art, it is necessary to continuously develop a DNA polymerase having increased gene mutation specificity and an optimal reaction buffer in which a plurality of substances are mixed so that the DNA polymerase can exert its functions. The present inventors have made an effort to develop a novel DNA polymerase capable of improving gene mutation-specific PCR amplification selectivity and a reaction buffer for improving the activity thereof, and as a result, confirmed that gene mutation specificity is significantly increased when mutation is induced to amino acid residues at specific positions of Taq polymerase, and KCl, (NH) in a PCR buffer composition is controlled 4 ) 2 SO 4 And/or TMAC (tetramethylammonium chloride), the activity of the DNA polymerase having increased gene mutation specificity is enhanced, thereby completing the present invention.
The present invention provides an optimal PCR buffer composition that enables a DNA polymerase having increased gene mutation specificity to perform its function, and provides a DNA polymerase having increased gene mutation specificity, which is used together with the PCR buffer composition, thereby significantly improving the activity of the DNA polymerase and realizing reliable gene mutation-specific amplification. In addition, the kit comprising the PCR buffer composition of the present invention and/or the DNA polymerase having increased gene specificity can effectively detect gene mutation or SNP, and thus can be effectively used for medical diagnosis of diseases and research of recombinant DNA.
Hereinafter, terms used herein will be described.
As used herein, "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 naturally or genetically encoded α -amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), valine (Val or V).
Amino acids are typically organic acids and may comprise a substituted or unsubstituted amino group, a substituted or unsubstituted carboxyl group and at least one side chain (chain) or group (group), or any analogue of these groups. Exemplary side chains include sulfhydryl, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazino, cyano, halo, hydrazide, alkenyl, alkynyl, ether, borate, boronic acid, phospho, phosphino, phosphine, heterocycle, enone, imine, aldehyde, ester, thioacid, hydroxylamine, or any combination of these groups.
Other exemplary amino acids include, but are not limited to: amino acids containing photoactivatable cross-linkers, metal-bound amino acids, spin-labeled amino acids, fluorescent amino acids, metal-containing amino acids, amino acids with novel functional groups, amino acids that interact covalently or non-covalently with other molecules, photocagable and/or photoisomerizable amino acids, radioactive amino acids, amino acids containing biotin or biotin analogs, glycosylated amino acids, other carbohydrate-modified amino acids, amino acids containing polyethylene glycol or polyethers, heavy atom substituted amino acids, chemically and/or photolytically decomposable amino acids, carbon-linked sugar-containing amino acids, redox-active amino acids, sulfur carboxylic acid-containing amino acids, and amino acids containing at least one toxic moiety.
In the DNA polymerase of the present invention, the term "mutant" refers to a recombinant polypeptide comprising one or more amino acid substitutions relative to a naturally occurring or unmodified DNA polymerase.
The term "thermostable polymerase" (referring to an enzyme that is thermostable) refers to a polymerase that is sufficiently heat resistant, retains sufficient activity to effect a subsequent polynucleotide extension reaction, and does not irreversibly denature (inactivate) upon warming during the time required to effect denaturation of double-stranded nucleic acids. As used herein, it applies to cycling temperatures in reactions such as PCR. Irreversible denaturation here means a complete loss of permanent enzyme activity. For thermostable polymerases, enzymatic activity refers to catalyzing the combination of nucleotides in a suitable manner to form a complementary polynucleotide extension product on a template nucleic acid strand. Thermophilic bacteria-derived thermostable DNA polymerases include, for example: DNA polymerases from Thermotoga maritima (Thermotoga maritime), thermus aquaticus (Thermus aquaticus), thermus thermophilus (Thermus thermophilus), thermus flavus (Thermus flavus), thermus filiformis (Thermus filiformis), thermus species Sps17, thermus species Z05, thermus firmus (Thermus caldophilus), bacillus caldotena (Bacillus caldotenax), thermopongata (Thermotoga neapolia), and Thermotoga africana (Thermosipho africana).
The term "thermally active" refers to an enzyme that retains its catalytic properties at temperatures typically used during the reverse transcription or annealing/extension phase of RT-PCR and/or PCR reactions (i.e., 45-80 ℃). Thermostable enzymes are enzymes that are not irreversibly inactivated or denatured when treated with the high temperatures required for denaturation of nucleic acids. Whereas a thermoactive enzyme may or may not be thermostable. A thermoactive DNA polymerase may include, but is not limited to, DNA or RNA dependent on a thermophilic or mesophilic species.
The term "host cell" refers to unicellular prokaryotic and eukaryotic organisms (e.g., bacteria, yeast, and actinomycetes) and unicells from higher plants or animals for culture in cell culture media.
The term "vector" is a DNA molecule such as a plasmid, phage, artificial chromosome, etc., which is capable of being replicated and which is capable of delivering foreign DNA to recipient cells, e.g., a gene. "plasmid", "vector" or "plasmid vector" are used interchangeably herein.
The term "nucleotide" refers to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single strand or double strand form, and may include analogs of natural nucleotides, unless otherwise specified.
The term "nucleic acid" or "polynucleotide" refers to a polymer of DNA or RNA, or analogs thereof. The nucleic acid may be, for example, a chromosome or chromosome fragment, a vector (e.g., an expression vector), an expression cassette, a naked DNA or RNA polymer, a Polymerase Chain Reaction (PCR) product, an oligonucleotide, a probe or primer, or comprise such. The nucleic acid may be, for example, single-stranded, double-stranded, or triple-stranded, but is not limited to any particular length. Unless otherwise indicated, a particular nucleic acid sequence includes, or can encode, a complementary sequence in addition to any sequence specified.
The term "primer" refers to a polynucleotide that can serve as a point of initiation of nucleic acid synthesis in the direction of the template when the polynucleotide is under conditions of initiation of extension. Primers can also be used in a variety of other oligonucleotide-mediated synthesis processes, including de novo synthesis of RNA and as a promoter for in vitro transcription-related processes. The primer is typically a single-stranded oligonucleotide (e.g., an oligodeoxyribonucleotide). Suitable lengths for primers are generally in the range of 6 to 40 nucleotides, more typically 15 to 35 nucleotides in length and depending on the intended use. Short primer molecules generally require lower temperatures to form sufficiently stable hybridization complexes with the template. The primer need not reflect the correct sequence of the template, but the primer must be sufficiently complementary to hybridize to the template to be extended.In particular embodiments, the term "primer pair" refers to a primer set comprising a 5 'sense primer that hybridizes complementarily 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 sequence to be amplified. If necessary, the primers can be labeled by incorporating a label that is detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful markers include: 32 p, fluorescent dyes, electron density reagents, enzymes (typically used in ELISA assays), biotin or haptens, and proteins for which antisera or monoclonal antibodies may be used.
The term "5 '-nucleic acid hydrolase (nuclease) probe" refers to an oligonucleotide comprising at least one luminescent label moiety for performing a 5' -nucleic acid hydrolase reaction to detect a target nucleic acid. In some embodiments, for example, a 5' -nucleolytic enzyme probe comprises only a single luminescent moiety (e.g., a fluorescent dye, etc.). In certain embodiments, the 5' -nucleolytic enzyme 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 labeled moieties, one of the two labels is separated or cleaved from the oligonucleotide, and the radiation intensity is increased and released. In certain embodiments, the 5' -nucleolytic enzyme probe is labeled with two different fluorescent dyes, e.g., a 5' -terminal reporter dye and a 3' -terminal quencher dye or moiety. In some embodiments, the 5' -nuclease probe is further labeled on the end or labeled at one or more positions other than the end. When the probe is intact, energy transfer typically occurs between the two fluorescent materials such that the fluorescent emission from the reporter dye is quenched by more than a certain fraction. During the extension step of the polymerase chain reaction, for example, the 5' -nucleolytic enzyme probe bound to the template nucleic acid has an activity such that the fluorescent emission of the reporter dye is no longer quenched, e.g., by the 5' to 3' -nucleolytic enzyme activity of Taq polymerase or other polymerases. In some embodiments, the 5 '-nucleolytic enzyme probe can be labeled with two or more different reporter dyes and 3' -terminal quencher dyes or moieties.
The term "FRET "or" fluorescence resonance energy transfer "or" forest resonance energy transfer "refers to the energy transfer between two or more chromophores, a donor chromophore and an acceptor chromophore (referred to as a quencher). The donor is typically excited by emitting light of the appropriate wavelength to transfer energy to the acceptor. The receptor typically re-emits the moving energy in the form of re-emission of light at a different wavelength. When the acceptor is a "dark" quencher, the energy of movement is dispersed in a form other than light. Whether a particular fluorophore acts as a donor or acceptor depends on the nature of the FRET pair other members. Commonly used donor-acceptor pairs include FAM-TAMRA pairs. Common quenchers are DABCYL and TAMRA. Commonly used dark quenchers include: black hole quench TM (BHQ),(Biosearch Technologies,Inc.,Novato,Cal.),Iowa Black TM (Integrated DNA Tech.,Inc.,Coralville,Iowa),
Figure BDA0001712784320000091
BlackBerry TM Quencher 650(BBQ-650)(Berry&Assoc.,Dexter,Mich.)。
The terms "conventional" or "natural" when referring to a nucleobase, nucleoside triphosphate or nucleotide mean that it occurs naturally in the polynucleotide (i.e., dATP, dGTP, dCTP, dTTP for DNA) and dITP and 7-deaza-dGTP are often used in place of dGTP and may be used in place of dATP in vitro DNA synthesis reactions such as sequencing.
The terms "unconventional" or "modified," when referring to a nucleic acid base, nucleoside, or nucleotide, includes modifications, derivatives, or analogs of the conventional bases, nucleosides, or nucleotides that occur naturally in the particular polynucleotide. In contrast to conventional dNTPs, a particular unconventional nucleotide is modified at the 2' position of the ribose. Thus, even if the naturally occurring nucleotide of an RNA is a ribonucleotide (i.e., ATP, GTP, CTPUTP, collectively rNTP), the nucleotide has a hydroxyl group at the 2' position of the sugar, and the dNTP is relatively absent, and thus, as used herein, a ribonucleotide is an unconventional nucleotide that serves as a substrate for DNA polymerase. As used herein, unconventional nucleotides include, but are not limited to, compounds that function as terminators for nucleic acid sequencing. Exemplary terminalStopple compounds include, but are not limited to, compounds having a 2',3' -dideoxy structure, known as dideoxynucleoside triphosphates. The dideoxynucleoside triphosphates ddATP, ddTTP, ddCTP and ddGTP, collectively referred to as ddNTPs. Other examples of terminator compounds include 2' -PO of ribonucleotides 4 And the like. Other unconventional nucleotides include, but are not limited to, thiodNTPs ([ [ alpha ] ])]-S]dNTP)、5'-[α]-boron (borano) -dNTP, [ alpha ]]-methyl-phosphonic acid dNTP, ribonucleoside triphosphate (rNTP). Unconventional bases may be replaced by radioisotopes such as 32 P、 33 P or 35 S; fluorescence labeling; a chemiluminescent label; a bioluminescent marker; hapten labels such as biotin; or an enzymatic label such as streptavidin or avidin. Fluorescent labels may include negatively charged dyes such as dyes of the fluorescein family, or neutrally charged dyes such as dyes of the rhodamine family, or positively charged dyes such as the cyanine family. The fluorescein family of dyes include, for example, FAM, HEX, TET, JOE, NAN, and ZOE. Dyes of the rhodamine family include Texas Red, ROX, R110, R6G, and TAMRA. Various dyes or nucleotides labeled with FAM, HEX, TET, JOE, NAN, ZOE, ROX, R110, R6G, texas Red, and TAMRA are marketed by Perkin-Elmer (Boston, mass.), applied Biosystems (Foster, calif.), invitrogen/Molecular Probes (Uygur., oreg.). Cyanine family dyes, including Cy2, cy3, cy5, and Cy7, are marketed by GE Healthcare UK Limited (Amersham Place, little Chalfount, white gold Hanshire, england).
The term "mismatch discrimination" refers to the biocatalytic (e.g., polymerase, ligase, etc.) ability to distinguish perfectly complementary sequences from mismatch-containing sequences when a nucleic acid (e.g., a primer or other oligonucleotide) is extended in a template-dependent manner by attaching (e.g., covalently) one or more nucleotides to the nucleic acid. The term "mismatch discrimination" refers to the biocatalytic ability of an extended nucleic acid (e.g., a primer or other oligonucleotide) to distinguish between fully complementary sequences from a mismatch-containing (about complementary) sequence that is mismatched at the 3' end of the nucleic acid as compared to the template to which the nucleic acid hybridizes. In some embodiments, the extended nucleic acid comprises a mismatch at the 3' end 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 from the terminus to the fully complementary sequence.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
The present invention relates to a PCR buffer composition for DNA polymerase activity enhancement with increased gene mutation specificity, the composition comprising 25 to 100mM KCI;1 to 15mM of (NH) 4 ) 2 SO 4 (ii) a The final pH was 8.0 to 9.0.
The polymerase used in the PCR should use an optimal reaction buffer in which various substances are mixed in order to perform its function. The reaction buffer usually contains a factor that stabilizes the pH, a metal ion as a cofactor (cofactor) and a stabilizing component that prevents denaturation of the polymerase.
The KCl can be used as an essential factor for stabilizing the enzyme, and helps the primer pair with the target DNA (pairing). In the present invention, the optimum concentration is determined by adjusting the concentration of KCl in the reaction buffer to confirm a high cation concentration that maximally delays amplification caused by mismatch while not decreasing amplification efficiency caused by match.
The mismatched amplification delay effect according to the change of KCl concentration in the reaction buffer was confirmed using E507K, E507K/R536K, E507K/R660V Taq polymerase, and E507K/R536K/R660V Taq polymerase, respectively, and as a result, the mismatched amplification delay effect was excellent even when KCl was not present in the reaction buffer for E507K/R536K/R660V Taq polymerase, as shown in FIG. 9; at 50mM for E507K/R536 and E507K/R660V; as a control E507K, the mismatch amplification delaying effect was excellent at 100 mM. As a result, E507K/R536K/R660V Taq polymerase was the lowest for the threshold KCl concentration, and E507K/R536K and E507K/R660V were lower than E507K.
To further deduce the appropriate KCI concentration, (NH) in reaction buffer was added using E507K/R536K/R660V Taq polymerase 4 ) 2 SO 4 The concentration was fixed at a certain value and amplification was performed with various concentrations of KCl. Confirmation of the results of amplification products by electrophoresisAs shown in FIG. 10, it was confirmed that the appropriate KCI concentration was 75mM.
Therefore, the KCl concentration in the buffer composition for PCR reaction of the present invention may be 25 to 100mM, preferably 60 to 90mM, more preferably 70 to 80mM, most preferably 75mM.
When the concentration of KCl is less than 25mM, there is no effect on the amplification of the general target, but the difference between the amplification by the matched primer and the amplification by the mismatched primer can be reduced, and when the concentration is more than 100mM, there is a problem that the amplification efficiency of the general target is reduced.
(NH) for PCR reaction buffer composition 4 ) 2 SO 4 Is a necessary cofactor for enzymatic activity, and is used with Tris to increase the activity of the polymerase. In one embodiment of the present invention, based on the above-mentioned derivation, the KCl concentration in the reaction buffer was fixed at 75mM and (NH) 4 ) 2 SO 4 Was varied from 2.5mM to 25mM to confirm appropriateness of (NH) 4 ) 2 SO 4 The concentration of (c).
The results are shown in FIG. 11, at 2.5mM to 15mM (NH) 4 ) 2 SO 4 At a concentration of (D), the amplification product was confirmed to be appropriate (NH) 4 ) 2 SO 4 The concentration of (2) is 5mM.
Further on (NH) 4 ) 2 SO 4 As shown in FIG. 12, when the concentration of (C) was close to 5mM (2.5 mM, 5mM, 10mM, respectively), AS-qPCR was carried out, and the difference in Ct value was the largest at 10mM, but the Ct value was slightly delayed in the matched amplification and the peak was downward-tilted, so that the optimum (NH) was determined 4 ) 2 SO 4 Is 5mM.
Therefore, (NH) in the buffer composition for PCR reaction of the present invention 4 ) 2 SO 4 The concentration of (b) may be 1 to 15mM, preferably 2.5 to 8mM, more preferably 4 to 6mM, most preferably 5mM.
When (NH) 4 ) 2 SO 4 Concentrations below 1mM had no effect on amplification of the general target, but resulted in reduced differences between amplification with matched primers and amplification with mismatched primers, and concentrations above 1mMAt 15mM, the amplification efficiency of the general target is lowered.
Thus, an optimal PCR buffer composition of the invention may comprise 70 to 80mM KCl;4 to 6mM of (NH) 4 ) 2 SO 4 (ii) a The final pH was 8.0 to 9.0.
The PCR buffer composition of the present invention may further comprise 5 to 80mM TMAC (Tetra methyl ammonium chloride).
TMAC is commonly used to reduce amplification due to mismatches or to increase stringency of hybridization reactions (stringency). In one embodiment of the present invention, the KCl concentration in the reaction buffer is fixed at 75mM, (NH) 4 ) 2 SO 4 Was fixed at 5mM, and the TMAC concentration was varied from 0mM to 80mM to confirm the appropriate TMAC concentration.
As a result, as shown in FIG. 13, it was confirmed that the TMAC concentration was appropriate and 70mM for E507K/R536K Taq polymerase; 25mM for E507K/R536K/R660V Taq polymerase. Further TMAC concentration was fixed at 25mM, (NH) 4 ) 2 SO 4 The KCl concentration was adjusted to 20, 40, 60 and 80mM and amplification was performed while fixing the concentration of (2.5 mM), and it was confirmed that the appropriate KCl concentration was 60mM for SNPs rs1015362 and rs4911414, as shown in FIG. 14.
When the concentration of TMAC exceeds 80mM, the amplification efficiency decreases, and therefore TMAC is preferably used in the above range.
Therefore, when the PCR buffer composition of the present invention comprises 5 to 80mM TMAC, the concentration of KCl may be 40 to 90mM, preferably 50 to 80mM, (NH) 4 ) 2 SO 4 The concentration of (B) may be 1 to 7mM, preferably 1.5 to 6mM.
Thus, when TMAC is included, an optimal PCR buffer composition of the present invention may include 15 to 70mM TMAC;50 to 80mM KCl;1.5 to 6mM of (NH) 4 ) 2 SO 4 (ii) a The final pH was 8.0 to 9.0.
The PCR buffer composition of the present invention may further comprise Tris. Cl and MgCl 2 Further, tween 20 and Bovine Serum Albumin (BSA) may be included.
The present invention provides a PCR kit for detecting gene mutation or SNP comprising the PCR buffer composition.
The PCR kit of the present invention may further comprise a PCR primer consisting of SEQ ID NO:1, the DNA polymerase can comprise (a) the amino acid sequence of SEQ ID NO:1, the substitution of the 507 amino acid residue in the amino acid sequence of seq id no; and (b) SEQ ID NO:1, the substitution of the 536 th amino acid residue, the substitution of the 660 th amino acid residue, or the substitution of the 536 th and 660 th, i.e., two, amino acid residues.
According to another preferred embodiment of the present invention, the substitution of 507 amino acid residue is a substitution of glutamic acid (E) with lysine (K), the substitution of 536 amino acid residue is a substitution of arginine (R) with lysine (K), and the substitution of 660 amino acid residue is a substitution of arginine (R) with valine (V).
The PCR kit of the present invention may include any reagents or other elements known to those of ordinary skill in the art for use in primer extension.
According to another preferred embodiment of the present invention, the PCR kit comprises more than one matched primer, more than one mismatched primer or more than one matched primer and more than one mismatched primer, wherein the more than one matched primer and the more than one mismatched primer hybridize to the target sequence, and the mismatched primer comprises an atypical (non-canonical) nucleotide at the 3' end to the 7 th base position of the hybridized target sequence.
The PCR kit of the present invention may further comprise: a) A nucleoside triphosphate; b) A quantification reagent that binds to double-stranded DNA; c) A polymerase blocking antibody; d) One or more control values or control sequences; e) One or more templates.
The "Taq polymerase", which is a thermostable DNA polymerase named according to the name of Thermus aquaticus (Thermus aquaticus), is originally isolated from the thermophilic bacterium. Thermus aquaticus is a bacterium that inhabits in hot springs and hot water spray ports, and Taq polymerase is an enzyme that is recognized as an enzyme capable of withstanding the conditions of protein denaturation (high temperature) required in the PCR process. The optimal activity temperature of Taq polymerase is 75-80 ℃, the half-life period is more than 2 hours at 92.5 ℃, 40 minutes at 95 ℃ and 9 minutes at 97.5 ℃, and 1000 base pairs of DNA can be copied within 10 seconds at 72 ℃. This lacks 3 → '5' exonuclease (exouclase) correcting activity and the error rate was measured in about 1 out of 9000 nucleotides. For example, when thermostable Taq is used, PCR can be performed at a high temperature (60 ℃ or higher). For Taq polymerase, SEQ ID NO:1 as a reference sequence.
In the present invention, SEQ ID NO:1, the Taq polymerase in which glutamic acid (E) at the 507 th amino acid residue is substituted with lysine (K) is named "E507K" (SEQ ID NO:2, base sequence SEQ ID NO: 7); SEQ ID NO:1, the Taq polymerase in which glutamic acid (E) at the 507 th amino acid residue is substituted with lysine (K) and arginine (R) at the 536 th amino acid residue is substituted with lysine (K) is named "E507K/R536K" (SEQ ID NO:3, base sequence SEQ ID NO: 8); the amino acid sequence of SEQ ID NO:1, the Taq polymerase in which glutamic acid (E) at the 507 th amino acid residue is substituted with lysine (K) and arginine (R) at the 660 th amino acid residue is substituted with valine (V) is named "E507K/R660V" (SEQ ID NO:4, base sequence SEQ ID NO: 9); finally, SEQ ID NO:1, glutamic acid (E) at the 507 amino acid residue was substituted with lysine (K), arginine (R) at the 536 amino acid residue was substituted with lysine (K), and Taq polymerase in which arginine (R) at the 660 amino acid residue was substituted with valine (V) was named "E507K/R536K/R660V" (SEQ ID NO:5, base sequence SEQ ID NO: 10).
According to one embodiment of the present invention, the DNA polymerase distinguishes between a matched primer and a mismatched primer, which hybridize to a target sequence, and the mismatched primer, which may include an atypical nucleotide at its 3' end with respect to the hybridized target sequence.
The mismatch primer is a hybridizing oligonucleotide, must be sufficiently complementary to hybridize to 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 (Watson-Crick) base pairs, A-U, A-T, and G-C.
The "atypical nucleotide" or "non-complementary nucleotide" refers to A-C, A-G, G-U, G-T, T-C, T-U, A, G-G, T-T, U-U, C-C, C-U, except Watson-Crick base pairs.
According to a preferred embodiment of the invention, the amplification of the target sequence comprising the matched primer may show a comparatively lower Ct value compared to the amplification of the target sequence comprising the mismatched primer.
For example, by covalently attaching one or more nucleotides to a primer, a DNA polymerase can extend a matched primer more efficiently than a primer that mismatches in a target-sequence dependent manner. Here, higher efficiency can be observed by the following example, as in RT-PCR, the Ct value of the matched primers is lower compared to the mismatched primers. The difference in Ct values between the matched and mismatched primers is 10 or more, preferably 10 to 20, or there is no amplicon synthesis caused by the mismatched primer.
For example, using a matched forward primer and reverse primer in a first reaction and a mismatched forward primer and matched reverse primer in the same experimental setup in a second reaction, the first reaction is larger than the second reaction by standard PCR-generated products.
The Ct (threshold cross cycle) value represents the DNA quantification method of quantitative PCR, depending on the fluorescence over the number of cycles of the plotted log phase. The threshold for DNA-based fluorescence detection is set at least slightly above background. The cycle number at which fluorescence exceeds the threshold is called Ct or Cq according to the MIQE criterion (quantification cycle). The Ct value for a given reaction is defined as the number of cycles at which the fluorescence emission crosses the fixed limit. For example, SYBR Green I and fluorescent probes can be used for real-time PCR for template DNA quantification. Fluorescence from the sample was collected during PCR at each cycle and plotted against cycle number. The initial template concentration is inversely proportional to the time at which the fluorescent signal is initially displayed. The higher the template concentration, the earlier the signal appears (at lower cycle numbers).
The invention also relates to nucleic acid sequences encoding the above DNA polymerases and vectors and host cells comprising the nucleic acid sequences. Various vectors can be prepared using the nucleic acid encoding the DNA polymerase of the present invention. Any vector may be used which includes replicon and control sequences derived from a species interchangeable with the host cell. The vector of the present invention may be an expression vector and comprise a nucleic acid region controlling transcription and translation operably linked to a nucleic acid sequence encoding a DNA polymerase of the present invention. Control sequences refer to the DNA sequences required for expression of an operably linked coding sequence in a particular host organism. For example, suitable regulatory sequences for prokaryotes include a promoter, any operational sequences and an alc ribosome binding site. In addition, the vector may contain a "Positive Regulatory Element (PRE)" to enhance the half-life of the transcribed mRNA. The nucleic acid regions that control transcription and translation are generally suitable for use in a host cell for expression of a polymerase. Various types of suitable expression vectors and suitable regulatory sequences for use in various host cells are known in the art. Generally, transcriptional and translational regulatory sequences can include, for example, promoter sequences, ribosome binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activating sequences. In typical embodiments, the regulatory sequences include a promoter and transcription initiation and termination sequences. The vector will also typically contain a polylinker region containing several restriction sites for insertion of foreign DNA. In certain embodiments, a "fusion tag" is used to facilitate purification, and if desired, the tag/tag (e.g., "histidine-tag") is subsequently removed. However, these are generally not necessary when purifying a thermoactive and/or thermostable protein from a mesophilic host (e.g. E.coli) using a "heating step". Appropriate vectors containing coding DNA replication sequences, regulatory sequences, phenotype selection genes, and the mutant polymerase of interest are prepared using standard recombinant DNA techniques. As is known in the art, isolated plasmids, viral vectors and DNA fragments are broken down and cut and ligated together in a specific order to produce the desired vector.
In a preferred embodiment of the invention, the expression vector contains a selectable marker gene for selection of transformed host cells. Selection genes are known in the art and will vary depending on the host cell used. Suitable selection genes may include genes encoding ampicillin and/or tetracycline resistance, whereby cells cultured with these vectors can be transformed in the presence of these antibiotics.
In a preferred embodiment of the invention, the nucleic acid sequence encoding the DNA polymerase of the invention is introduced into the cell alone or in combination with a vector. 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,
Figure BDA0001712784320000151
Electrophoresis, viral infection, and the like.
Prokaryotes are used as host cells for the initial cloning steps of the invention. This is particularly useful in order to prepare large quantities of DNA rapidly, to prepare single stranded DNA templates for generating site-directed mutations, to screen many mutants simultaneously, and to sequence the resulting mutants for DNA. Suitable host cells for prokaryotic cells include e.coli K12 strain 94 (ATCC No.31,446), e.coli 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; coli, such as HB101, JM101, NM522, NM538, NM539, and other species, including the bacillaceae family, such as Bacillus subtilis, other enterobacteriaceae family, such as Salmonella typhimurium, serratia marcescens, and various prokaryotic genera of Pseudomonas species (Pseudomonas sp.) may be used as hosts, typically plasmids used to transform e.coli (e.coli) include pBR322, pUCI8, pucscriri 9, pucc 8, pucc 119, and bluept m13, however, many other suitable vectors may be used.
The present invention also provides a method for preparing a DNA polymerase, comprising the steps of: a step of culturing the host cell; a step of isolating 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 the DNA polymerase under appropriate conditions to 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. A suitable host cell for the preparation of polymerases with plasmid vectors containing the lambda pL promoter is the E.coli strain DG116 (ATCC No. 53606). Depending on the expression, the polymerase can be collected and isolated.
Once purified, mismatch discrimination of the DNA polymerase of the present invention can be determined. For example, mismatch discrimination activity is measured by comparing the amplification of a target sequence with a perfect match of a primer to the amplification of a target sequence with a single base mismatch at the 3' end of the primer. Amplification can be detected in real time, for example, by using TaqMan TM And (3) a probe. The ability of the polymerase to distinguish between two target sequences can be estimated by comparing the Ct of the two reactions.
The present invention provides a method for detecting at least one gene mutation or SNP in vitro (in vitro) from more than one template using the PCR kit.
The term "SNP (Single Nucleotide Polymorphisms)" refers to a genetic change or mutation showing a difference in one Nucleotide sequence (A, T, G or C) in a DNA Nucleotide sequence.
In the in vitro (in vitro) SNP detection method of the invention, the target sequence may be present in the test sample and comprises DNA, cDNA or RNA, preferably genomic DNA. The test sample may be a cell lysate prepared from bacteria, a bacterial culture or a cell culture. In addition, the test sample may be comprised in an animal, preferably in a vertebrate, more preferably in a human subject. The target sequence may be comprised in genomic DNA, preferably in genomic DNA of an individual, more preferably in a bacterium or a vertebrate, most preferably in genomic DNA of a human subject.
The SNP detection method of the invention may include melting temperature analysis using a double strand specific dye such as SYBR Green I.
Melting temperature profile analysis can be performed in real-time PCR equipment such as ABI 5700/7000 (96-well format) or ABI 7900 (384-well format) including software (one board software SDS 2.1). Alternatively, the melting temperature profile analysis may be performed as an endpoint analysis.
"dye that binds to double-stranded DNA" or "double-strand specific dye" may be used in the case where it has high fluorescence when bound to double-stranded DNA compared to when it is not bound to double-stranded DNA. Examples of such dyes include SOYTO-9, SOYTO-13, SOYTO-16, SOYTO-60, SOYTO-64, SYTO-82, bromoethane (EtBr), SYTOX Orange, TO-PRO-1, SYBR Green I, TO-PRO-3 or EvaGreen. These dyes have been used for real-time application testing, except EtBr and EvaGreen (Quiagen).
The in vitro (in vitro) SNP detection method of the invention can be carried out by real-time PCR, agarose gel analysis after standard PCR, gene mutation-specific amplification or allele-specific amplification by real-time PCR, four-primer amplification hindered mutation system PCR or isothermal amplification.
By "standard PCR" is meant a technique known to the skilled artisan for amplifying a single or multiple copies of DNA or cDNA. Almost all PCR uses Taq polymerase or a thermostable DNA polymerase such as Klen Taq. DNA polymerases enzymatically assemble a new DNA strand from nucleotides by using single-stranded DNA as a template and using oligonucleotides (primers). Amplicons produced by PCR can be analyzed on agarose gels.
The "real-time PCR" can monitor the process of PCR in real time as it is performed. Thus, data is collected throughout the PCR, rather than at the end of the PCR. In real-time PCR, the reaction is characterized by the point in the cycle at which amplification is first detected, rather than the target amount accumulated after a fixed number of cycles. Quantitative PCR is performed mainly using two methods, dye-based detection and probe-based detection.
The "Allele Specific Amplification (ASA)" is an Amplification technique in which PCR primers are designed to distinguish between other templates by a single nucleotide residue.
The "gene mutation-specific amplification or allele-specific amplification by real-time PCR" can detect a gene mutation or SNP in an efficient manner. Unlike most other methods of detecting gene mutations or SNPs, pre-amplification of the target genetic material is not necessary. ASA combines amplification and detection in a single reaction based on the difference between matched and mismatched primer/target sequence complexes. The increase in amplified DNA during the reaction can be monitored in real time by the increase in fluorescent signal caused by a dye such as SYBR Green I, which emits light when bound to double stranded DNA. By genetic mutation-specific amplification or allele-specific amplification by real-time PCR, a delay in the appearance of a fluorescent signal or absence of a signal occurs when a mismatch occurs. In gene mutation or SNP detection, this provides information about the presence or absence of a gene mutation or SNP.
The four-primer amplification hindered mutation system PCR and a control fragment amplify wild type and mutant alleles together in a single-tube PCR reaction. The non-allele-specific control amplicon was amplified by two universal (outer) primers flanking the mutation region. The two allele-specific (internal) primers are designed in the opposite direction to the universal primers and amplify both wild-type and mutant amplicons with the universal primers. As a result, the two allele-specific amplicons, which are of different lengths due to the asymmetric position of the mutation relative to the universal (outer) primer, can be easily separated by standard gel electrophoresis. The control amplicon provides an internal control for false negatives and amplification failures, and at least one of the two allele-specific amplicons is consistently present in the four-primer amplification hindered mutation system PCR.
By "isothermal amplification" is meant that amplification of nucleic acids is performed at lower temperatures, independent of the thermal cycler, preferably temperatures that do not need to be changed during amplification. The temperature used in isothermal amplification may be between room temperature (22-24 ℃) and about 65 ℃, or at ambient temperatures of about 60-65 ℃, 45-50 ℃, 37-42 ℃, or 22-24 ℃. Isothermal amplification products can be detected by gel electrophoresis, ELISA, elaos (Enzyme linked oligonucleotide assay), real-time PCR, ECL (modified chemiluminescence), chip-based capillary electrophoresis device bioanalyzer (bioanalyzer) for analysis of RNA, DNA, and proteins or turbidity.
In one embodiment of the invention, it is confirmed whether the mismatch primer extension ability is reduced for a template comprising a SNP (rs 1408799, rs1015362 and/or rs 4911414) using E507K/R536K, E507K/R660V or E507K/R536K/R660V Taq polymerase.
As a result, as shown in FIGS. 6 to 8, it was confirmed that the amplification delay caused by the mismatched primer was most significant in E507K/R536K/R660V Taq polymerase compared to E507K Taq polymerase, and the effect was most significant in E507K/R536K/R660V Taq polymerase.
This confirmed that the DNA polymerase of the present invention has higher mismatch extension selectivity compared to conventional Taq polymerase (E507K). Therefore, the DNA polymerase of the present invention is expected to be effectively applied to medical diagnosis of diseases and research of recombinant DNA.
According to another preferred embodiment of the present invention, the PCR kit comprises more than one matched primer, more than one mismatched primer or more than one matched primer and more than one mismatched primer, wherein the more than one matched primer and the more than one mismatched primer hybridize to the target sequence, and the mismatched primer comprises an atypical (non-canonical) nucleotide at the 3' end to the 7 th base position of the hybridized target sequence.
The PCR kit of the present invention may further comprise nucleoside triphosphates.
The PCR kit of the present invention further comprises a) one or more buffers; b) A quantification reagent that binds to double-stranded DNA; c) A polymerase blocking antibody; d) One or more control values or control sequences; e) One or more templates; as shown in the figure.
The present invention will be described in detail by examples. These embodiments are illustrative of the present invention and those having ordinary skill in the art will understand that the scope of the present invention is not limited to the embodiments.
Example 1 Induction of Taq polymerase mutations
1-1 fragment PCR
In this example, SEQ ID NO was prepared as follows: 1 (1) in the amino acid sequence thereof, taq DNA polymerase in which the 536 th amino acid residue of arginine is substituted with lysine (hereinafter referred to as "R536K"), taq DNA polymerase in which the 660 th amino acid residue of arginine is substituted with valine (hereinafter referred to as "R660V"), and Taq DNA polymerase in which the 536 th amino acid residue of arginine is substituted with lysine and the 660 th amino acid residue of arginine is substituted with valine (hereinafter referred to as "R536K/R660V").
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.
TABLE 1
Figure BDA0001712784320000181
TABLE 2
Figure BDA0001712784320000182
Figure BDA0001712784320000191
The PCR product was confirmed by electrophoresis, and as a result, as shown in FIG. 1 (b), the band of each fragment was confirmed, and thus it was confirmed that the target fragment was amplified.
1-2 overlap (overlap) PCR
Each fragment amplified in 1-1 was used as a template, and the full length was amplified at both ends using primers (Eco-F and Xba-R primers). The reaction conditions are shown in tables 3 and 4.
TABLE 3
Figure BDA0001712784320000192
TABLE 4
Figure BDA0001712784320000193
As a result, as shown in FIG. 1 (c), it was confirmed that "R536K", "R660V", "R536K/R660V" Tag polymerase was amplified.
1-3. Connection (ligation)
pUC19 was digested with restriction enzymes EcoRI/XbaI at 37 ℃ for 4 hours under the conditions shown in Table 5 and the DNA was purified, and the purified DNA was treated with SAP at 37 ℃ for 1 hour under the conditions shown in Table 6 to prepare vectors.
TABLE 5
Figure BDA0001712784320000201
TABLE 6
Figure BDA0001712784320000202
For the insert (insert), the overlap PCR product of example 1-2 was purified and digested with restriction enzymes EcoRI/XbaI at 37 ℃ for 3 hours under the conditions shown in Table 7, followed by gel extraction with the prepared vector (FIG. 2).
TABLE 7
Figure BDA0001712784320000203
Coli DH5 α was transformed and screened on ampicillin-containing medium after 2 hours of ligation at Room Temperature (RT) under the conditions shown in table 8. Plasmids prepared from the obtained colonies were sequenced to obtain Taq DNA polymerase mutants ("R536K", "R660V" and "R536K/R660V") into which the desired mutation was introduced.
TABLE 8
Figure BDA0001712784320000204
Figure BDA0001712784320000211
Example 2 introduction of E507K mutation
2-1 fragment PCR
The activities of "R536K", "R660V" and "R536K/R660V" Taq DNA polymerases prepared in example 1 were tested, and as a result, it was confirmed that the activities were decreased (data not shown), and E507K mutation was introduced into "R536K", "R660V" and "R536K/R660V", respectively (in the amino acid sequence of SEQ ID NO:1, the glutamic acid at the 507 th amino acid residue was substituted with lysine), and as a control group, E507K mutation was also introduced into wild-type (WT) Taq DNA polymerase. The Taq DNA polymerase introduced with the E507K mutation was prepared in the same manner as in example 1.
Taq DNA polymerase fragments (F6 to F7) were amplified by PCR using the mutation-specific primers shown in Table 9, as shown in FIG. 3. The reaction conditions are shown in Table 10.
TABLE 9
Figure BDA0001712784320000212
Watch 10
Figure BDA0001712784320000213
* Template plasmid: pUC19-Tag (WT)
pUC19-Tag(R536K)
pUC19-Tag(R660V)
pUC19-Tag(R536K/R660V)
2-2 overlap (overlap) PCR
Each fragment amplified in 2-1 was used as a template, and the full length was amplified at both ends using primers (Eco-F and Xba-R primers). The reaction conditions are shown in Table 11.
TABLE 11
Figure BDA0001712784320000221
2-3. Connection (ligation)
pUC19 was digested with restriction enzymes EcoRI/XbaI at 37 ℃ for 4 hours under the conditions shown in Table 5 and the DNA was purified, and the purified DNA was treated with SAP at 37 ℃ for 1 hour under the conditions shown in Table 6 to prepare vectors.
For the insert (insert), the overlapped PCR product of example 2-2 was purified and digested with restriction enzymes EcoRI/XbaI at 37 ℃ for 3 hours under the conditions shown in Table 7, followed by gel extraction together with the prepared vector (FIG. 4).
Coli DH5 α or DH10 β was transformed and screened on ampicillin-containing medium after 2 hours of ligation at Room Temperature (RT) under the conditions shown in table 8. 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 desired mutation was introduced.
Example 3 qPCR Using the DNA polymerase of the present invention
Using Taq polymerases each containing the mutations "E507K/R536K", "E507K/R660V" and "E507K/R536K/R660V" obtained in the above example 2, it was confirmed whether or not the ability to extend the mismatch primer was reduced with respect to the SNP-containing template. As a control group, "E507K" Taq polymerase containing the E507K mutation was used.
The SNP-containing templates used in the present invention were rs1408799, rs1015362, and rs4911414, and the genotype of each template and the sequence information of its specific primer (IDT, usa) are shown in tables 12 and 13.
TABLE 12
Figure BDA0001712784320000222
Watch 13
Figure BDA0001712784320000231
The qPCR conditions (Applied Biosystems 7500 Fast) are shown in Table 14 below.
TABLE 14
Figure BDA0001712784320000232
The probes were double-labeled as shown in Table 15 below.
Watch 15
Figure BDA0001712784320000233
The oral epithelial cells were collected using an oral epithelial cell collection kit purchased from Noble Bio, dissolved in 500 μ l of lysate (lysis solution), and centrifuged at 12,000 × g for 3 minutes. The supernatant was transferred to a new tube, 1. Mu.l each time (FIG. 5).
The reaction conditions are shown in Table 16, and the composition of the reaction buffer is shown in Table 17.
TABLE 16
Figure BDA0001712784320000234
Figure BDA0001712784320000241
TABLE 17
Figure BDA0001712784320000242
Except for the specific primers shown in table 13, other reaction solutions were prepared in the same manner in two test tubes, and qPCR was performed by adding each allele-specific primer. At this point, the fluorescence signals detected in each tube were pooled to calculate and analyze the difference in cycle (Ct) values for reaching a threshold (threshold) fluorescence value on AB7500 software (v 2.0.6). The longer the time for which the Ct value is delayed in amplification by the mismatch primer, the better the gene mutation specificity or the allele specificity can be judged.
As a result of AS-qPCR in rs1408799, rs1015362 and rs4911414, AS shown in FIGS. 6 to 8, it was confirmed that the effect of the amplification delay caused by the mismatch primer was most significant in the E507K/R536K/R660V mutation in the case of Taq polymerase containing the E507K/R536K, E507K/R660V mutation, or E507K/R536K/R660V mutation, AS compared to the control group E507K.
This confirms that the Taq DNA polymerase comprising the E507K/R536K, E507K/R660V or E507K/R536K/R660V mutation of the present invention has more excellent mismatch extension selectivity compared to the Taq polymerase comprising the E507K mutation. Therefore, the Taq DNA polymerase of the present invention is expected to be effectively applied to medical diagnosis of diseases and research of recombinant DNA.
EXAMPLE 4 optimization of KCl concentration in reaction buffer
In this example, in order to find a high cation concentration at which amplification by mismatch is maximally delayed without decreasing amplification efficiency by matching, the KCl concentration in the PCR reaction buffer is adjusted to determine the optimum concentration of KCl.
Taq polymerases obtained in example 2 comprising the mutations "E507K/R536K", "E507K/R660V" or "E507K/R536K/R660V", respectively, were used and the KCl concentration threshold was compared to the Taq polymerase comprising the E507K mutation.
For the template containing the SNP, rs1408799 was used, the genotype of the template was TT, and rs1408799 primer described in table 12 was used as the primer. The qPCR conditions (Applied Biosystems 7500 Fast) were performed under the conditions shown in Table 14, 1408799-FAM shown in Table 15 was used for the double-labeled probe, the reaction conditions are shown in Table 18, and the composition of the reaction buffer is shown in Table 19.
Watch 18
Figure BDA0001712784320000251
Watch 19
Figure BDA0001712784320000252
As shown in FIG. 9, the KCI concentration threshold values of E507K/R536K/R660V Taq polymerase were the lowest, and the KCl concentration threshold values of E507K/R536K and E507K/R660V were lower than that of E507K.
Based on the above results, further experiments were performed using E507K/R536K/R660V Taq polymerase to determine the appropriate KCl concentration, using rs1408799-T specific primers shown in Table 13. The qPCR conditions (applied biosystems 7500 Fast) were performed for 35 cycles under the conditions of Table 14, and the reaction conditions are shown in Table 20.
Watch 20
Figure BDA0001712784320000253
The composition of the reaction buffer of the control group was the same as that of Table 21, and the composition of the reaction buffer of the experimental group was the same as that of Table 8, (NH) 4 ) 2 SO 4 The concentration was fixed at 2.5mM, and various changes were made to the KCl concentration.
TABLE 21
Figure BDA0001712784320000261
As a result of confirming the PCR product by electrophoresis after amplification under the above-described conditions, it was confirmed that the KCl concentration was 75mM, which was suitable for minimizing the delay of amplification due to mismatch without reducing the amplification efficiency due to match, as shown in FIG. 10.
EXAMPLE 5 of reaction buffer (NH) 4 ) 2 SO 4 Concentration optimization
This example was carried out based on the results of example 4, in which the KCl concentration in the reaction buffer was fixed at 75mM for (NH) 4 ) 2 SO 4 The concentration was varied to confirm the optimum (NH) 4 ) 2 SO 4 And (4) concentration. The primers used rs1408799-T specific primers shown in Table 13, qPCR conditions (Applied Biosystems 7500 Fast) were performed for 35 cycles under the conditions shown in Table 14, the reaction conditions are shown in Table 20, and the composition of the reaction buffer in the control group is the same as that in Table 21.
As a result, as shown in FIG. 11, (NH) was appropriate 4 ) 2 SO 4 The concentration was 5mM.
Based on the results, the KCl concentration in the reaction buffer was fixed at 75mM and (NH) 4 ) 2 SO 4 The concentration of (2) was set to about 5mM (2.5 mM, 5mM, and 10mM, respectively), and the effect of amplification delay due to mismatch was confirmed.
The primers used were rs1408799 primer described in Table 13, the double-labeled probe used was 1408799-FAM shown in Table 15, and the reaction conditions are shown in Table 22.
TABLE 22
Figure BDA0001712784320000262
As a result, (NH) is shown in FIG. 12 4 ) 2 SO 4 The concentration of (C) was found to have the greatest difference in Ct value at 10mM, but it was confirmed that the Ct value of amplification caused by matching was slightly delayed and the peak was downward-shifted, thereby determining the appropriate value (NH) 4 ) 2 SO 4 The concentration of (2) is 5mM.
By combining the results of examples 4 and 5, it was confirmed that the optimum reaction buffer combination contained 50mM Tris. Cl and 2.5mM MgCl 2 75mM KCl, 5mM (NH) 4 ) 2 SO 4 0.1% Tween 20 and 0.01% BSA.
Example 6 addition of TMAC to reaction buffer and optimization of its concentration
In this example, TMAC was added to the reaction buffer and the optimum concentration thereof was confirmed. Based on the results of examples 4 and 5, the KCl concentration in the reaction buffer was fixed at 75mM, (NH) 4 ) 2 SO 4 The concentration was fixed at 5mM, and the concentration of TMAC was variedThus, the optimal TMAC concentration was confirmed.
Using E507K/R536K or E507K/R536K/R660V Taq polymerase, rs1408799 was used as a template containing SNP, TT was used as the genotype of the template, and rs1408799 primers shown in Table 12 were used as primers. The qPCR conditions (applied biosystems 7500 Fast) were performed under the conditions of Table 14, the dual labeled probes were 1408799-FAM of Table 15, and the reaction conditions are shown in Table 22.
As shown in FIG. 13, the appropriate TMAC concentration was confirmed to be 60mM for E507K/R536K Taq polymerase; the concentration of Taq polymerase was 25mM for E507K/R536K/R660V, and it was confirmed that the amplification efficiency decreased when the concentration of TMAC was too high.
EXAMPLE 7 KCl, (NH) of reaction buffer 4 ) 2 SO 4 And TMAC concentration optimization
In this example, based on the results confirmed in example 6, E507K/R536K/R660V Taq polymerase was used to confirm the optimum KCl, (NH) in the reaction buffer 4 ) 2 SO 4 And TMAC concentration.
Specifically, the TMAC concentration in the reaction buffer was fixed at 25mM, (NH) 4 ) 2 SO 4 The concentration was fixed at 2.5mM, and the KCl concentration was adjusted to 20, 40, 60, and 80mM, respectively. Experiments were performed for two SNPs, rs1015362 and rs4911414, with the genotypes of the templates as described in Table 12, primers rs1015362 and rs4911414 as described in Table 13, qPCR conditions (Applied Biosystems 7500 Fast) as described in Table 14, dual labeled probes as 1408799-FAM as described in Table 15, and reaction conditions as described in Table 22.
As a result of the experiment, as shown in FIG. 14, it was found that the amplification efficiency was decreased when the KCl concentration was appropriately 60mM and 80mM for the two SNPs.
From the above results, it was confirmed that the optimum KCl concentration in the reaction buffer was 60mM, (NH) 4 ) 2 SO 4 The concentration was 2.5mM and the TMAC concentration was 25mM. More specifically, it was confirmed that KCl was used at a concentration of 75mM and (NH) was used at a concentration of 5mM for E507K/R536K polymerase 4 ) 2 SO 4 60mM TMAC was most effective; for E507K/R536K/R660V polymerase, KCl at a concentration of 60mM, 2.5mM, was used(NH 4 ) 2 SO 4 TMAC at 25mM was most effective.
Sequence listing
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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 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 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 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 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 (Artificial Sequence)
<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 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 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 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 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> 2499
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
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> 7
<211> 2499
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
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> 8
<211> 2499
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
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> 9
<211> 2499
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<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 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> 10
<211> 2499
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<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 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> 11
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ggggtacctc atcaccccgg 20
<210> 12
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
cttggtgagc tccttgtact gcaggat 27
<210> 13
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
atcctgcagt acaaggagct caccaag 27
<210> 14
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gatggtcttg gccgccacgc gcatcagggg 30
<210> 15
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
cccctgatgc gcgtggcggc caagaccatc 30
<210> 16
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gctctagact atcactcctt ggcggagagc ca 32
<210> 17
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
cttgccggtc tttttcgtct tgccgat 27
<210> 18
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
atcggcaaga cgaaaaagac cggcaag 27
<210> 19
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
ccagtgttag gttatttcta acttg 25
<210> 20
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
gctcggagca catggtcaa 19
<210> 21
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gctcggagca catggtcag 19
<210> 22
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
tgaagagcag gaaagttctt ca 22
<210> 23
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
actgtgtgtc tgaaacagtg 20
<210> 24
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
actgtgtgtc tgaaacagta 20
<210> 25
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
gtaagtcttt gctgagaaat tcattg 26
<210> 26
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
gtaagtcttt gctgagaaat tcattt 26
<210> 27
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
agtatccagg gttaatgtga aag 23
<210> 28
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
agatatttgt aaggtattct ggcct 25
<210> 29
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
tgctgaacaa atagtcccga ccag 24
<210> 30
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
tttctctagt tgcctttaag attt 24

Claims (8)

1. A PCR kit comprising a DNA polymerase and a PCR buffer composition, characterized in that the PCR buffer composition comprises 60 to 90mM KCl;2.5 to 8mM of (NH) 4 ) 2 SO 4 (ii) a A final pH of 8.0 to 9.0;
wherein the DNA polymerase comprises:
(a) A substitution of the 507 th amino acid residue in the amino acid sequence of SEQ ID NO. 1; and
(b) Substitution of 536 th and 660 th amino acid residues in the amino acid sequence of SEQ ID NO. 1; wherein, the 507 th amino acid residue is substituted by lysine (K) for glutamic acid (E), the 536 th amino acid residue is substituted by lysine (K) for arginine (R), and the 660 th amino acid residue is substituted by valine (V) for arginine (R).
2. The PCR kit of claim 1, wherein the concentration of KCl is 70 to 80mM, and the concentration of (NH) 4 ) 2 SO 4 Is in a concentration of 4 to 6mM.
3. The PCR kit of claim 1, wherein the PCR buffer composition further comprises Tris-Cl and MgCl 2 。
4. A PCR kit comprising a DNA polymerase and a PCR buffer composition, wherein the composition comprises 5 to 80mM of TMAC, KCl and (NH) 4 ) 2 SO 4 (ii) a A final pH of 8.0 to 9.0; the concentration of KCl is 50 to 80mM, the (NH) 4 ) 2 SO 4 Is in a concentration of 1.5 to 6mM;
wherein the DNA polymerase comprises:
(a) A substitution of the 507 th amino acid residue in the amino acid sequence of SEQ ID NO. 1; and
(b) Substitution of 536 th and 660 th amino acid residues in the amino acid sequence of SEQ ID NO. 1; wherein, the 507 th amino acid residue is substituted by lysine (K) for glutamic acid (E), the 536 th amino acid residue is substituted by lysine (K) for arginine (R), and the 660 th amino acid residue is substituted by valine (V) for arginine (R).
5. The PCR kit according to claim 4, wherein the concentration of TMAC is 25mM, the concentration of KCl is 60mM, and the concentration of (NH) 4 ) 2 SO 4 Was 2.5mM.
6. The PCR kit of claim 5, wherein the PCR buffer composition further comprises Tris-Cl and MgCl 2 。
7. The PCR kit according to any one of claims 1 to 6, further comprising:
a) A nucleoside triphosphate;
b) A quantification reagent that binds to double-stranded DNA;
d) A polymerase blocking antibody;
d) One or more control values or control sequences;
e) One or more templates.
8. A method for in vitro detection of at least one genetic mutation or SNP from more than one template using the PCR kit of any one of claims 1 to 7.
CN201810691172.6A 2017-07-12 2018-06-28 PCR buffer composition for enhancing DNA polymerase activity with increased gene mutation specificity Active CN109251965B (en)

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KR102286025B1 (en) * 2019-01-11 2021-08-05 주식회사 진캐스트 Mass spectrometry using dna polymerases with increased mutation specificity
WO2020145715A1 (en) * 2019-01-11 2020-07-16 주식회사 진캐스트 Dna polymerase for detecting tert mutation and kit comprising same
KR102370550B1 (en) * 2019-01-11 2022-03-08 주식회사 진캐스트 DNA polymerase for detecting EGFR mutation and kit comprising the same
TWI715997B (en) * 2019-06-13 2021-01-11 新加坡商克雷多生物醫學私人有限公司 Pcr apparatus for real-time detecting multiplex fluorescent signals
KR102247419B1 (en) 2020-03-24 2021-05-03 정지원 Buffer composition and analysis method using the same
KR102264902B1 (en) 2021-03-05 2021-06-14 주식회사 모노바이오 PCR premix composition with improved stability and its preparation method

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