CN109251965A - A kind of DNA polymerase activity enhancing PCR buffer composition with increased gene mutation specificity - Google Patents

A kind of DNA polymerase activity enhancing PCR buffer composition with increased gene mutation specificity Download PDF

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CN109251965A
CN109251965A CN201810691172.6A CN201810691172A CN109251965A CN 109251965 A CN109251965 A CN 109251965A CN 201810691172 A CN201810691172 A CN 201810691172A CN 109251965 A CN109251965 A CN 109251965A
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CN109251965B (en
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李炳哲
朴日铉
李辉皓
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Gene Caster Co Ltd
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    • C12Q2600/156—Polymorphic or mutational markers

Abstract

The DNA polymerase activity enhancing PCR buffer composition and application thereof with increased gene mutation specificity that the present invention relates to a kind of, more specifically, the present invention provides the PCR buffer composition for increasing the DNA polymerase activity enhancing of gene mutation specificity in the induced mutation of specific amino acids position, archaeal dna polymerase comprising the PCR buffer composition and/or with increased gene mutation specificity for detecting gene mutation or the PCR kit of SNP, and external (in vitro) detects at least one gene mutation or the detection method of SNP from more than one template using the kit.

Description

It is a kind of with increased gene mutation specificity DNA polymerase activity enhancing use PCR Buffer composition
Technical field
The present invention relates to a kind of, and the DNA polymerase activity with increased gene mutation specificity enhances with PCR buffer Composition and application thereof, more specifically, the present invention are provided in the induced mutation of specific amino acids position to increase gene mutation spy Anisotropic archaeal dna polymerase enhancement PCR buffer composition living increases comprising the PCR buffer composition and/or have Gene mutation specificity archaeal dna polymerase for detect gene mutation or SNP PCR kit and utilize the reagent Box external (in vitro) from more than one template detects at least one gene mutation or the detection method of SNP.
Background technique
Since first man genoid group sequence is found, the research of researcher, which is mainly concentrated, explores single nucleotide mutation On hereditary difference between the individual such as (single nucleotide polymorphism, SNPs).Single nucleotide mutation and various diseases in genome Different drug resistances or susceptible constitution are related, this is more and more clear, therefore become major concern object.Future medicine correlation core The knowledge of thuja acid variation is applicable to the treatment method of individual inheritance supply, and can prevent invalid or the drug of side effect is caused to be controlled It treats (Shi, Expert Rev.Mol.Diagn.1,363-365 (2001)).Efficient nucleotide diversity in terms of time and cost The exploitation of identification technology will bring the further development of pharmacogenetics.
SNPs occupies main hereditary variation in human genome, can induce 90% of inter-individual difference or more. (Kwok,Annu.Rev.Genomics Hum,Genet.2,235-258(2001)；Kwok and Chen,Curr.Issues Mol.Biol.5,43-60(2003)；Twyman and Primrose,Pharmacogenomics 4,67-79(2003)).For Other variances such as these hereditary variations and mutation are detected, can be used various methods.For example, can be by appropriate The nucleic acid samples being analysed under hybridization conditions hybridize to realize to target with the hybridized primer for having sequence variants specificity The identification (Guo et al., Nat.Biotechnol.15,331-335 (1997)) of the variant of nucleic acid.
It has however been found that this hybridizing method, is especially unable to satisfy clinical needs in terms of required sensitivity in measurement.Cause This, PCR is widely used the diagnosis detection in abrupt climatic changes such as molecular biology, SNP and other allelic sequences variants In method (Saiki et al., Science 239,487-490 (1988)), wherein in view of the presence of variant, lead to before hybridization It crosses polymerase chain reaction (PCR) and expands target nucleic acid to be detected.For the hybridized primer of this measurement, usually using single-stranded widow Nucleotide.The specific example of the modification of the measurement includes using fluorogenic hybridization probe (Livak, Genet.73-5 2017-07- 12Anal.14,143-149(1999)).In general, having attempted to the measuring method automation for making SNP and other sequences make a variation (Gut,Hum.Mutat.17,475-492(2001))。
The countermeasure of sequence variations specific hybrid known in the art is mentioned by so-called gene mutation specific amplification For.In the detection method, in amplification procedure, variation specificity amplification primer, the end the 3'- tool of usual primer are used There is so-called otherness terminal nucleotide residue, residue is only complementary to a specificity variation of target nucleic acid to be detected.In the party In method, by measuring nucleotide variants with the existence or non-existence of DNA product after PCR amplification.Gene mutation specific amplification Principle be based on forming typical (canonical) in gene mutation specificity amplification primer end or atypia primer-template is multiple Close object.In the 3' prime end of perfect match, amplification caused by archaeal dna polymerase is generated, and extends in mismatched primers end and is pressed down System.
For example, U.S. Patent No. 5,595,890 disclose gene mutation specific amplification method and its application, such as disclose For detecting the application method of the clinically relevant point mutation in k-ras oncogene (point mutation).In addition, the U.S. Patent the 5,521,301st allele-specific amplification method for disclosing the genotyping for ABO blood group system. On the contrary, U.S. Patent No. 5,639,611 disclose and lead to the related equipotential base of the point mutation of herrik syndrome with detection The application method of cause-specific amplification.However, gene mutation specific amplification or allele specific amplification have low selection The problem of property, it is therefore desirable to the optimization step of more complicated, time and cost-intensive.
It is described for detection sequence variation, polymorphism and the method for concentrating on point mutation, when sequence variations to be detected with When the dominance variance of identical nucleic acid segment (or mutually homogenic) compares insufficient, it is especially desirable to gene-specific amplification (or gene Mutation specific amplification).
For example, when detecting Sporadic tumors in body fluids such as blood, serum or blood plasma by gene mutation specific amplification This thing happens when cell (U.S. Patent No. 5,496,699).For this purpose, DNA is first from body fluid such as blood, serum or blood plasma In separate, and DNA is made of insufficient Sporadic tumors cell and excessive non-proliferative cell.Therefore, excessive In the presence of wild type DNA, it should be detected from multiple duplications in the important mutation of the Tumour DNA in k-ras gene It arrives.
All methods for gene mutation specific amplification disclosed in the prior art, although using 3'- otherness core Thuja acid residue, even if target nucleic acid not exclusively matches with sequence variants to be detected, in suitable archaeal dna polymerase existence condition Under, have the shortcomings that the primer extend of reduced levels occurs.Particularly, when specific sequence variants are by the inclusion of another sequence The excessive background nucleic of variant and be detected, will lead to false positive results.Known method, which has another disadvantage that, to be needed Use the end 3'- otherness oligonucleotide residues.Disadvantage present in the method for this based on PCR, mainly due to for filling Subregion divides the unworthiness of polymerase used in the method for base mismatch and generates.Therefore, it still can not directly be obtained by PCR It obtains about whether the clear information that there is mutation.So far, intensive purifying and measurement are required additional time and costs Method is come mutation of clarifying a diagnosis.Therefore, the selectivity that raising gene mutation specificity or allele-specific PCR expand New method can bring very big influence to the confidence level of direct gene mutation or snp analysis by PCR and potent property.
There is the archaeal dna polymerase of increased gene mutation specificity therefore, it is necessary to lasting research and the DNA can be made to polymerize Enzyme plays its function, mixing many kinds of substance optimum response buffer.
The present inventor, which is dedicated to exploitation, can be improved gene mutation-specific PCR amplification selectivity Novel DNA polymerase And it for improving its active reaction buffer, as a result confirms to induce the amino acid residue of Taq polymerase specific location prominent When change, gene mutation specificity is dramatically increased, the KCl, (NH in control PCR buffer composition4)2SO4And/or TMAC (four Ammonio methacrylate, Tetra methyl ammonium chloride) concentration when, it is described have increased gene mutation it is special The increased activity of the archaeal dna polymerase of property, thereby completing the present invention.
Summary of the invention
The present invention proposes to solve the technical task, provides a kind of with increased gene mutation specificity DNA polymerase activity enhancing PCR buffer composition.
It is a further object to provide dash forward comprising the PCR buffer composition and/or with increased gene The archaeal dna polymerase of change specificity is used to detect gene mutation or the PCR kit of SNP.
It is a further object to provide utilize the PCR kit external (in from more than one template Vitro at least one gene mutation or the detection method of SNP) are detected.
In order to realize the purpose, the present invention provides, and there is the DNA polymerase activity of increased gene mutation specificity to increase PCR buffer composition is used by force, and the composition includes 25 to 100mM KCl；1 to 15mM (NH4)2SO4；Final pH is 8.0 to 9.0.
According to a preferred embodiment of the present invention, the concentration of the KCl is 60 to 90mM.
According to a further advantageous embodiment of the invention, the described (NH4)2SO4Concentration be 2 to 8mM.
Another preferred embodiment according to the present invention, the concentration of the KCl are 70 to 80mM, (the NH4)2SO4It is dense Degree is 4 to 6mM.
The present invention provides the DNA polymerase activity enhancing PCR buffers combinations with increased gene mutation specificity Object, the PCR buffer composition further include 5 to 80mM TMAC (tetramethyl ammonium chloride, Tetra methyl ammonium chloride)。
According to a preferred embodiment of the present invention, the concentration of the KCl is 40 to 90mM.
According to a further advantageous embodiment of the invention, the described (NH4)2SO4Concentration be 1 to 7mM.
Another preferred embodiment according to the present invention, TMAC (tetramethyl ammonium chloride, the Tetra methyl Ammonium chloride) concentration be 15 to 70mM, the concentration of the KCl is 50 to 80mM, (the NH4)2SO4Concentration It is 1.5 to 6mM.
According to a further advantageous embodiment of the invention, the PCR buffer composition can further include Tris Cl and MgCl2
The present invention provide it is a kind of comprising the PCR buffer composition for detecting gene mutation or the PCR reagent of SNP Box.
Preferred embodiment in accordance with the present invention, the PCR kit can further include by the amino acid of SEQ ID NO:1 The archaeal dna polymerase of the Taq polymerase of sequence (base sequence of SEQ ID NO:6) composition, the archaeal dna polymerase may include (a) The substitution of 507th amino acid residue in the amino acid sequence of SEQ ID NO:1；And (b) amino acid sequence of SEQ ID NO:1 In the substitution of the 536th amino acid residue, the substitution of the 660th amino acid residue or the 536th and the 660th i.e. two ammonia The substitution of base acid residue.
According to a further advantageous embodiment of the invention, the substitution of the 507th amino acid residue is glutamic acid (E) quilt Lysine (K) replaces, and the substitution of the 536th amino acid residue is that arginine (R) is replaced (K) by lysine, and the described 660th The substitution of a amino acid residue is that arginine (R) is replaced by valine (V).
According to a further advantageous embodiment of the invention, the PCR kit further includes: a) nucleoside triphosphate；b) Quantitative reagent in conjunction with double-stranded DNA；C) polymerase blocking antibody；D) one or more control values or control sequence；E) one Or multiple template.
The present invention also provides a kind of, and using the PCR kit, external (in vitro) is detected from more than one template The detection method of at least one gene mutation or SNP.
Compared with prior art, the present invention have it is following the utility model has the advantages that
The present invention, which provides one kind, can make the archaeal dna polymerase with increased gene mutation specificity play its function most Good PCR buffer composition is used together with the archaeal dna polymerase with increased gene mutation specificity, is significantly improved described The activity of archaeal dna polymerase is, it can be achieved that reliable gene mutation-specific amplification.In addition, including PCR buffer group of the invention Gene mutation or SNP can be effectively detected in the kit for closing object and/or the archaeal dna polymerase with increased gene specific, Therefore the medical diagnosis in disease and the research of recombinant DNA can be effectively utilized.
Detailed description of the invention
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention, Objects and advantages will become more apparent upon:
Fig. 1 describes the preparation of the Taq archaeal dna polymerase respectively containing R536K, R660V and R536K/R660V mutation Journey, wherein (a) is the schematic diagram of segment PCR and over-lap PCR, and (B) is shown in the electrophoresis confirmation of the amplified production in segment PCR As a result, the result of amplified production (c) is confirmed after display over-lap PCR amplification overall length by electrophoresis；
Fig. 2 is shown, in order to extract gel, the pUC19 carrier of SAP processing is carried out after being decomposed with restriction enzyme EcoRI/XbaI Result is confirmed with the electrophoresis of the overlapping PCR products of Fig. 1 (c) of purifying；
Fig. 3 is to prepare to separately include E507K, E507K/R536K, E507K/R660V and E507K/R536K/R660V mutation Taq archaeal dna polymerase during segment PCR and over-lap PCR schematic diagram；
Fig. 4 is shown, in order to extract gel, the pUC19 carrier of SAP processing is carried out after being decomposed with restriction enzyme EcoRI/XbaI Result is confirmed with the electrophoresis of the overlapping PCR products of Fig. 3 of purifying；
Fig. 5 is to show by collecting mouth epithelial cells to prepare the schematic diagram of the process of pcr template；
Fig. 6 shows poly- using E507K/R536K, E507K/R660V and E507K/R536K/R660V Taq of the invention Result of the synthase to the rs1408799 AS-qPCR carried out；As a control group, using the Taq polymerase for including E507K mutation；
Fig. 7 shows poly- using E507K/R536K, E507K/R660V and E507K/R536K/R660V Taq of the invention Result of the synthase to the rs1015362 AS-qPCR carried out；As a control group, using the Taq polymerase for including E507K mutation；
Fig. 8 shows poly- using E507K/R536K, E507K/R660V and E507K/R536K/R660V Taq of the invention Result of the synthase to the rs4911414 AS-qPCR carried out；As a control group, using the Taq polymerase for including E507K mutation；
Fig. 9, which is shown, uses E507K, E507K/R536K, E507K/R660V and E507K/R536K/R660V of the invention Taq polymerase confirms the chart of the carryover effects of mispairing amplification according to the variation of KCl concentration in reaction buffer；
Figure 10 is shown, in order to determine optimal KCl concentration in reaction buffer, fixed (NH4)2SO4Concentration and variation After the concentration of KCl is expanded, the result of PCR product is confirmed by electrophoresis；
Figure 11 is shown, in order to determine optimal (NH in reaction buffer4)2SO4Concentration fixes the concentration of KCl and variation (NH4)2SO4Concentration expanded after, pass through electrophoresis confirm PCR product result；
Figure 12 is shown, according to (NH in reaction buffer4)2SO4The variation of concentration, the carryover effects of confirmation mispairing amplification Chart；
Figure 13 is shown, KCl and (NH are fixed in reaction buffer4)2SO4Concentration, according to the variation of TMAC concentration, really Admit one's mistake and matches the chart of the carryover effects of amplification；
Figure 14 is shown, TMAC and (NH are fixed in reaction buffer4)2SO4Concentration, according to the variation of KCl concentration, really Admit one's mistake and matches the chart of the carryover effects of amplification.
Specific embodiment
The present invention is described in detail below.
As described above, the shortcomings that in order to improve the gene mutation disclosed in conventional art-specific amplification method, need to hold The continuous mixing for developing the archaeal dna polymerase with increased gene mutation specificity and the archaeal dna polymerase can be made to play its function The optimum response buffer of many kinds of substance, the exploitation of these methods, by the direct gene mutation or snp analysis to PCR is passed through Confidence level and potent property bring very big influence.The present inventor, which is dedicated to exploitation, can be improved gene mutation-specific PCR amplification Selectivity Novel DNA polymerase and for improving its active reaction buffer, as a result confirm to Taq polymerase specific position When the amino acid residue Mutation induction at place, gene mutation specificity is dramatically increased, when control PCR buffer composition in KCl, (NH4)2SO4And/or TMAC (tetramethyl ammonium chloride, Tetra methyl ammonium chloride) concentration when, the tool There is the increased activity of the archaeal dna polymerase of increased gene mutation specificity, thereby completing the present invention.
The present invention provides the best PCR that the archaeal dna polymerase with increased gene mutation specificity can be made to play its function Buffer composition is used together with the archaeal dna polymerase with increased gene mutation specificity, and it is poly- to significantly improve the DNA The activity of synthase is, it can be achieved that reliable gene mutation-specific amplification.In addition, including PCR buffer composition of the invention And/or gene mutation or SNP can be effectively detected in the kit of the archaeal dna polymerase with increased gene specific, therefore The medical diagnosis in disease and the research of recombinant DNA can be effectively utilized.
In the following, will be illustrated to terms used herein.
" amino acid " refers to any monomeric unit that can be imported into peptide, polypeptide or protein herein.Such as this paper institute It uses, term " amino acid " includes following 20 kinds natural or genetic coding a-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 acid is usually organic acid, may include substituteding or unsubstituted amino, substituted or unsubstituted carboxyl With any analog of at least one side chain (chain) or group (group) or these groups.Illustrative side chain includes mercapto Base, seleno, sulfonyl, alkyl, aryl, acyl group, ketone group, azido, hydroxyl, diazanyl, cyano, halogen, hydrazides, alkenyl, alkynyl, Ether, borate, boronate, phospho, phosphono and sulfonic base, phosphino-, heterocycle, ketenes, imines, aldehyde radical, ester group, thio-acid, hydroxyl Any combination of amine or these groups.
Other exemplary amino acids include but is not limited to: containing can photoactivated cross-linking agent amino acid, metal bonding amino Acid, spin labeling amino acid, Fluorescent amino acid, metalliferous amino acid, the amino acid with novel functional group and other molecules The amino acid that covalently or non-covalently interacts, can light cage and/or can photoisomerization amino acid, radioactive amino acids, contain The amino acid of biotin or biotin analog, glycosylated amino acid, other carbohydrate modifications amino acid, contain poly- second The amino acid of glycol or polyethers, heavy atom substituted amino acid, can chemical breakdown and/or can photodegradation amino acid, carbon connection Containing glycoprotein amino acid, redox active amino acids, the amino acid containing thionothiolic acid and contain the amino acid of at least one toxin part.
In archaeal dna polymerase of the invention, term " mutant " refers to relative to naturally occurring or unmodified DNA polymerization Enzyme includes the recombinant polypeptide of one or more amino acid substitutions.
Term " thermostable polymerases " (referring to heat stable enzyme) refers to enough thermal resistances, keeps enough work Property to realize subsequent polynucleotides extension, and heating treatment in the time course needed for realizing double-strandednucleic acid denaturation Shi Buhui is irreversibly denaturalized (inactivation).As used herein, it suitable for PCR etc. reaction circulating temperature.Here can not Contravariance refers to completely losing for permanent enzymatic activity.For thermostable polymerases, enzymatic activity refers to suitable mode Catalysis nucleotide combination on template nucleic acid chain to form complementary polynucleotides extension products.Heat derived from thermophilic bacteria is steady Qualitative archaeal dna polymerase includes for example: coming from Thermotoga maritima (Thermatoga maritime), thermus aquaticus (Thermus Acquaticus), thermus thermophilus (Thermus thermophilus), yellow Thermus (Thermusflavus), filiform are dwelt heat Hard Thermus (the Thermus of bacterium (Thermus filiformus), Thermus kind Sps17, Thermus kind Z05, heat Caldophilus), Bacillus caldotenax (Bacillus caldotenax), new Apollo are dwelt thermobacillus (Thermotoga Neapolitana), Africa is dwelt the archaeal dna polymerase of hot chamber bacterium (Thermosipho africanus).
Term " thermal activities " refer to RT-PCR and/or PCR reaction in reverse transcription or annealing/extension stage it is usually used Temperature (that is, 45~80 DEG C) under keep the enzyme of its catalytic performance.Thermal stability enzyme is at the high temperature needed for nucleic acid denaturation The enzyme that irreversibly will not be inactivated or be denaturalized when reason.And thermal activities enzyme may be heat-staple or may not be heat-staple.Heat Active dna polymerase can include but is not limited to DNA or RNA dependent on thermophilic species or mesophilic property species.
Term " host cell " refers to the unicellular original from higher plant or animal for cultivating in cell culture medium Core biology and eukaryotic organisms (such as bacterium, yeast and actinomyces) and unicellular.
Term " carrier (vector) " is can be replicated, and exogenous DNA can be transmitted to recipient cell by gene etc. DNA molecular such as plasmid, bacteriophage and artificial chromosome etc.." plasmid ", " carrier " or " plasmid vector " is interchangeable herein It uses.
Term " nucleotide (nucleotide) " refers to single-stranded (single strand) or double-strand (double Strand) (the deoxyribonucleic acid of DNA existing for form；) or ribonucleic acid DNA (ribonucleic acid；It RNA), and unless otherwise specified, may include the analog of natural nucleotide.
Term " nucleic acid " or " polynucleotides " refer to DNA or RNA polymer or the polymer of its analog.Nucleic acid can be with It is such as chromosome or chromosome segment, carrier (such as expression vector), expression cassette, exposed DNA or RNA polymer, polymerization Enzyme chain reaction (PCR) product, oligonucleotides, probe or primer, or include the substance.Nucleic acid can be for example single-stranded, double Chain or three chains, but it is not limited to any specific length.Unless otherwise indicated, specific nucleic acid sequence is in addition to comprising specified any Further include complementary series except sequence or it can be encoded.
Term " primer " refers to that the nucleic acid that can be served as when polynucleotides when extending under entry condition on template direction closes At the polynucleotides of starting point.Primer can also be used for various other oligonucleotide mediated synthesis processes, including de novo formation (de Novo) RNA and the promoter as transcription correlated process in test tube.Primer is usually single-stranded oligonucleotide (for example, few deoxidation core Ribotide).The suitable length of primer is usually in 6 to 40 nucleotide ranges, and more typical length is in 15 to 35 nucleosides In acid range and depend on desired use.It is sufficiently stable miscellaneous to be formed with template that Short primer molecules usually require lower temperature Hand over compound.Primer does not need the correct sequence of reflection template, but primer must have enough complementarity extended with need Template is hybridized.In specific embodiments, term " primer pair " refers to including the end 5' with nucleic acid sequence to be amplified The 5' sense primer of Complementary hybridization, the primer sets of the 3'- antisense primer hybridized with the end 3' of sequence to be amplified.If it is necessary, It can be drawn by incorporation by the label that spectrum, photochemistry, biochemistry, immunochemistry or chemical means detect to mark Object.For example, useful label includes:32P, fluorescent dye, electron dense reagents, enzyme (being commonly used in elisa assay), biotin Or haptens and the protein that antiserum or monoclonal antibody can be used.
Term " 5'- hydrolase nucleic acid (nuclease, nuclease) probe " refers to comprising at least one for carrying out 5'- core The luminous marker part of sour water solution enzyme reaction is to detect the oligonucleotides of target nucleic acid.In some embodiments, for example, 5'- Nucleic acid hydrolysis enzyme probe only includes single luminous component (such as fluorescent dye etc.).In certain embodiments, 5'- nucleic acid hydrolysis Enzyme probe includes self complementary region, and probe is allowed to form hairpin structure in the selection conditions.In some embodiments, 5'- nucleic acid hydrolysis enzyme probe includes two or more mark parts, and one in two labels separates or divides from oligonucleotides Solution, radiation intensity increase and are released.In certain embodiments, 5'- nucleic acid hydrolysis enzyme probe is contaminated with two different fluorescence Material label, such as the end 5'- report dyestuff and the end 3'- quencher dyes or part mark.In some embodiments, 5'- core Sour enzyme probe labels again on end or labels in one or more positions in addition to end.When probe is intact, energy Amount transfer usually occur between two kinds of fluorescent materials so that from report dyestuff fluorescent emission be quenched definite part with On.During the extension step of polymerase chain reaction, for example, the 5'- nucleic acid hydrolysis enzyme probe in conjunction with template nucleic acid has So that the activity that the fluorescent emission of report dyestuff is no longer quenched, such as by Taq polymerase or 5' to the 3'- core of other polymerases Sour hydrolytic enzyme activities decompose.In some embodiments, 5'- nucleic acid hydrolysis enzyme probe can use two or more different reports Road dyestuff and the end 3'- quencher dyes or part mark.
Term " FRET " or " fluorescence resonance energy transfer " or " forester Resonance energy transfer " refer to two or more A chromophore, the energy transfer between donor chromophore and acceptor chromaphore (referred to as quencher).It is suitable that donor usually passes through transmitting When wavelength light and excite, energy is transferred to receptor.Receptor will move usually in the form of the light for re-emitting different wave length Energy launch again.When receptor is " dark " quencher, mobile energy is dispersed in the form other than light.One Whether specific fluorescent material serves as donor or receptor depends on FRET to the property of other members.Common Donor Acceptor pair Including FAM-TAMRA pairs.Common quencher is DABCYL and TAMRA.Common dark quencher includes: BlackHole QuenchersTM(BHQ),(Biosearch Technologies,Inc.,Novato,Cal.),Iowa BlackTM (Integrated DNA Tech.,Inc.,Coralville,Iowa),BlackBerryTMQuencher 650(BBQ- 650)(Berry&Assoc.,Dexter,Mich.)。
When referring to nucleic acid base, nucleoside triphosphate or nucleotide, term " conventional " or " natural " refer to natural hair It is born in the polynucleotides (that is, being dATP, dGTP, dCTP, dTTP for DNA), and dITP and 7-deaza-dGTP are frequent For replacing dGTP, can be used to replace dATP in synthesis reaction of DNA in the test tubes such as sequencing.
When referring to nucleic acid base, nucleosides or nucleotide, term " unconventional " or " modification " are included in specific polynucleotides The base of the routine naturally occurred, the modification of nucleosides or nucleotide, derivative or the like.It is specific compared with conventional dNTP Unconventional nucleotide be modified in the position 2' of ribose.Therefore, even if the nucleotide of RNA naturally occurred is ribonucleotide Acid (that is, ATP, GTP, CTPUTP, general designation rNTP), nucleotide has hydroxyl group in sugared 2 ' positions, and comparatively dNTP is not deposited , therefore it is as used herein, ribonucleotide is unconventional nucleotide as archaeal dna polymerase substrate.As used herein, non- Common nucleotides include but is not limited to the compound for being used as nucleic acid sequencing terminator.Exemplary terminator compound includes but unlimited In with 2', the compound of 3'- dideoxy structure, referred to as dideoxyribonucleoside triphosphate.Dideoxyribonucleoside triphosphate ddATP, DdTTP, ddCTP and ddGTP are collectively referred to as ddNTP.The other examples of terminator compound include the 2'-PO of ribonucleotide4 Analog.Very regulatory nucleotide includes but is not limited to thio dNTP ([[α]-S] dNTP), 5'- [α]-boron (borano)-for other DNTP, [α]-methyl-phosphonate dNTP, ribonucleotide triphosphate (rNTP).Unconventional base can be with radioactive isotope such as32P 、33P or35S；Fluorescent marker；Chemiluminescent labeling；Bioluminescence marker；Haptens label such as biotin；Or enzyme label such as strepto- Avidin or avidin mark.Fluorescent marker may include negatively charged dyestuff such as fluorescein family Dyestuff or the dyestuff such as dyestuff of rhodamine family or positively charged dyestuff such as cyanine family with neutral charge.Fluorescein man The dyestuff of race includes such as FAM, HEX, TET, JOE, NAN and ZOE.The dyestuff of rhodamine family include Texas Red, ROX, R110, R6G and TAMRA.With FAM, HEX, TET, JOE, NAN, ZOE, ROX, R110, R6G, Texas Red and TAMRA label Various dyestuffs or nucleotide pass through Perkin-Elmer (Boston, Massachusetts), Applied Biosystems (Foster City, California), Invitrogen/Molecular Probes (Eugene, Oregon) market sale.Cyanine family dyestuff includes Cy2, Cy3, Cy5 and Cy7 pass through GE Healthcare UK Limited (Amersham Place, Little Chalfont, Buckinghamshire, England) market sale.
Term " differentiation of mispairing ", refer to by nucleic acid by one or more nucleosides acid adhesions (such as covalently), When extending nucleic acid (such as primer or other oligonucleotides) with template dependent manner, for including that sequence has been distinguished from mispairing- The ability of the biocatalysis (such as the enzymes such as polymerase, ligase) of complete complementary sequence.Term " differentiation of mispairing ", refers to extension Nucleic acid (such as primer or other oligonucleotides) compared with the template that nucleic acid hybridizes, from the end 3' nucleic acid have mispairing mispairing- The biocatalysis ability of the sequence of complete complementary is distinguished comprising (about complementary) sequence.In some embodiments, the nucleic acid of extension Mispairing is included in the end 3' for fully-complementary sequence.In some embodiments, the nucleic acid of extension is for fully-complementary sequence It include mispairing on the position (N-1) 3'- of end second and/or the position N-2.
Unless otherwise indicated, all technologies and scientific term used in this specification have and ordinary skill people The normally understood identical meaning of member.
The present invention relates to the DNA polymerase activity enhancing PCR buffers combinations with increased gene mutation specificity Object, the composition include 25 to 100mM KCI；1 to 15mM (NH4)2SO4；Final pH is 8.0 to 9.0.
Polymerase used in PCR should use the optimum response buffer for mixing various substances, to play its function.Reaction Buffer usually contains the factor of stable pH, as confactor (cofactor) metal ion and prevent polymerization enzyme denaturation Stabilize ingredient.
The KCl can be used as the necessary factor of stable enzyme, help primer and target DNA pairing (pairing).In the present invention In, optium concentration is determined by adjusting the concentration of KCl in reaction buffer, is utmostly postponed as caused by mispairing with confirmation Amplification, while the high cation concentration of the amplification efficiency caused by matching is not reduced.
E507K, E507K/R536K, E507K/R660V and E507K/R536K/R660V Taq polymerase are used respectively, really Recognize and carryover effects are expanded according to the mispairing that the variation of KCl concentration generates in reaction buffer, as a result as shown in figure 9, for E507K/ R536K/R660V Taq polymerase, even if not having KCl in reaction buffer, it is also very outstanding that mispairing expands carryover effects；For E507K/R536 and E507K/R660V, in 50mM；E507K as a control group, in 100mM, it is excellent that mispairing expands carryover effects It is elegant.As a result from the point of view of, for the threshold value of KCl concentration, E507K/R536K/R660V Taq polymerase is minimum, E507K/R536K and E507K/R660V ratio E507K is low.
It is using E507K/R536K/R660V Taq polymerase, reaction is slow in order to further derive KCI concentration appropriate (NH in fliud flushing4)2SO4Concentration is fixed on certain numerical value, is expanded using a variety of KCl concentration.Confirm that amplification produces by electrophoresis Object as a result, as shown in Figure 10, can be confirmed that KCI concentration appropriate is 75mM.
Therefore, KCl concentration can be 25~100mM in PCR reaction buffer composition of the invention, preferably 60~ 90mM, more preferably 70~80mM, most preferably 75mM.
When KCl concentration is lower than 25mM, the amplification of general target is not influenced, but can make to be generated by matching primer Amplification reduces with the amplification difference generated by mismatched primers, when concentration is higher than 100mM, under the amplification efficiency for generating general target The problem of drop.
For PCR reaction buffer the composition, (NH4)2SO4For the necessary confactor of enzymatic activity, together with Tris For improving the activity of polymerase.In one embodiment of the present of invention, based on above-mentioned derivation result, by reaction buffer Middle KCl concentration is fixed on 75mM, by (NH4)2SO4Concentration various change is carried out from 2.5mM to 25mM, it is appropriate to confirm (NH4)2SO4Concentration.
Its result is as shown in figure 11, in (the NH of 2.5mM to 15mM4)2SO4Concentration under, confirm amplified production, and appropriate (NH4)2SO4Concentration be 5mM.
Further in (NH4)2SO4Concentration close in the case where 5mM (respectively use 2.5mM, 5mM, 10mM), carry out AS- QPCR, as a result as shown in figure 12, although the different maximum of Ct value difference at 10mM, confirmation matching amplification in Ct be slightly delayed, peak to Under topple over, it is thus determined that (NH appropriate4)2SO4Concentration be 5mM.
Therefore, (NH in PCR reaction buffer composition of the invention4)2SO4Concentration can be 1~15mM, preferably 2.5~8mM, more preferably 4~6mM, most preferably 5mM.
As (NH4)2SO4When concentration is lower than 1mM, the amplification of general target is not influenced, but can make to be produced by matching primer Raw amplification reduces with the amplification difference generated by mismatched primers, when concentration is higher than 15mM, generates the amplification effect of general target The problem of rate declines.
Therefore, best PCR buffer composition of the invention may include 70 to 80mM KCl；4 to 6mM (NH4)2SO4；Final pH is 8.0 to 9.0.
PCR buffer composition of the invention can further include 5 to 80mM TMAC (tetramethyl ammonium chloride, Tetra methyl ammonium chloride)。
TMAC expands as caused by mispairing or improves the preciseness (stringency) of hybridization reaction commonly used in reducing.This In one embodiment of invention, based on above-mentioned derivation result, KCl concentration in reaction buffer is fixed on 75mM, (NH4)2SO4Concentration be fixed on 5mM, TMAC concentration is subjected to various change from 0mM to 80mM, to confirm that TMAC appropriate is dense Degree.
Its result is as shown in figure 13, it is thus identified that TMAC concentration appropriate is for E507K/R536K Taq polymerase 70mM；It is 25mM for E507K/R536K/R660V Taq polymerase.TMAC concentration is further fixed on 25mM, (NH4)2SO4Concentration be fixed on 2.5mM, KCl concentration is adjusted separately as 20,40,60,80mM, is expanded, as a result such as Figure 14 institute Show, for SNP rs1015362 and rs4911414, KCl concentration appropriate is 60mM for confirmation.
When TMAC concentration is more than 80mM, amplification efficiency is reduced, therefore TMAC is used preferably in the range.
Therefore, when PCR buffer composition of the invention includes the TMAC of 5~80mM, the concentration of KCl can be 40~ 90mM, preferably 50~80mM, (NH4)2SO4Concentration can be 1~7mM, preferably 1.5~6mM.
Therefore, when comprising TMAC, best PCR buffer composition of the invention may include 15 to 70mM TMAC； 50 to 80mM KCl；1.5 to 6mM (NH4)2SO4；Final pH is 8.0 to 9.0.
PCR buffer composition of the invention can further include TrisCl and MgCl2, can also further wrap Include Tween 20 and bovine serum albumin(BSA) (BSA).
The present invention is provided to be used to detect gene mutation or the PCR kit of SNP comprising the PCR buffer composition.
PCR kit of the invention can further include the Taq polymerase being made of the amino acid sequence of SEQ ID NO:1 Archaeal dna polymerase, the archaeal dna polymerase may include the 507th amino acid residue in the amino acid sequence of (a) SEQ ID NO:1 Substitution；And (b) substitution of the 536th amino acid residue in the amino acid sequence of SEQ ID NO:1, the 660th amino acid The substitution of residue or the 536th and the 660th i.e. substitution of two amino acid residues.
According to a further advantageous embodiment of the invention, the substitution of the 507th amino acid residue is glutamic acid (E) quilt Lysine (K) replaces, and the substitution of the 536th amino acid residue is that arginine (R) is replaced (K) by lysine, and the described 660th The substitution of a amino acid residue is that arginine (R) is replaced by valine (V).
PCR kit of the invention may include those skilled in the art's known any agent used in primer extend process Or other element.
According to a further advantageous embodiment of the invention, the PCR kit includes more than one matching primer, one Above mismatched primers include more than one matching primer and more than one mismatched primers simultaneously, it is one more than Matching primer and more than one mismatched primers hybridize with target sequence, the mismatched primers, for hybridizing target sequence, at the end its 3' End to the 7th base positions include atypia (non-canonical) nucleotide.
PCR kit of the invention also can further include: a) nucleoside triphosphate；B) the quantitative examination in conjunction with double-stranded DNA Agent；C) polymerase blocking antibody；D) one or more control values or control sequence；E) one or more templates.
" Taq polymerase ", be according to the name of thermophilic bacteria thermus aquaticus (Thermus aquaticus) come The Taq DNA polymerase of name is initially separated from the bacterium.Thermus aquaticus is inhabited in hot spring and hot water Bacterium in ejiction opening, Taq polymerase are identified as being able to bear albuminous degeneration condition (high temperature) needed for PCR process Enzyme.The optimum activity temperature of Taq polymerase is 75~80 DEG C, and half-life period is 2 hours or more under the conditions of 92.5 DEG C, at 95 DEG C Under the conditions of be under the conditions of 40 minutes, 97.5 DEG C be 9 minutes, reproducible 1000 base pair dnas in 10 seconds under the conditions of 72 DEG C.This Shortage 3 → ' 5' exonuclease (exonuclease) proofreading activity, and in 9000 nucleotide, about measured in 1 Make mistake rate.For example, PCR can be carried out under high temperature (60 DEG C or higher) when using heat-resisting Taq.For Taq polymerase, Use amino acid sequence shown in SEQ ID NO:1 as consensus sequence.
In the present invention, in the amino acid sequence of SEQ ID NO:1, the glutamic acid (E) of the 507th amino acid residue is taken It is named as " E507K " (SEQ ID NO:2, base sequence are SEQ ID NO:7) on behalf of the Taq polymerase of lysine (K)； In the amino acid sequence of SEQ ID NO:1, the glutamic acid (E) of the 507th amino acid residue is substituted by lysine (K), and the 536th The Taq polymerase that the arginine (R) of a amino acid residue is substituted by lysine (K) is named as " E507K/R536K " (SEQ ID NO:3, base sequence are SEQ ID NO:8)；In the amino acid sequence of SEQ ID NO:1, the 507th amino acid residue Glutamic acid (E) is substituted by lysine (K), and the arginine (R) of the 660th amino acid residue is substituted by the Taq of valine (V) Polymerase is named as " E507K/R660V " (SEQ ID NO:4, base sequence are SEQ ID NO:9)；Finally, SEQ ID In the amino acid sequence of NO:1, the glutamic acid (E) of the 507th amino acid residue is substituted by lysine (K), the 536th amino The arginine (R) of sour residue is substituted by lysine (K), and the arginine (R) of the 660th amino acid residue is substituted by valine (V) Taq polymerase be named as " E507K/R536K/R660V " (SEQ ID NO:5, base sequence be SEQ ID NO: 10)。
According to one embodiment of present invention, the archaeal dna polymerase distinguishes matching primer and mismatched primers, the matching are drawn Object and mismatched primers hybridize with target sequence, the mismatched primers, may include atypia core in its end 3' for hybridizing target sequence Thuja acid.
The mismatched primers are hybridization oligonucleotides, it is necessary to which sufficiently complementation with target sequence to hybridize, but does not reflect target sequence Exact nucleotide sequence.
" canonical nucleotide (the canonical nucleotide) " or " complementary nucleotide " refer in standard Watson-gram Gram (Watson-Crick) base-pair, A-U, A-T and G-C.
" atypia nucleotide (the non-canonical nucleotide) " or " non-complementary nucleotide " refer to except fertile A-C, A-G, G-U, G-T, T-C, T-U, A, G-G, T-T, U-U, C-C other than gloomy-Crick (Watson-Crick) base-pair, C-U。
According to a preferred embodiment of the present invention, comprising matching the amplification of the target sequence of primer, and include mismatched primers The amplification of target sequence compare, the archaeal dna polymerase can express than lower Ct value.
For example, archaeal dna polymerase can be than being relied on by the way that one or more nucleotide are covalently attached to primer with target sequence Extend matched primer to the primer higher efficiency of property mode mispairing.Herein, higher efficiency can be observed by following example It arrives, the Ct value as compared with mismatched primers, matched primer in RT-PCR is lower.Match the Ct value between primer and mismatched primers Difference is 10 or higher, preferably 10~20, or synthesizes there is no the amplicon caused by mismatched primers.
For example, using matched forward primer and reverse primer in the first reaction, in the second reaction, set with identical experiment It sets, using the forward primer and matched reverse primer of mispairing, the product formed by standard PCR, first reaction ratio second A reaction is bigger.
Ct (threshold crossings circulation) value indicates the DNA quantitative approach of quantitative PCR, depends on drawing in logarithm phase loop number Fluorescence.The threshold value of fluorescence detection based on DNA is set as at least more slightly higher than background.Fluorescence is more than that the recurring number of threshold value is known as Ct Or Cq (quantitative circulation, quantification cycle) is known as according to MIQE criterion.The Ct value of given reaction is defined as fluorescence Emit the cycle-index of cross-fixing limiting value.For example, SYBR Green I and fluorescence probe can be used for the quantitative reality of template DNA When PCR.Fluorescence of each circulating collection from sample during PCR, and compare cycle-index drawing.Initial template concentration with it is glimmering The fluorescence signal of optical signal is initially displayed the time and is inversely proportional.Template concentrations are higher, and signal is more early to be occurred (counting in lower circulation It is existing).
Carrier the invention further relates to the nucleic acid sequence for encoding above-mentioned archaeal dna polymerase and comprising the nucleic acid sequence and host are thin Born of the same parents.Various carriers can be prepared using the nucleic acid for encoding archaeal dna polymerase of the invention.It can be used including from can be with host The replicon for the species that cell exchanges and any carrier of control sequence.Carrier of the invention can be expression vector and including The nucleic acid region for the control transcription and translation being operably connected with the nucleic acid sequence for encoding archaeal dna polymerase of the invention.Regulation DNA sequence dna needed for sequence refers to the coded sequence expressed and be operably connected in specific host organism.For example, being suitable for The regulating and controlling sequence of prokaryotes includes promoter, any sequence of operation and alc ribosome bind site.In addition, carrier can contain There is " positive regulating factor (Positive Retroregulatory Element, PRE) " to enhance the half-life period of the mRNA of transcription. Control transcription and translation nucleic acid region is generally applicable to the host cell of expression polymerase.It is known in the art to be used for various places Various types of suitable expression vectors of chief cell and suitable regulating and controlling sequence.In general, transcription and translation regulating and controlling sequence can be with Including such as promoter sequence, ribosome bind site, transcription initiation and termination sequence, translation initiation and termination sequence and increasing Hadron or activation sequence.In a typical implementation, adjusting empty sequence includes promoter and transcription initiation and termination sequence.Carrier Usually also comprising the polylinker region containing several restriction sites for being used to be inserted into exogenous DNA.In certain embodiments In, promote to purify using " fusion mark ", and if desired, then remove label/mark (such as " histidine tag (His-tag)").However, working as from mesophilic host (such as Escherichia coli) the purifying thermal activities and/or heat for using " heating stepses " When stability albumen, these are usually necessary.Contain coding DNA replication sequence, tune using the preparation of standard recombinant dna technology Control sequence, the suitable carrier of Phenotypic Selection gene and the mutant polymerase of concern.As it is known in the art, isolated plasmid, virus Carrier and DNA fragmentation are decomposed and shear and linked together with particular order to generate desired carrier.
In a preferred embodiment of the invention, expression vector contains the selected marker for selecting conversion host cell Gene.Selection gene is well known in the art, and will be different according to used host cell.Suitable selection Gene may include the gene of encoding ampicillin and/or tetracyclin resistance, it is possible thereby to there are the feelings of these antibiotic The cell of these carriers is cultivated in conversion under condition.
In a preferred embodiment of the invention, encode archaeal dna polymerase of the invention nucleic acid sequence individually or with load Body combination imports in cell.It imports or its equivalent expressions refers to that nucleic acid is suitable for the mode of subsequent integration, amplification and/or expression Into cell.Introduction method includes such as CaPO4Precipitating, liposome fusion,Electrophoresis, virus infection etc..
Prokaryotes are used as the host cell of initial cloning steps of the present invention.In order to quickly prepare a large amount of DNA, in order to make It is ready for use on and generates site-orientation mutation single-stranded DNA templates, in order to screen many mutant simultaneously, in order to generated prominent Variant carries out DNA sequencing, this is particularly useful.The suitable host cell of prokaryotic cell includes E. coli K12 Bacterial strain 94 (ATCC No.31,446), E. coli bacterial strain W3110 (ATCC No.27,325), E. coli K12 bacterial strain DG116 (ATCC No.53,606), E. coli X1776 (ATCC No.31,537) and Escherichia coli E.coli B；Many different bacterial strains such as HB101, JM101, NM522, NM538, NM539 of E. coli, Yi Jiqi His species, including Bacillaceae such as bacillus subtilis ((Bacillus subtilis), other enterobacteriaceaes such as mouse typhus Salmonella (Salmonella typhimurium), serratia marcescens (Serratia marcesans) and various The prokaryotes category of pseudomonad kind (pseudomonas, Pseudomonas sp.) can be by as host.Typically, for turning The plasmid for changing Escherichia coli (E.coli) includes pBR322, pUCI8, pUCI9, pUCIl8, pUC 119 and Bluescript M13.It is also possible, however, to use many other suitable carriers.
The present invention also provides the methods for preparing archaeal dna polymerase, comprising the following steps: the step of cultivating the host cell； The step of archaeal dna polymerase is separated from culture and its culture supernatant.
Archaeal dna polymerase of the invention is by the way that under appropriate conditions of inducing or archaeal dna polymerase is caused to be expressed, culture is by containing There is the host cell of the expression vector conversion of the nucleic acid sequence of encoding DNA polymerase to prepare.It is being suitable for protein expression Under the conditions of cultivate conversion the method for host cell be known in the art.For with the plasmid vector system containing λ pL promoter The Suitable host cells of standby polymerase are coli strain DG116 (ATCC No.53606).According to expression, Ke Yishou Collect simultaneously separation of polymeric enzyme.
Once purifying, so that it may which the mispairing of measurement archaeal dna polymerase of the present invention is distinguished.For example, by comparing primer complete The amplification of the target sequence of the single base mismatch of the amplification and 3 ' end of primer for the target sequence matched is active to measure mispairing difference.Amplification It can be detected in real time, such as by using TaqManTMProbe.Polymerase area can be estimated by comparing the Ct of two kinds of reactions It is divided to the ability of two kinds of target sequences.
The present invention provides external (in vitro) detection at least one from more than one template using the PCR kit The detection method of kind gene mutation or SNP.
Term " SNP (single nucleotide polymorphism, Single Nucleotide Polymorphisms) " refers in DNA core The heredity variation or mutation of the difference of a nucleotide sequence (A, T, G or C) are shown in nucleotide sequence.
In external (in vitro) SNP detection method of the invention, target sequence can reside in test sample and wrap DNA, cDNA or RNA are included, genomic DNA is preferably included.Test sample can be from bacterium, bacterial cultures or cell culture A kind of cell lysate of preparation.In addition, test sample may be embodied in animal, it preferably is contained in vertebrate, it is more excellent Choosing is included in people experimenter.Target sequence may be embodied in genomic DNA, preferably be contained in the genomic DNA of individual, more It preferably is contained in bacterium or vertebrate, is most preferably comprised in the genomic DNA of people experimenter.
SNP detection method of the invention may include the melting temperature using double-stranded specific dyestuff such as SYBR Green I Analysis.
Melting temperature tracing analysis can be in the ABI 5700/ comprising software (onboard software SDS 2.1) It is carried out in the real time PCR equipments such as 7000 (96 hole formats) or ABI 7900 (384 hole format).Alternatively, melting temperature tracing analysis It can be executed with end point analysis.
" in conjunction with the dyestuff of double-stranded DNA " or " double-stranded specific dyestuff ", and not compared with when double-stranded DNA combines, with double-strand It can be used in the case that there is high fluorescence when DNA is combined.The example of this dyestuff 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.In addition to EtBr and EvaGreen (Quiagen), these dyestuffs have been used for applying in real time Test.
External (in vitro) SNP detection method of the invention can pass through the Ago-Gel after real-time PCR, standard PCR Analysis, gene mutation specific amplification or allele specific amplification by real-time PCR, four primer amplification Refracting Mutation systems System PCR or isothermal duplication carry out.
So-called " standard PCR " is the known single copy or multicopy for being used for DNA amplification or cDNA of those skilled in the art Technology.Almost all of PCR uses Taq polymerase or heat-stable DNA polymerase such as Klen Taq.Archaeal dna polymerase is by making Single stranded DNA is used to assemble new DNA chain from nucleotide enzymatic as template and using oligonucleotides (primer).It is generated by PCR Amplicon, can be in analysed on agarose gel.
" the real-time PCR " can monitor its process in real time when executing PCR.Therefore, number is collected during entire PCR According to, rather than collected at the end of PCR.In real-time PCR, reaction is characterized in detecting the time of amplification in circulation for the first time Point, rather than the aim parameter accumulated after fixed cycles number.It is main to use the detection based on dyestuff and the detection based on probe two Kind of method executes quantitative PCR.
" allele specific amplification (Allele the Specific Amplification, ASA) " is a kind of amplification Technology, PCR primer, which is designed to single nucleotide acid residue, can distinguish other templates.
Described " passing through the gene mutation specific amplification or allele specific amplification of real-time PCR " can be with high efficacious prescriptions Formula detects gene mutation or SNP.It is different from the other methods of most of detection gene mutations or SNP, target gene material it is pre- Amplification is not required.ASA is tied in single reaction according to the difference between matching and primer/target sequence compound of mispairing Close amplification and detection.The increase of DNA amplification can be by glimmering as caused by such as dyestuff of SYBR Green I in reaction process The increase of optical signal carrys out real-time monitoring, and the dyestuff shines when in conjunction with double-stranded DNA.It is special by the genetic mutation of real-time PCR Specific amplification or allele specific amplification occur the delay of fluorescence signal occur when mispairing or signal are not present.In gene In mutation or SNP detection, this is provided about whether there are the information of gene mutation or SNP.
" the four primer amplification Refracting Mutation System PCR " expands together with control segment wild in single tube PCR reaction Type and mutant allele.Non-allelic genes Specificity control amplicon is drawn by two general (outsides) of saltation zone side Object amplification.The design direction of two allele-specific (inside) primers is with universal primer on the contrary, expanding together with universal primer Increase wild type and saltant type amplicon.As a result, two allele specific amplification are since the position of mutation is relative to general (outside) primer is asymmetric, therefore has different length, can be easily separated by standard gel electrophoresis.It is described right The internal contrast of false negative and amplification failure is provided according to amplicon, and in two allele specific amplification at least One is present in always in four primer amplification Refracting Mutation System PCR.
" isothermal duplication " refers to that the amplification of nucleic acid carries out at a lower temperature, and independent of thermal cycler, preferably Temperature does not need to change during amplification.Temperature used in isothermal duplication can room temperature (22~24 DEG C) to about 65 DEG C it Between, or in about 60~65 DEG C, 45~50 DEG C, 37 DEG C~42 DEG C or 22~24 DEG C of room temperature.Isothermal duplication product can be by solidifying Gel electrophoresis, ELISA, ELOSA (enzyme-linked oligonucleotides test, Enzyme linked oligosorbent assay), in real time The capillary electrophoresis based on chip of PCR, ECL (improved chemiluminescence), analysis RNA, DNA and protein or turbidity is raw Object analyzer (bioanalyzer) detection.
In one embodiment of the invention, using E507K/R536K, E507K/R660V or E507K/R536K/R660V Taq polymerase confirms for the template comprising SNP (rs1408799, rs1015362 and/or rs4911414), mismatched primers Whether extension ability is reduced.
As a result, as shown in Fig. 6 to 8, compared with E507K Taq polymerase, it can be verified that E507K/R536K, E507K/ R660V or E507K/R536K/R660V Taq polymerase expands delay as caused by mismatched primers, and effect is in E507K/ It is the most obvious in R536K/R660V Taq polymerase.
Which demonstrate compared with conventional Taq polymerase (E507K), there is archaeal dna polymerase of the invention higher mispairing to prolong Stretch selectivity.Thus, it is expected that archaeal dna polymerase of the invention can be effectively applied to the medical diagnosis of disease and the research of recombinant DNA.
According to a further advantageous embodiment of the invention, the PCR kit includes more than one matching primer, one Above mismatched primers include more than one matching primer and more than one mismatched primers simultaneously, it is one more than Matching primer and more than one mismatched primers hybridize with target sequence, the mismatched primers, for hybridizing target sequence, at the end its 3' End to the 7th base positions include atypia (non-canonical) nucleotide.
PCR kit of the invention can further include nucleoside triphosphate.
PCR kit of the invention further includes a) one or more buffers；B) quantitative reagent in conjunction with double-stranded DNA； C) polymerase blocking antibody；D) one or more control values or control sequence；E) one or more templates；As shown in the figure.
It below will the present invention is described in detail by embodiment.These embodiments are to exemplary theory of the invention Bright, there is the people of general knowledge should be understood that interest field of the invention is not limited to the embodiment for this field.
Embodiment 1 induces Taq polymerase mutation
1-1. segment PCR
In the present embodiment, the 536th amino acid in the amino acid sequence of SEQ ID NO:1 is prepared in accordance with the following methods Taq archaeal dna polymerase (hereinafter referred to as " R536K ") that the arginine of residue is replaced by lysine, the 660th amino acid residue The smart ammonia of Taq archaeal dna polymerase (hereinafter referred to as " R660V ") and the 536th amino acid residue that arginine is replaced by valine Acid by lysine replaces and the arginine of the 660th amino acid residue is replaced by valine Taq archaeal dna polymerase (hereinafter referred to as For " R536K/R660V ").
Firstly, using mutant-specific primers shown in table 1 by PCR amplification Taq archaeal dna polymerase segment (F1 to F5), As shown in Fig. 1 (a).Reaction condition is as shown in table 2.
Table 1
Table 2
PCR product is confirmed by electrophoresis, as a result as shown in Fig. 1 (b), it is thus identified that the band of each segment confirms target piece with this Section has been amplified.
1-2. is overlapped (overlap) PCR
Using each segment expanded in the 1-1 as template, both ends are expanded complete using primer (Eco-F and Xba-R primer) It is long.Reaction condition is as shown in Table 3 and Table 4.
Table 3
Table 4
As a result, as shown in Fig. 1 (c), it is thus identified that " R536K ", " R660V ", " R536K/R660V " Tag polymerase are amplified.
1-3. connection (ligation)
Under the conditions shown in Table 5, pUC19 is decomposed 4 hours and is purified at 37 DEG C with restriction enzyme EcoRI/XbaI The DNA of purifying is handled 1 hour with SAP at 37 DEG C under the conditions shown in Table 6 to prepare carrier by DNA.
Table 5
Table 6
For insert (insert), the overlapping PCR products of the embodiment 1-2 are purified, and the condition shown in table 7 Under, it is decomposed at 37 DEG C 3 hours with restriction enzyme EcoRI/XbaI, gel extraction (figure is then carried out together with the carrier of preparation 2)。
Table 7
Under the conditions shown in Table 8, after being connected 2 hours at room temperature (RT), convert E. coli DH5 α and It is screened on culture medium containing ampicillin.The plasmid prepared from the bacterium colony obtained is sequenced, and acquisition is prominent needed for introducing The Taq DNA polymerase mutant body (" R536K ", " R660V " and " R536K/R660V ") of change.
Table 8
Embodiment 2 introduces E507K mutation
2-1. segment PCR
Work to " R536K ", " R660V " and " R536K/R660V " the Taq archaeal dna polymerase prepared in the embodiment 1 Property tested, as a result confirmation activity decline (data are not shown), respectively to " R536K ", " R660V " and " R536K/R660V " Being introduced into E507K mutation, (in the amino acid sequence of SEQ ID NO:1, the glutamic acid of the 507th amino acid residue is taken by lysine Generation), as a control group, E507K mutation is also introduced into wild type (WT) Taq archaeal dna polymerase.Introduce the Taq of E507K mutation Archaeal dna polymerase preparation method is same as Example 1.
Using mutant-specific primers shown in table 9 by PCR amplification Taq archaeal dna polymerase segment (F6 to F7), such as Fig. 3 It is shown.Reaction condition is as shown in table 10.
Table 9
Table 10
* template plasmid: pUC19-Tag (WT)
pUC19-Tag(R536K)
pUC19-Tag(R660V)
pUC19-Tag(R536K/R660V)
2-2. is overlapped (overlap) PCR
Using each segment expanded in the 2-1 as template, both ends are expanded complete using primer (Eco-F and Xba-R primer) It is long.Reaction condition is as shown in table 11.
Table 11
2-3. connection (ligation)
Under the conditions shown in Table 5, pUC19 is decomposed 4 hours and is purified at 37 DEG C with restriction enzyme EcoRI/XbaI The DNA of purifying is handled 1 hour with SAP at 37 DEG C under the conditions shown in Table 6 to prepare carrier by DNA.
For insert (insert), the overlapping PCR products of the embodiment 2-2 are purified, and the condition shown in table 7 Under, it is decomposed at 37 DEG C 3 hours with restriction enzyme EcoRI/XbaI, gel extraction (figure is then carried out together with the carrier of preparation 4)。
Under the conditions shown in Table 8, after being connected 2 hours at room temperature (RT), convert E. coli DH5 α or DH10 β is simultaneously screened on the culture medium containing ampicillin.The plasmid prepared from the bacterium colony obtained is sequenced, and is introduced Taq DNA polymerase mutant body (" E507K/R536K ", " E507K/R660V " and " E507K/R536K/ of required mutation R660V”)。
Embodiment 3 carries out qPCR using archaeal dna polymerase of the invention
Using respectively containing " E507K/R536K ", " E507K/R660V " and " E507K/ obtained in the embodiment 2 The Taq polymerase of R536K/R660V " mutation, for the template containing SNP, it extends whether mismatched primers ability is reduced for confirmation. As a control group, using " E507K " Taq polymerase being mutated containing E507K.
The template comprising SNP being used in the present invention is rs1408799, rs1015362 and rs4911414, each template Genotype and its specific primer (IDT, the U.S.) sequence information as shown in table 12 and table 13.
Table 12
Table 13
QPCR condition (Applied Biosystems 7500Fast) is as shown in table 14 below.
Table 14
Probe carries out double labeling, as shown in table 15 below.
Table 15
Kit is collected using the mouth epithelial cells purchased from Noble Bio and collects mouth epithelial cells, is dissolved in In 500 μ l lysates (lysis solution), and with 12,000 × g is centrifuged 3 minutes.Supernatant is transferred in new pipe, often It is secondary to use 1 μ l (Fig. 5).
Reaction condition such as table 16 indicates that the composition of reaction buffer such as table 17 indicates.
Table 16
Table 17
Other than the specific primer shown in the table 13, other reaction solutions are prepared in two test tubes in the same way, are led to It crosses and adds each allele-specific primers progress qPCR.At this point, merge the fluorescence signal that is detected in each test tube with Calculated on AB7500 software (v 2.0.6) and analysis reach threshold value (threshold)) difference of circulation (Ct) value of fluorescent value. The time of Ct value delay in the amplification as caused by mismatched primers is longer, can determine whether as gene mutation specificity or allele spy It is anisotropic good.
AS-qPCR's as a result, as shown in Fig. 6 to 8 is carried out on rs1408799, rs1015362 and rs4911414, and it is right It is compared according to a group E507K, it can be verified that the Taq comprising E507K/R536K, E507K/R660V or E507K/R536K/R660V mutation In the case where polymerase, amplification caused by mismatched primers postpones, and effect is the brightest in E507K/R536K/R660V mutation It is aobvious.
Which demonstrate compared with the Taq polymerase being mutated comprising E507K, that of the invention includes E507K/R536K, E507K/ There is the Taq archaeal dna polymerase of R660V or E507K/R536K/R660V mutation more excellent mispairing to extend selectivity.Therefore, in advance Counting Taq archaeal dna polymerase of the invention can be effectively applied to the research of medical diagnosis and recombinant DNA of disease.
The KCl concentration of 4 reaction buffer of embodiment optimizes
In the present embodiment, the amplification efficiency as caused by matching is made not reduce and expand most as caused by mispairing in order to find The high cation concentration postponed to limits adjusts the KCl concentration in PCR reaction buffer to determine the optium concentration of KCl.
" E507K/R536K ", " E507K/R660V " or " E507K/R536K/ are separately included using what is obtained in embodiment 2 The Taq polymerase of R660V " mutation, and the KCl concentration threshold compared with the Taq polymerase for including E507K mutation.
Template comprising SNP uses rs1408799, the genotype of template is TT, and primer in table 12 using recording Rs1408799 primer.QPCR condition (Applied Biosystems 7500Fast) is carried out with the condition of table 14, double labeling Probe uses the 1408799-FAM of table 15, and reaction condition is as shown in table 18, reaction buffer composition such as table 19.
Table 18
Table 19
As a result as shown in figure 9, the KCI concentration threshold of E507K/R536K/R660V Taq polymerase is minimum, E507K/ The KCl concentration threshold ratio E507K of R536K, E507K/R660V are low.
Based on the above results, in order to determine KCl concentration appropriate, it is polymerize using E507K/R536K/R660V Taq Enzyme carries out further experiment, and primer uses the rs1408799-T specific primer recorded in table 13.QPCR condition (AppliedBiosystems 7500Fast) carries out 35 circulations with the condition of table 14, and reaction condition is as shown in table 20.
Table 20
The reaction buffer of control group forms, the composition of the reaction buffer of experimental group such as table 8 identical as table 21, will (NH4)2SO4Concentration is fixed on 2.5mM, carries out various change to KCl concentration.
Table 21
With after condition amplification by electrophoresis confirmation PCR product as a result, as shown in Figure 10, can be confirmed can both make by The KCl concentration appropriate that amplification postpones to the maximum extent and reduces the amplification efficiency caused by matching not caused by mispairing 75mM。
(the NH of 5 reaction buffer of embodiment4)2SO4Concentration optimizes
The present embodiment is fixed on 75mM based on the result of the embodiment 4, by KCl concentration in reaction buffer, right (NH4)2SO4Concentration carries out various change, confirms optimal (NH with this4)2SO4Concentration.Primer is using recording in table 13 Rs1408799-T specific primer, qPCR condition (Applied Biosystems 7500Fast) carry out 35 with the condition of table 14 A circulation, reaction condition is as shown in table 20, and the reaction buffer composition of control group is identical as table 21.
As a result, as shown in figure 11, (NH appropriate4)2SO4Concentration is 5mM.
Based on the result, KCl concentration in reaction buffer is fixed on 75mM, by (NH4)2SO4Concentration set It is scheduled on 5mM or so (respectively 2.5mM, 5mM and 10mM), further confirms that and expands carryover effects as caused by mispairing.
Primer uses the 1408799-FAM of table 15 using the rs1408799 primer recorded in table 13, double labeling probe, Reaction condition is as shown in table 22.
Table 22
As a result, as shown in figure 12, (NH4)2SO4Concentration at 10mM the different maximum of Ct value difference, but confirmation caused by matching Amplification Ct be slightly delayed, peak is toppled over downwards, it is thus determined that (NH appropriate4)2SO4Concentration be 5mM.
Integrated embodiment 4 and embodiment 5 as a result, confirmation optimum response buffers combinations include 50mM TrisCl, The MgCl of 2.5mM2, 75mM KCl, 5mM (NH4)2SO4, 0.1%Tween 20 and 0.01%BSA.
TMAC is added in 6 reaction buffer of embodiment and its concentration optimizes
In the present embodiment, TMAC is added in reaction buffer, and confirms its optium concentration.With the embodiment 4 and Based on the result of embodiment 5, KCl concentration in reaction buffer is fixed on 75mM, (NH4)2SO4Concentration is fixed on 5mM, right The concentration of TMAC carries out various change, confirms optimal TMAC concentration with this.
Using E507K/R536K or E507K/R536K/R660V Taq polymerase, the template comprising SNP is used The genotype of rs1408799, template are TT, and primer uses the rs1408799 primer recorded in table 12.QPCR condition (AppliedBiosystems 7500Fast) is carried out with the condition of table 14, and double labeling probe uses the 1408799- of table 15 FAM, reaction condition are as shown in table 22.
Experimental result is as shown in figure 13, it is thus identified that TMAC concentration appropriate is for E507K/R536K Taq polymerase 60mM；It is 25mM for E507K/R536K/R660V Taq polymerase, and if confirming TMAC excessive concentration, amplification efficiency reduction.
The KCl, (NH of 7 reaction buffer of embodiment4)2SO4And TMAC concentration optimizes
In the present embodiment, based on the embodiment 6 confirmation as a result, being polymerize using E507K/R536K/R660V Taq Enzyme confirms optimal KCl, (NH in reaction buffer4)2SO4And TMAC concentration.
Specifically, TMAC concentration in reaction buffer is fixed on 25mM, (NH4)2SO4Concentration is fixed on 2.5mM, will KCl concentration adjusts separately as 20,40,60,80mM.Two SNP, that is, rs1015362 and rs4911414 are tested, template Genotype it is as described in Table 12, primer uses rs1015362 the and rs4911414 primer recorded in table 13, qPCR condition (Applied Biosystems 7500Fast) is carried out with the condition of table 14, and double labeling probe uses the 1408799- of table 15 FAM, reaction condition are as shown in table 22.
Experimental result, as shown in figure 14, for two SNP, KCl concentration appropriate is that 60mM can be with when concentration is 80mM Observe amplification efficiency reduction.
By result above, confirm that optimal KCl concentration is 60mM, (NH in reaction buffer4)2SO4Concentration is 2.5mM, TMAC concentration is 25mM.More specifically, confirmation uses KCl, 5mM's that concentration is 75mM for E507K/R536K polymerase (NH4)2SO4, the TMAC of 60mM it is most effective；For E507K/R536K/R660V polymerase, using the KCl that concentration is 60mM, (the NH of 2.5mM4)2SO4Concentration, the TMAC of 25mM are most effective.
Sequence table
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Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro
805 810 815
Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu
820 825 830
<210> 4
<211> 832
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu
1 5 10 15
Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly
20 25 30
Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala
35 40 45
Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val
50 55 60
Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly
65 70 75 80
Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu
85 90 95
Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu
100 105 110
Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys
115 120 125
Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp
130 135 140
Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly
145 150 155 160
Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro
165 170 175
Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn
180 185 190
Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu
195 200 205
Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu
210 215 220
Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys
225 230 235 240
Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val
245 250 255
Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe
260 265 270
Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu
275 280 285
Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly
290 295 300
Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp
305 310 315 320
Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val 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 (16)

1. a kind of DNA polymerase activity enhancing PCR buffer composition with increased gene mutation specificity, described group Close the KCl that object includes 25 to 100mM；1 to 15mM (NH4)2SO4；Final pH is 8.0 to 9.0.
2. PCR is used in the DNA polymerase activity enhancing according to entitlement requests the 1st with increased gene mutation specificity Buffer composition, it is characterized in that, the KCl concentration is 60 to 90mM.
3. PCR is used in the DNA polymerase activity enhancing according to entitlement requests the 1st with increased gene mutation specificity Buffer composition, it is characterized in that, (the NH4)2SO4Concentration is 2.5 to 8mM.
4. PCR is used in the DNA polymerase activity enhancing according to entitlement requests the 1st with increased gene mutation specificity Buffer composition, it is characterized in that, the KCl concentration is 70 to 80mM, (the NH4)2SO4Concentration is 4 to 6mM.
5. PCR is used in the DNA polymerase activity enhancing according to entitlement requests the 1st with increased gene mutation specificity Buffer composition, it is characterized in that, further include 5 to 80mM TMAC.
6. PCR is used in the DNA polymerase activity enhancing according to entitlement requests the 5th with increased gene mutation specificity Buffer composition, it is characterized in that, the KCl concentration is 40 to 90mM.
7. PCR is used in the DNA polymerase activity enhancing according to entitlement requests the 5th with increased gene mutation specificity Buffer composition, it is characterized in that, (the NH4)2SO4Concentration is 1 to 7mM.
8. PCR is used in the DNA polymerase activity enhancing according to entitlement requests the 5th with increased gene mutation specificity Buffer composition, it is characterized in that, the TMAC concentration is 15 to 70mM, and the KCl concentration is 50 to 80mM, (the NH4)2SO4Concentration is 1.5 to 6mM.
9. PCR is used in the DNA polymerase activity enhancing according to entitlement requests the 1st with increased gene mutation specificity Buffer composition, it is characterized in that, the PCR buffer composition further includes TrisCl and MgCl2。
10. a kind of gene that is used to detect comprising any one of entitlement requests the 1st to the 9th PCR buffer composition is dashed forward The PCR kit of change or SNP.
11. according to entitlement requests the 10th for detecting gene mutation or the PCR kit of SNP, it is characterized in that, the PCR Kit further includes the archaeal dna polymerase for the Taq polymerase being made of the amino acid sequence of SEQ ID NO:1, and the DNA is poly- Synthase includes the substitution of the 507th amino acid residue in the amino acid sequence of (a) SEQ ID NO:1；And (b) SEQ ID NO:1 Amino acid sequence in the substitution of the 536th amino acid residue, the substitution of the 660th amino acid residue or the 536th and The 660 i.e. substitution of two amino acid residues.
12. according to entitlement requests the 11st for detecting gene mutation or the PCR kit of SNP, it is characterized in that, described the The substitution of 507 amino acid residues is that glutamic acid (E) is replaced by lysine (K), and the substitution of the 536th amino acid residue is Arginine (R) is replaced (K) by lysine, and the substitution of the 660th amino acid residue is that arginine (R) is taken by valine (V) Generation.
13. according to entitlement requests the 10th for detecting gene mutation or the PCR kit of SNP, it is characterized in that, the PCR Kit further includes:
A) nucleoside triphosphate；
B) quantitative reagent in conjunction with double-stranded DNA；
C) polymerase blocking antibody；
D) one or more control values or control sequence；
E) one or more templates.
14. it is a kind of using entitlement requests the 10th PCR kit from more than one template vitro detection at least one base Because of mutation or the detection method of SNP.
15. according to the 14th the method for entitlement requests, it is characterized in that, the PCR kit is further included by SEQ ID The archaeal dna polymerase of the Taq polymerase of the amino acid sequence composition of NO:1, the archaeal dna polymerase include (a) SEQ ID NO:1's The substitution of 507th amino acid residue in amino acid sequence；And (b) the 536th ammonia in the amino acid sequence of SEQ ID NO:1 I.e. two amino acid residues of the substitution of base acid residue, the substitution of the 660th amino acid residue or the 536th and the 660th take Generation.
16. according to the 15th the method for entitlement requests, it is characterized in that, the substitution of the 507th amino acid residue is paddy Propylhomoserin (E) is replaced by lysine (K), and the substitution of the 536th amino acid residue is that arginine (R) by lysine replaces (K), The substitution of 660th amino acid residue is that arginine (R) is replaced by valine (V).
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WO2020145754A1 (en) * 2019-01-11 2020-07-16 주식회사 진캐스트 Mass spectrometry using dna polymerase with increased genetic mutation specificity
KR102310819B1 (en) * 2019-01-11 2021-10-12 주식회사 진캐스트 DNA polymerase for detecting TERT mutation and kit comprising the 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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US20030180741A1 (en) * 2001-12-21 2003-09-25 Holly Hogrefe High fidelity DNA polymerase compositions and uses therefor
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WO2015082449A2 (en) * 2013-12-02 2015-06-11 Universitaet Konstanz Mutated dna polymerases with high selectivity and activity
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US20030180741A1 (en) * 2001-12-21 2003-09-25 Holly Hogrefe High fidelity DNA polymerase compositions and uses therefor
EP2554665A2 (en) * 2011-08-03 2013-02-06 Fermentas UAB DNA polymerases
WO2015082449A2 (en) * 2013-12-02 2015-06-11 Universitaet Konstanz Mutated dna polymerases with high selectivity and activity
CN106520997A (en) * 2016-12-14 2017-03-22 南京诺唯赞医疗科技有限公司 Single cell gene expressions quantitative analysis method

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