TWI688656B - PCR buffer composition for enhancing DNA polymerase activity with increased specificity of gene mutation - Google Patents

Abstract

The present invention provides a PCR buffer composition for enhancing DNA polymerase activity with increased gene mutation specificity and uses thereof. More specifically, the present invention provides mutation induction at specific amino acid positions to increase gene mutation specificity PCR buffer composition for enhancing DNA polymerase activity, PCR kit for detecting gene mutation or SNP containing the PCR buffer composition and/or DNA polymerase with increased gene mutation specificity, And a method for detecting at least one gene mutation or SNP in vitro from more than one template by using the kit.

Description

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

The present invention relates to a PCR buffer composition for enhancing DNA polymerase activity with increased gene mutation specificity and its use, in particular to a DNA polymerase activity for inducing mutation at a specific amino acid position to increase gene mutation specificity PCR buffer composition for enhancement, PCR kit for detecting gene mutation or SNP containing the PCR buffer composition and/or DNA polymerase with increased gene mutation specificity, and using the kit A method for detecting at least one gene mutation or SNP in vitro from more than one template.

Since the discovery of the first human genome sequence, researchers have focused on exploring genetic differences between individuals such as single nucleotide mutations (single nucleotide polymorphisms, SNPs). Single nucleotide mutations in the genome are associated with different drug resistance or susceptibility of various diseases, which has become increasingly clear, so it has become the main concern. The knowledge of future medical-related nucleotide variations can be applied to the treatment of genetic supply of individuals, and can prevent ineffective or cause side effects of drug treatment (Shi, Expert Rev. Mol. Diagn. 1, 363-365 (2001)). The development of efficient nucleotide variation identification technology in time and cost will bring further development of pharmacogenetics.

SNPs occupy the main genetic variation in the human genome and can induce more than 90% of the differences between individuals. (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)). In order to detect these genetic variations and mutations and other nucleic acid variations, various methods can be used. For example, the identification of a variant of a target nucleic acid can be achieved by hybridizing a nucleic acid sample to be analyzed with a hybridization primer specific for sequence variants under appropriate hybridization conditions (Guo et al., Nat. Biotechnol. 15, 331-335 (1997)).

However, it has been found that this hybridization method cannot meet the clinical needs particularly in terms of the sensitivity required for measurement. Therefore, PCR has been widely used in diagnostic tests for mutation detection in molecular biology, SNP, and other allelic sequence variants (Saiki et al., Science 239, 487-490 (1988)), where changes are considered Before the hybridization, the target nucleic acid to be detected is amplified by polymerase chain reaction (PCR) before hybridization. For the hybridization primer used for this assay, a single-stranded oligonucleotide is usually used. Specific examples of modifications of the assay include the use of fluorescent hybridization probes (Livak, Genet. 73-5 2017-07-12 Anal. 14, 143-149 (1999)). In general, attempts have been made to automate SNP and other sequence variation determination methods (Gut, Hum. Mutat. 17, 475-492 (2001)).

Strategies for specific hybridization of sequence variations known in the art are provided by so-called gene mutation-specific amplification. In this detection method, mutation-specific amplification primers have been used in the amplification process. Usually, the 3'-end of the primer has a so-called differential terminal nucleotide residue, and the residue is only one of the target nucleic acids to be detected. Specific mutations are complementary. In this method, nucleotide variants are measured by the presence or absence of DNA products after PCR amplification. The principle of gene mutation-specific amplification is based on the formation of canonical or atypical primer-template complexes at the ends of gene mutation-specific amplification primers. At the end of the precisely matched 3'primer, amplification caused by DNA polymerase is generated, while extension at the end of the mismatched primer is inhibited.

For example, US Patent No. 5,595,890 discloses a gene mutation-specific amplification method and its application, such as a method for detecting clinically relevant point mutations in k-ras tumor genes. In addition, US Patent No. 5,521,301 discloses an allele-specific amplification method for genotype analysis of the ABO blood group system. In contrast, US Patent No. 5,639,611 discloses the use of allele-specific amplification related to the detection of point mutations that cause sickle-type anemia. However, gene mutation-specific amplification or allele-specific amplification has the problem of low selectivity, so more complicated, time- and cost-intensive optimization steps are required.

The method for detecting sequence variation, polymorphism, and focus on point mutations requires gene-specificity when the sequence variation to be detected is inadequate compared with the dominant variation of the same nucleic acid fragment (or the same gene) Amplification (or gene mutation specific amplification).

For example, this occurs when sporadic tumor cells are detected in body fluids such as blood, serum, or plasma by specific amplification of gene mutations (US Patent No. 5,496,699). To this end, DNA is first separated from body fluids such as blood, serum, or plasma, and DNA consists of insufficient sporadic tumor cells and excessive non-proliferating cells. Therefore, in the presence of excess wild-type DNA, important mutations in tumor DNA in the k-ras gene should be detected from multiple replications.

All methods disclosed in the prior art for the specific amplification of gene mutations, although using 3'-differential nucleotide residues, even if the target nucleic acid does not completely match the sequence variant to be detected, the appropriate In the presence of DNA polymerase, it has the disadvantage of lower primer extension. In particular, when a specific sequence variant is detected by an excess of background nucleic acid containing another sequence variant, a false positive result may result. Another disadvantage of the known method is the need to use 3'-terminal differential oligonucleotide residues. The disadvantages of this PCR-based method are mainly due to the unsuitability of the polymerase used in the method for sufficiently distinguishing mismatched bases. Therefore, it is not yet possible to obtain clear information about the existence of mutations directly by PCR. To date, additional time and cost-intensive purification and assay methods are needed to clearly diagnose mutations. Therefore, a new method to increase the specificity of gene mutation specificity or allele-specific PCR amplification will greatly affect the reliability and potency of direct gene mutation or SNP analysis by PCR.

Therefore, there is a need to continuously study DNA polymerases with increased specificity of gene mutations and optimal reaction buffers that can mix the multiple substances that enable the DNA polymerases to perform their functions.

The present inventors are devoted to the development of a novel DNA polymerase capable of improving the selectivity of gene mutation-specific PCR amplification and a reaction buffer used to increase its activity, and the results confirmed the induction of amino acid residues at specific positions of Taq polymerase During mutation, the specificity of gene mutation increases significantly. When the concentration of KCl, (NH ₄ ) ₂ SO ₄ and/or TMAC (Tetra methyl ammonium chloride) in the PCR buffer composition is controlled, The activity of the DNA polymerase with increased gene mutation specificity is enhanced, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a PCR buffer composition for enhancing DNA polymerase activity with increased specificity of gene mutations.

Another object of the present invention is to provide a PCR kit for detecting gene mutations or SNPs comprising the PCR buffer composition.

Another object of the present invention is to provide a method for detecting at least one gene mutation or SNP in vitro from more than one template using the PCR kit.

In order to achieve the stated object, the present invention provides a PCR buffer composition for enhancing DNA polymerase activity with increased gene mutation specificity, wherein the composition contains 25 to 100 mM KCl; 1 to 15 mM (NH ₄ ) ₂ SO ₄ ; final pH is 8.0 to 9.0.

In an embodiment of the invention, the KCl concentration is 60 to 90 mM.

In yet another embodiment of the present invention, the (NH ₄ ) ₂ SO ₄ concentration is 2.5 to 8 mM.

In still another embodiment of the present invention, wherein the KCl concentration is 70 to 80 mM, and the (NH ₄ ) ₂ SO ₄ concentration is 4 to 6 mM.

The present invention provides a PCR buffer composition for enhancing DNA polymerase activity with increased gene mutation specificity, wherein the PCR buffer composition further comprises 5 to 80 mM TMAC (Tetra methyl ammonium chloride) .

In an embodiment of the invention, the KCl concentration is 40 to 90 mM.

In still another embodiment of the present invention, wherein the (NH ₄ ) ₂ SO ₄ concentration is 1 to 7 mM.

In another embodiment of the present invention, wherein the TMAC (Tetra methyl ammonium chloride) concentration is 15 to 70 mM, the KCl concentration is 50 to 80 mM, and the (NH ₄ ) ₂ SO ₄ concentration 1.5 to 6 mM.

As in another embodiment of the present invention, wherein the PCR buffer composition further comprises Tris. Cl and MgCl ₂ .

The invention provides a PCR kit for detecting gene mutation or SNP containing the PCR buffer composition.

In still another embodiment of the present invention, wherein the PCR kit may further comprise a DNA polymerase of Taq polymerase consisting of the amino acid sequence of SEQ ID NO: 1 (base sequence of SEQ ID NO: 6), The DNA polymerase may comprise (a) substitution of the 507th amino acid residue in the amino acid sequence of SEQ ID NO: 1; and (b) 536th in the amino acid sequence of SEQ ID NO: 1 Substitution of amino acid residues, substitution of the 660th amino acid residue, or substitution of the 536th and 660th, ie, two amino acid residues.

In still another embodiment of the present invention, wherein the substitution of the 507th amino acid residue is glutamic acid (E) replaced by lysine (K), the substitution of the 536th amino acid residue It is that arginine (R) is replaced by lysine (K), and the substitution of the 660th amino acid residue is that arginine (R) is replaced by valine (V).

In yet another embodiment of the present invention, wherein the PCR kit further comprises: (i) nucleoside triphosphate; (ii) quantitative reagents that bind to double-stranded DNA; (iii) polymerase blocking antibody; (iv) One or more control values or control sequences; (v) one or more templates.

The invention also provides a detection method for detecting at least one gene mutation or SNP in vitro from more than one template by using the PCR kit.

Compared with the prior art, the present invention has the following beneficial effects: The present invention provides an optimal PCR buffer composition that enables DNA polymerases with increased gene mutation specificity to exert their functions, and is specific to those with increased gene mutations When used together, the DNA polymerase significantly improves the activity of the DNA polymerase and can achieve reliable gene mutation-specific amplification. In addition, the kit containing the PCR buffer composition of the present invention and/or DNA polymerase with increased gene specificity can effectively detect gene mutations or SNPs, and thus can be effectively used for medical diagnosis of diseases and recombinant DNA the study.

The following detailed description of the non-limiting embodiments makes the other features, objects, and advantages of the present invention more obvious:

1 is a description of the preparation process of Taq DNA polymerase containing R536K, R660V and R536K/R660V mutations, respectively, (a) is a schematic diagram of fragment PCR and overlapping PCR, (b) shows the electrophoresis of amplified products in fragment PCR As a result of confirmation, (c) shows the result of confirming the amplified product by electrophoresis after amplifying the full length by overlapping PCR.

Fig. 2 shows the results of electrophoretic confirmation of the pUC19 vector subjected to SAP treatment and the purified overlapping PCR product of Fig. 1(c) after being decomposed with the restriction enzyme EcoRI/XbaI for extracting the gel.

3 is a schematic diagram of fragment PCR and overlap PCR in the preparation of Taq DNA polymerase containing E507K, E507K/R536K, E507K/R660V, and E507K/R536K/R660V mutations, respectively.

Fig. 4 shows the results of electrophoresis confirmation of the pUC19 vector subjected to SAP treatment and the purified overlapping PCR product of Fig. 3 after being decomposed with the restriction enzyme EcoRI/XbaI in order to extract the gel.

FIG. 5 is a schematic diagram showing the process of preparing a PCR template by collecting oral epithelial cells.

6 is a graph showing the results of AS-qPCR of rs1408799 using the E507K/R536K, E507K/R660V, and E507K/R536K/R660V Taq polymerases of the present invention; as a control group, Taq polymerase including the E507K mutation was used.

7 is a graph showing the results of AS-qPCR performed on rs1015362 using E507K/R536K, E507K/R660V, and E507K/R536K/R660V Taq polymerase of the present invention; as a control group, Taq polymerase including E507K mutation was used.

8 is a graph showing the results of AS-qPCR of rs4911414 using E507K/R536K, E507K/R660V, and E507K/R536K/R660V Taq polymerases of the present invention; as a control group, Taq polymerase including E507K mutation was used.

9 is a graph showing the delay effect of mismatch amplification based on the change in KCl concentration in the reaction buffer using the E507K, E507K/R536K, E507K/R660V, and E507K/R536K/R660V Taq polymerases of the present invention.

FIG. 10 shows the results of confirming the PCR product by electrophoresis after fixing the concentration of (NH ₄ ) ₂ SO ₄ and changing the concentration of KCl to determine the optimal KCl concentration in the reaction buffer.

11 is to determine the optimal reaction buffer ₍₄ NH) ₄ SO ₂ concentration, and changes in the concentration of KCl fixed (NH _{4) 2} SO ₄ concentration after the amplification, the PCR product to confirm the results by electrophoresis.

FIG. 12 is a graph showing the delay effect of mismatch amplification confirmed according to the change in (NH ₄ ) ₂ SO ₄ concentration in the reaction buffer.

13 is a graph showing the effect of fixing the concentration of KCl and (NH ₄ ) ₂ SO ₄ in the reaction buffer, and confirming the delay effect of mismatch amplification according to the change in TMAC concentration.

FIG. 14 is a graph showing the effect of fixing the TMAC and (NH ₄ ) ₂ SO ₄ concentration in the reaction buffer, and confirming the delay effect of mismatch amplification based on the change in KCl concentration.

The present invention will be described in detail below.

As described above, in order to improve the shortcomings of the gene mutation-specific amplification method disclosed in the prior art, it is necessary to continue to develop a DNA polymerase with increased gene mutation specificity and a mixed variety that enables the DNA polymerase to exert its function The optimal reaction buffer of the substance, the development of these methods will have a great influence on the reliability and potency of direct gene mutation or SNP analysis by PCR. The present inventors are devoted to the development of a novel DNA polymerase capable of improving the selectivity of gene mutation-specific PCR amplification and a reaction buffer used to increase its activity, and the results confirmed the induction of amino acid residues at specific positions of Taq polymerase During mutation, the specificity of gene mutation increases significantly. When the concentration of KCl, (NH ₄ ) ₂ SO ₄ and/or TMAC (Tetra methyl ammonium chloride) in the PCR buffer composition is controlled, the The activity of the DNA polymerase with increased gene mutation specificity is enhanced, thereby completing the present invention.

The present invention provides an optimal PCR buffer composition that enables a DNA polymerase with increased gene mutation specificity to perform its function, and is used together with a DNA polymerase with increased gene mutation specificity to significantly improve the DNA polymerase The activity can achieve reliable gene mutation-specific amplification. In addition, the kit containing the PCR buffer composition of the present invention and/or DNA polymerase with increased gene specificity can effectively detect gene mutations or SNPs, and thus can be effectively used for medical diagnosis of diseases and recombinant DNA the study.

The terminology used in this article will be explained below.

As used herein, "amino acid" refers to any monomer unit that can be introduced into a peptide, polypeptide, or protein. As used herein, the term “amino acid” includes the following 20 natural or genetically encoded α -amino acids: alanine (Ala or A), arginine (Arg or R), aspartame (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), amphetamine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) , Valine (Val or V).

Amino acids are usually organic acids and may contain substituted or unsubstituted amine groups, substituted or unsubstituted carboxyl groups, and at least one side chain (chain) or group (group), or any analogs of these groups . Exemplary side chains include mercapto, seleno, sulfonyl, alkyl, aryl, acetyl, keto, azido, hydroxy, hydrazino, cyano, halo, hydrazide, alkenyl, alkynyl , Ether group, boric acid ester, boric acid group, phosphorous dioxygen group, phosphine sulfonic acid group, phosphine group, heterocyclic ring, enone, imine, aldehyde group, ester group, thioacid, hydroxylamine or any of these groups combination.

Other exemplary amino acids include, but are not limited to: amino acids containing photoactivatable crosslinking agents, metal-bound amino acids, spin-labeled amino acids, fluorescent amino acids, metal-containing amino acids, Amino acids with novel functional groups, amino acids covalently or non-covalently interacting with other molecules, photocageable and/or photoisomerizable amino acids, radioactive amino acids, containing biotin or biotin Amino acids of analogs, glycosylated amino acids, amino acids modified with other carbohydrates, amino acids containing polyethylene glycols or polyethers, amino acids substituted with heavy atoms, chemically decomposable and/or Photolytic amino acids, carbon-linked sugar-containing amino acids, redox-active amino acids, sulfur-containing carboxylic acid-containing amino acids, and amino acids containing at least one toxic moiety.

In the DNA polymerase of the present invention, the term "mutant" refers to a recombinant polypeptide containing one or more amino acid substitutions relative to a naturally occurring or unmodified DNA polymerase.

The term "thermostable polymerase" (referring to a thermostable enzyme) means having sufficient heat resistance, maintaining sufficient activity to achieve the subsequent polynucleotide extension reaction, and the time course required to achieve denaturation of double-stranded nucleic acids It will not be irreversibly denatured (inactivated) during the medium temperature treatment. As used herein, it is suitable for cycling temperatures in reactions such as PCR. Irreversible here refers to the complete loss of permanent enzyme activity. For thermostable polymerases, enzyme activity refers to catalyzing the combination of nucleotides in a suitable manner to form complementary polynucleotide extension products on the template nucleic acid strand. Thermostable bacteria-derived thermostable DNA polymerases include, for example, from Thermatoga maritime , Thermus acquaticus , Thermus thermophilus , Thermusflavus ), Thermus filiformus , Thermus filiformus , Thermus sp.17 , Thermus sp. Z05 , Thermus caldophilus , Bacillus caldotenax , New Apollo thermus ( Thermotoga neapolitana ), the DNA polymerase of Thermosipho africanus ( Thermosipho africanus ).

The term "thermal activity" refers to an enzyme that maintains its catalytic properties at temperatures (ie, 45-80°C) commonly used in the reverse transcription or binding/extension phases of RT-PCR and/or PCR reactions. Thermostable enzymes are enzymes that are not irreversibly inactivated or denatured when treated with the high temperatures required for denaturation of nucleic acids. The thermally active enzyme may or may not be thermally stable. Thermally active DNA polymerases can include, but are not limited to, DNA or RNA dependent on thermophilic or mesophilic species.

The term "host cell" refers to single-cell prokaryotic and eukaryotic organisms (such as bacteria, yeast, and actinomycetes) and single cells from higher plants or animals used for cultivation in cell culture media.

The term "vector" is a DNA molecule such as plastid, bacteriophage, artificial chromosome, etc. capable of being replicated and capable of transferring genes and the like to foreign DNA to recipient cells. "Plastid", "carrier" or "plastidic carrier" are used interchangeably herein.

The term "nucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in the form of single strands or double strands, and unless Unless otherwise specified, analogs of natural nucleotides may be included.

The term "nucleic acid" or "polynucleotide" refers to polymers of DNA or RNA polymers, or analogs thereof. Nucleic acids can be, for example, chromosomes or chromosome fragments, vectors (such as expression vectors), expression cassettes, naked DNA or RNA polymers, polymerase chain reaction (PCR) products, oligonucleotides, probes or primers, or contain Described substance. The nucleic acid can be, for example, single-stranded, double-stranded, or triple-stranded, but is not limited to any particular length. Unless otherwise specified, a specific nucleic acid sequence includes a complementary sequence or can encode it in addition to any specified sequence.

The term "primer" refers to a polynucleotide that can serve as a starting point for nucleic acid synthesis in the direction of the template when the polynucleotide is under extension initiation conditions. Primers can also be used in a variety of other oligonucleotide-mediated synthesis processes, including de novo RNA and as a promoter for transcription-related processes in test tubes. Primers are usually single-stranded oligonucleotides (eg, oligodeoxyribonucleotides). The appropriate length of the primer is usually in the range of 6 to 40 nucleotides, and the more typical length is in the range of 15 to 35 nucleotides and depends on the intended use. Short primer molecules usually require lower temperatures to form a sufficiently stable hybrid complex with the template. The primer does not need to reflect the correct sequence of the template, but the primer must have sufficient complementarity to hybridize with the template to be extended. In certain embodiments, the term "primer pair" refers to a 3'-antisense primer that includes a 5'sense primer that hybridizes complementary to the 5'end of the nucleic acid sequence to be amplified and a 3'end that hybridizes to the 3'end of the sequence to be amplified Intro group. If necessary, the primer can be labeled by incorporating a label detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include: 32P, fluorescent dyes, electron density reagents, enzymes (usually used for ELISA analysis), biotin or haptens, and proteins that can use antisera or monoclonal antibodies.

The term "5'-nuclease probe" refers to an oligonucleotide comprising at least one luminescent label moiety for performing a 5'-nucleolytic enzyme reaction to detect a target nucleic acid. In some embodiments, for example, the 5'-nucleolytic enzyme probe contains only a single luminescent moiety (eg, fluorescent dye, etc.). In certain embodiments, the 5'-nucleolytic enzyme probe contains a self-complementary region so that the probe can form a hairpin structure under selected conditions. In some embodiments, the 5'-nucleic acid hydrolase probe contains two or more label moieties, one of the two labels is separated or decomposed from the oligonucleotide, and the radiation intensity is increased to be released. In certain embodiments, the 5'-nucleolytic enzyme probe is labeled with two different fluorescent dyes, such as a 5'-end reporter dye and a 3'-end quencher dye or partial label. In some embodiments, the 5'-nuclease probe is relabeled at the end or at one or more positions other than the end. When the probe is intact, energy transfer usually occurs between the two fluorescent materials, so that the fluorescent emission from the reporter dye is quenched by more than a certain amount. During the extension step of the polymerase chain reaction, for example, the 5'-nucleic acid hydrolase probe bound to the template nucleic acid has an activity such that the fluorescent emission of the reporter dye is no longer quenched, such as by Taq polymerase or other polymerization The enzyme's 5'to 3'-nucleolytic enzyme activity breaks down. In some embodiments, the 5'-nucleolytic enzyme probe may be labeled with two or more different reporter dyes and 3'-end quencher dyes or partially.

The term "FRET" or "fluorescence resonance energy transfer" or "forester resonance energy transfer" refers to two or more chromophores, donor chromophore and acceptor chromophore (called quencher) Energy transfer. The donor is usually excited by emitting light of an appropriate wavelength to transfer energy to the acceptor. Receptors usually re-emit moving energy in the form of re-emitting light of different wavelengths. When the receptor is a "dark" quencher, the moving energy is dispersed in a form other than light. Whether a particular fluorescent substance acts as a donor or acceptor depends on the nature of FRET to other members. Commonly used donor-acceptor pairs include FAM-TAMRA pairs. Commonly used quenchers are DABCYL and TAMRA. Commonly used dark quenchers include: BlackHole Quenchers ^TM (BHQ), (Biosearch Technologies, Inc., Novato, Cal.), Iowa Black ^TM (Integrated DNA Tech., Inc., Coralville, Iowa), BlackBerry ^TM Quencher 650 ( BBQ-650) (Berry & Assoc., Dexter, Mich.).

When referring to nucleic acid bases, nucleoside triphosphates or nucleotides, the term "conventional" or "natural" refers to naturally occurring in the polynucleotide (ie, dATP, dGTP, dCTP, dTTP for DNA ), and dITP and 7-deaza-dGTP are often used to replace dGTP, which can be used to replace dATP in DNA synthesis reactions such as sequencing.

When referring to nucleic acid bases, nucleosides, or nucleotides, the term "unconventional" or "modification" includes modifications, derivations of conventional bases, nucleosides, or nucleotides that occur naturally in a particular polynucleotide Thing or the like. Compared with conventional dNTPs, specific unconventional nucleotides are modified at the 2'position of ribose. Therefore, even if the naturally occurring nucleotide of RNA is a ribonucleotide (ie, ATP, GTP, CTP, UTP, collectively referred to as rNTP), the nucleotide has a hydroxyl group at the 2'position of the sugar, and relatively speaking, dNTP does not exist. Therefore, as used herein, ribonucleotides are unconventional nucleotides that serve as substrates for DNA polymerases. As used herein, unconventional nucleotides include but are not limited to compounds used as nucleic acid sequence terminators. Exemplary terminator compounds include, but are not limited to, compounds having a 2', 3'-dideoxy structure, known as dideoxynucleoside triphosphate. Dideoxynucleoside triphosphate ddATP, ddTTP, ddCTP and ddGTP are collectively referred to as ddNTP. Other examples of terminator compounds include 2'-PO4 analogs of ribonucleotides. Other unconventional nucleotides include but are not limited to thiodNTP ([[ α ]-S]dNTP), 5'-[ α ]-borano-dNTP, [ α ]-methyl-phosphonate dNTP, ribose Nucleoside triphosphate (rNTP). Unconventional bases can be radioisotopes such as 32P, 33P or 35S; fluorescent tags; chemiluminescent tags; bioluminescent tags; hapten tags such as biotin; or enzyme tags such as streptavidin or avidin mark. Fluorescent labels may include negatively charged dyes such as the fluorescein family of dyes, or neutrally charged dyes such as the rhodamine family of dyes, or positively charged dyes such as the cyanine family. Dyes of the luciferin family include, for example, FAM, HEX, TET, JOE, NAN, and ZOE. The dyes of the Rhodamine family include Texas Red, ROX, R110, R6G and TAMRA. Various dyes or nucleotides labeled with FAM, HEX, TET, JOE, NAN, ZOE, ROX, R110, R6G, Texas Red, and TAMRA through Perkin-Elmer (Boston, Massachusetts), Applied Biosystems (Foss Special City, California), Invitrogen/Molecular Probes (Eugene, Oregon) market. Cyanine family dyes including Cy2, Cy3, Cy5, and Cy7 are marketed through GE Healthcare UK Limited (Amersham Place, Little Chalfont, Buckinghamshire, England).

The term "distinguishment of mismatch" refers to the use of one or more nucleotides attached to a nucleic acid (eg, covalently) to extend the nucleic acid (eg, a primer or other oligonucleotide) in a template-dependent manner. From mismatches-the ability to distinguish biocatalysts (such as polymerases, ligases, etc.) from sequences that contain completely complementary sequences. The term "distinguishment of mismatches" refers to the fact that extended nucleic acids (such as primers or other oligonucleotides) have mismatches from the 3'end nucleic acid with mismatches-containing (approximately complementary) sequence distinctions compared to the template where the nucleic acid hybridizes The biocatalytic ability of a completely complementary sequence. In some embodiments, the extended nucleic acid contains a mismatch at the 3'end for a completely complementary sequence. In some embodiments, the extended nucleic acid contains a mismatch at the second (N-1) 3'-end and/or N-2 position of the terminal for a completely complementary sequence.

Unless otherwise stated, all technical and scientific terms used in this specification have the same meaning as commonly understood by those of ordinary skill in the art.

The present invention relates to a PCR buffer composition for enhancing DNA polymerase activity with increased gene mutation specificity, the composition comprising 25 to 100 mM KCI; 1 to 15 mM (NH ₄ ) ₂ SO ₄ ; the final pH is 8.0 To 9.0.

The polymerase used in PCR should use the best reaction buffer mixed with various substances in order to exert its function. The reaction buffer usually contains a factor that stabilizes the pH, metal ions as a cofactor and a stabilizing component that prevents denaturation of the polymerase.

The KCl can be used as an essential factor for stabilizing enzymes, helping primers pair with target DNA. In the present invention, the optimal concentration is determined by adjusting the concentration of KCl in the reaction buffer to confirm that the amplification caused by the mismatch is delayed to the greatest extent without reducing the high cation concentration of the amplification efficiency caused by the matching.

Using E507K, E507K/R536K, E507K/R660V, and E507K/R536K/R660V Taq polymerases, respectively, to confirm the mismatch amplification delay effect due to the change in KCl concentration in the reaction buffer, the results are shown in Figure 9, for E507K/R536K /R660V Taq polymerase, even if there is no KCl in the reaction buffer, its mismatch amplification delay effect is excellent; for E507K/R536 and E507K/R660V, at 50mM; as a control group E507K, at 100mM, mismatch amplification The delay effect is excellent. As a result, for the KCl concentration threshold, E507K/R536K/R660V Taq polymerase is the lowest, and E507K/R536K and E507K/R660V are lower than E507K.

In order to further deduce the appropriate KCI concentration, E507K/R536K/R660V Taq polymerase was used to fix the (NH ₄ ) ₂ SO ₄ concentration in the reaction buffer to a certain value, and amplification was performed using multiple KCl concentrations. As a result of confirming the amplified product by electrophoresis, as shown in FIG. 10, it was confirmed that the appropriate KCI concentration was 75 mM.

Therefore, the concentration of KCl in the PCR reaction buffer composition of the present invention may be 25-100 mM, preferably 60-90 mM, more preferably 70-80 mM, and most preferably 75 mM.

When the concentration of KCl is lower than 25mM, it has no effect on the amplification of the general target, but the difference between the amplification caused by the matched primer and the amplification caused by the mismatched primer can be reduced. When the concentration is higher than 100mM, the general The problem is that the amplification efficiency of the target decreases.

For the PCR reaction buffer composition, the (NH ₄ ) ₂ SO ₄ is a necessary cofactor for enzyme activity, and is used together with Tris to increase the activity of the polymerase. In one embodiment of the present invention, based on the above derivation results, the KCl concentration in the reaction buffer is fixed at 75 mM, and the concentration of (NH ₄ ) ₂ SO ₄ is varied from 2.5 mM to 25 mM to confirm the appropriate ( NH ₄ ) ₂ SO ₄ concentration.

As a result, as shown in FIG. 11, the amplification product was confirmed at a concentration of (NH ₄ ) ₂ SO ₄ from 2.5 mM to 15 mM, and an appropriate concentration of (NH ₄ ) ₂ SO ₄ was 5 mM.

Further, when the concentration of (NH ₄ ) ₂ SO ₄ was close to 5 mM (2.5 mM, 5 mM, and 10 mM, respectively), AS-qPCR was performed. The results are shown in FIG. 12, although the difference in Ct value was the largest at 10 mM. It was confirmed that Ct was slightly delayed in the matched amplification, and the peak was dumped downward, so it was determined that the appropriate concentration of (NH ₄ ) ₂ SO ₄ was 5 mM.

Therefore, the concentration of (NH ₄ ) ₂ SO ₄ in the PCR reaction buffer composition of the present invention may be 1-15 mM, preferably 2.5-8 mM, more preferably 4-6 mM, and most preferably 5 mM.

When the concentration of (NH ₄ ) ₂ SO ₄ is less than 1 mM, it has no effect on the amplification of the general target, but the difference between the amplification caused by the matched primer and the amplification caused by the mismatched primer can be reduced. When the concentration is high At 15 mM, there is a problem that the amplification efficiency of the general target decreases.

Therefore, the optimal PCR buffer composition of the present invention may contain 70 to 80 mM KCl; 4 to 6 mM (NH ₄ ) ₂ SO ₄ ; the final pH is 8.0 to 9.0.

The PCR buffer composition of the present invention may further contain 5 to 80 mM of TMAC (Tetra methyl ammonium chloride).

TMAC is commonly used to reduce amplification caused by mismatches or to improve stringency of hybridization reactions. In an embodiment of the present invention, based on the above derivation results, the KCl concentration in the reaction buffer is fixed at 75 mM, the (NH ₄ ) ₂ SO ₄ concentration is fixed at 5 mM, and the TMAC concentration is varied from 0 mM to 80 mM. To confirm the appropriate TMAC concentration.

As a result, as shown in FIG. 13, it was confirmed that an appropriate TMAC concentration was 70 mM for E507K/R536K Taq polymerase and 25 mM for E507K/R536K/R660V Taq polymerase. Further, the TMAC concentration was fixed at 25 mM, the (NH ₄ ) ₂ SO ₄ concentration was fixed at 2.5 mM, and the KCl concentration was adjusted to 20, 40, 60, and 80 mM, respectively, and the results were amplified. As shown in FIG. 14, it was confirmed that for SNP rs1015362 and rs4911414, the appropriate KCl concentration is 60mM.

When the TMAC concentration exceeds 80 mM, the amplification efficiency decreases, so it is preferable to use TMAC within the range.

Therefore, when the PCR buffer composition of the present invention contains 5-80 mM TMAC, the concentration of KCl may be 40-90 mM, preferably 50-80 mM, and the concentration of (NH ₄ ) ₂ SO ₄ may be 1-7 mM, It is preferably 1.5-6 mM.

Therefore, when TMAC is included, the optimal PCR buffer composition of the present invention may include TMAC of 15 to 70 mM; KCl of 50 to 80 mM; (NH ₄ ) ₂ SO _{4 of} 1.5 to 6 mM; and a final pH of 8.0 to 9.0.

The PCR buffer composition of the present invention may further include Tris. Cl and MgCl ₂ may further include Tween 20 and bovine serum albumin (BSA).

The present invention provides a PCR kit for detecting gene mutations or SNPs comprising the PCR buffer composition.

The PCR kit of the present invention may further include a DNA polymerase of Taq polymerase consisting of the amino acid sequence of SEQ ID NO: 1, and the DNA polymerase may include (a) the amino acid sequence of SEQ ID NO: 1 Substitution of the 507th amino acid residue in; and (b) the substitution of the 536th amino acid residue in the amino acid sequence of SEQ ID NO: 1 and the 660th amino acid residue or The 536th and 660th are the substitution of two amino acid residues.

According to another preferred embodiment of the present invention, the substitution of the 507th amino acid residue is the substitution of glutamic acid (E) by lysine (K), and the substitution of the 536th amino acid residue The substitution is substitution of arginine (R) with lysine (K), and the substitution of the 660th amino acid residue is substitution of arginine (R) with valine (V).

The PCR kit of the present invention may include any reagents or other elements known to those skilled in the art during the primer extension process.

According to another preferred embodiment of the present invention, the PCR kit includes more than one matched primer, more than one mismatched primer, or contains more than one matched primer and more than one mismatched primer, the more than one The matched primer and one or more mismatched primers hybridize to the target sequence, and the mismatched primers contain non-canonical nucleotides from the 3′ end to the 7th base position for the hybridized target sequence .

The PCR kit of the present invention may further comprise: (i) nucleoside triphosphate; (ii) quantitative reagents that bind to double-stranded DNA; (iii) polymerase blocking antibody; (iv) one or more control values or Control sequence; (v) one or more templates.

The "Taq polymerase" is a heat-resistant DNA polymerase named after the name of the thermophilic bacterium Thermus aquaticus and was originally isolated from the bacterium. The aquatic thermophilic bacteria are bacteria that inhabit hot springs and hot water spouts, and Taq polymerase is confirmed to be an enzyme that can withstand the protein denaturation conditions (high temperature) required during the PCR process. The optimal activity temperature of Taq polymerase is 75-80℃, the half-life is more than 2 hours at 92.5℃, 40 minutes at 95℃, 9 minutes at 97.5℃, and 10 seconds at 72℃ It can replicate 1000 base pairs of DNA. This lacks the 3→'5' exonuclease correction activity, and out of 9000 nucleotides, the error rate is measured in about 1 of them. For example, when heat-resistant Taq is used, PCR can be performed at a high temperature (60°C or higher). For Taq polymerase, the amino acid sequence shown in SEQ ID NO: 1 was used as a reference sequence.

In the present invention, in the amino acid sequence of SEQ ID NO: 1, the Taq polymerase in which the glutamic acid (E) of the 507th amino acid residue is replaced with lysine (K) is named "E507K "(SEQ ID NO: 2, the base sequence is SEQ ID NO: 7); in the amino acid sequence of SEQ ID NO: 1, the glutamic acid (E) of the 507th amino acid residue is replaced by Lai Amino acid (K), Taq polymerase where arginine (R) of the 536th amino acid residue was replaced with lysine (K) was named "E507K/R536K" (SEQ ID NO: 3, base The base sequence is SEQ ID NO: 8); in the amino acid sequence of SEQ ID NO: 1, the glutamic acid (E) of the 507th amino acid residue is replaced with lysine (K), the 660th Taq polymerase with amino acid residues of arginine (R) replaced by valine (V) was named "E507K/R660V" (SEQ ID NO: 4, base sequence is SEQ ID NO: 9) ; Finally, in the amino acid sequence of SEQ ID NO: 1, the glutamic acid (E) of the 507th amino acid residue was replaced with lysine (K), the refined of the 536th amino acid residue The amino acid (R) was replaced with lysine (K), and the 660th amino acid residue of arginine (R) was replaced with valine (V). Taq polymerase was named "E507K/R536K /R660V" (SEQ ID NO: 5, the base sequence is SEQ ID NO: 10).

According to an embodiment of the present invention, the DNA polymerase distinguishes between matched primers and mismatched primers, the matched primers and mismatched primers hybridize with the target sequence, and the mismatched primers, for the hybridized target sequence, in its 3 The'end may contain atypical nucleotides.

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

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

The "non-canonical nucleotide" or "non-complementary nucleotide" refers to AC, AG, GU, GT, except Watson-Crick base pairs. TC, TU, A, GG, TT, UU, CC, CU.

According to a preferred embodiment of the present invention, the amplification of the target sequence including the matching primers, compared with the amplification of the target sequence including the mismatching primers, the DNA polymerase may exhibit a lower Ct value .

For example, by covalently linking one or more nucleotides to a primer, the DNA polymerase can extend the matched primer more efficiently than a primer that is mismatched in a target sequence-dependent manner. Here, higher efficiency can be observed by the following example, as in RT-PCR, the Ct value of the matched primer is lower than that of the mismatched primer. The difference in Ct value between the matched primer and the mismatched primer is 10 or higher, preferably 10-20, or there is no amplicon synthesis caused by the mismatched primer.

For example, in the first reaction, matched forward primers and reverse primers are used. In the second reaction, using the same experimental settings, using mismatched forward primers and matched reverse primers, the product formed by standard PCR, the first One response is greater than the second response.

The Ct (threshold cross-cycle) value represents the DNA quantification method for quantitative PCR, depending on the fluorescence plotted on the number of log phase cycles. The threshold for DNA-based fluorescence detection is set at least slightly higher than the background. The number of cycles where the fluorescence exceeds the threshold is called Ct or Cq (quantification cycle) according to the MIQE criteria. The Ct value for a given reaction is defined as the number of cycles of fluorescent emission crossing a fixed limit. For example, SYBR Green I and fluorescent probes can be used for real-time PCR of template DNA quantification. The fluorescence from the sample was collected every cycle during PCR and plotted against the number of cycles. The initial template concentration is inversely proportional to the initial display time of the fluorescent signal. The higher the template concentration, the earlier the signal appears (appears at a lower number of cycles).

The present invention also relates to a nucleic acid sequence encoding the above DNA polymerase and a vector and host cell containing the nucleic acid sequence. Various vectors can be prepared using the nucleic acid encoding the DNA polymerase of the present invention. Any vector including replicons and control sequences derived from species interchangeable with the host cell can be used. The vector of the present invention may be an expression vector and includes a nucleic acid region that controls transcription and translation operably linked to the nucleic acid sequence encoding the DNA polymerase of the present invention. Regulatory sequence refers to the DNA sequence required to express the operably linked coding sequence in a particular host organism. For example, regulatory sequences suitable for prokaryotes include promoters, any operating sequences, and alc ribosome binding sites. In addition, the vector may contain "Positive Retroregulatory Element (PRE)" to enhance the half-life of transcribed mRNA. Nucleic acid regions that control transcription and translation are generally suitable for host cells used to express polymerases. Various types of suitable expression vectors and suitable regulatory sequences for various host cells are known in the art. Generally, transcription and translation control sequences can include, for example, promoter sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or promoter sequences. In typical embodiments, regulatory sequences include promoters and transcription initiation and termination sequences. The vector usually also contains a multi-site adapter region containing several restriction sites for insertion of foreign DNA. In certain embodiments, "fusion tags" are used to facilitate purification, and if necessary, tags/markers are subsequently removed (eg, "His-tag"). However, when thermally active and/or thermostable proteins are purified from mesophilic hosts (eg E. coli) using a "heating step", these are usually unnecessary. Standard recombinant DNA techniques are used to prepare suitable vectors containing the DNA replication sequences, regulatory sequences, phenotypic selection genes, and the mutant polymerase of interest. As is known in the art, isolated plastids, viral vectors, and DNA fragments are broken down and sheared and ligated together in a specific order to produce the desired vector.

In a preferred embodiment of the present invention, the expression vector contains a selection marker gene for selecting transformed host cells. Selection genes are known in the art and will vary depending on the host cell used. Suitable selection genes may include genes encoding ampicillin and/or tetracycline resistance, whereby cells that culture these vectors can be transformed in the presence of these antibiotics.

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

Prokaryotes are used as host cells in the initial transplantation step of the present invention. For the rapid preparation of large amounts of DNA, for the preparation of single-stranded DNA templates for generating site-oriented mutations, for the simultaneous screening of many mutants, and for the DNA sequencing of the resulting mutants, this is particularly useful. Suitable host cells for prokaryotic cells include E. coli E. coli K12 strain 94 (ATCC No. 31, 446), E. coli E. coli strain W3110 (ATCC No. 27, 325), E. coli E. coli K12 strain DG116 ( ATCC No. 53, 606), E. coli X1776 (ATCC No. 31, 537) and E. coli B; many different strains of E. coli such as HB101, JM101, NM522, NM538, NM539 , And other species, including Bacillus subtilis ( Bacillus subtilis ), other Enterobacteriaceae such as Salmonella typhimurium , Serratia marcesans , and various Pseudomonas Prokaryotes of the species ( Pseudomonas sp. ) can be used as hosts. Typically, plastids used to transform E. coli include pBR322, pUCI8, pUCI9, pUCI18, pUC 119, and Bluescript M13. However, many other suitable carriers can also be used.

The present invention also provides a method for preparing a DNA polymerase, which includes the following steps: a step of culturing the host cell; and a step of separating the DNA polymerase from the culture and its culture supernatant.

The DNA polymerase of the present invention is prepared by culturing a host cell transformed with an expression vector containing a nucleic acid sequence encoding DNA polymerase under appropriate conditions to induce or cause expression of the DNA polymerase. Methods for culturing transformed host cells under conditions suitable for protein expression are known in the art. A suitable host cell for preparing a polymerase with a plastid vector containing a λ pL promoter is E. coli strain DG116 (ATCC No. 53606). Depending on the expression, the polymerase can be collected and isolated.

Once purified, the mismatch discrimination of the DNA polymerase of the invention can be determined. For example, the mismatch discrimination activity is measured by comparing the amplification of a target sequence with a perfect match of a primer and the amplification of a single base mismatched target sequence at the 3'end of the primer. Amplification can be detected instantly, for example by using TaqManTM probes. The ability of the polymerase to distinguish between two target sequences can be estimated by comparing the Ct of the two reactions.

The invention provides a method for detecting at least one gene mutation or SNP in vitro from more than one template by using the PCR kit.

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

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

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

The melting temperature curve analysis can be performed in real-time PCR equipment such as ABI 5700/7000 (96-well format) or ABI 7900 (384-well format) containing software (onboard software SDS 2.1). Alternatively, melting temperature curve analysis may be performed as an endpoint analysis.

"Dye that binds double-stranded DNA" or "dual-strand specific dye" can be used when it has high fluorescence when it is bound to double-stranded DNA compared to when it is not bound to double-stranded DNA. Examples of such dyes include SOYTO-9, SOYTO-13, SOYTO-16, SOYTO-60, SOYTO-64, SYTO-82, bromoethane (EtBr), SYTOX Orange, TO-PRO-1, SYBR Green I, TO-PRO-3 or EvaGreen. In addition to EtBr and EvaGreen (Quiagen), these dyes have been used for immediate application testing.

The in vitro SNP detection method of the present invention can be blocked by real-time PCR, agarose gel analysis after standard PCR, gene mutation-specific amplification or allele-specific amplification by real-time PCR, and four-primer amplification are blocked Mutation system PCR or isothermal amplification.

The so-called "standard PCR" is a technique known to those of ordinary skill for amplifying single or multiple copies of DNA or cDNA. Almost all PCRs use Taq polymerase or thermostable DNA polymerase such as Klen Taq. DNA polymerase enzymatically assembles new strands of DNA from nucleotides by using single-stranded DNA as a template and using oligonucleotides (primers). Amplicons generated by PCR can be analyzed on agarose gel.

The "instant PCR" can monitor its process in real time when performing PCR. Therefore, data is collected throughout the PCR process, not at the end of the PCR. In real-time PCR, the characteristic of the reaction is the time point when the amplification is first detected in the cycle, rather than the target amount accumulated after a fixed number of cycles. Quantitative PCR is mainly performed using two methods, dye-based detection and probe-based detection.

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

The "gene mutation-specific amplification or allele-specific amplification by real-time PCR" can detect gene mutations or SNPs in an efficient manner. Unlike most other methods for detecting gene mutations or SNPs, pre-amplification of target genetic material is not necessary. ASA combines amplification and detection in a single reaction based on the distinction between matched and mismatched primer/target sequence complexes. The increase in amplified DNA during the reaction can be monitored in real time by the increase in the fluorescent signal caused by a dye such as SYBR Green I, which emits light when bound to the double-stranded DNA. Through real-time PCR-based genetic mutation-specific amplification or allele-specific amplification, there is a delay in the fluorescent signal or the absence of the signal when a mismatch occurs. In gene mutation or SNP testing, this provides information about whether there is a gene mutation or SNP.

The "four primer amplification hindered mutation system PCR" amplifies wild-type and mutant alleles together with control fragments in a single-tube PCR reaction. Non-allele-specific control amplicons are amplified by two universal (outer) primers flanking the mutation region. The design direction of the two allele-specific (internal) primers is opposite to the universal primer, and the wild-type and mutant amplicons are amplified together with the universal primer. As a result, the two allele-specific amplicons are asymmetrical with respect to the universal (external) primer because of the mutation position, and therefore have different lengths, and can be easily separated by standard gel electrophoresis. The control amplicon provides an internal control for false negatives and failed amplification, and at least one of the two allele-specific amplicons is always present in the four primer amplification hindered mutation system PCR.

The "isothermal amplification" refers to that the amplification of nucleic acid is performed at a lower temperature without relying on a thermal cycler, and the preferred temperature does not need to be changed during amplification. The temperature used in the isothermal amplification can be between room temperature (22-24°C) and about 65°C, or normal temperature of about 60-65°C, 45-50°C, 37°C-42°C or 22-24°C . Isothermal amplification products can be analyzed by gel electrophoresis, ELISA, ELOSA (Enzyme linked oligosorbent assay), real-time PCR, ECL (improved chemiluminescence), analysis of RNA, DNA and protein or turbidity Wafer-based capillary electrophoresis device bioanalyzer (bioanalyzer) to detect.

In one embodiment of the present invention, using E507K/R536K, E507K/R660V or E507K/R536K/R660V Taq polymerase to confirm whether the mismatched primer extension ability of the template containing SNP (rs1408799, rs1015362 and/or rs4911414) cut back.

As a result, as shown in FIGS. 6 to 8, compared with E507K Taq polymerase, the amplification delay caused by mismatched primers of the E507K/R536K, E507K/R660V, or E507K/R536K/R660V Taq polymerase can be confirmed, and its effect It is most obvious in E507K/R536K/R660V Taq polymerase.

This confirms that the DNA polymerase of the present invention has a higher selectivity for mismatch extension than conventional Taq polymerase (E507K). Therefore, it is expected that the DNA polymerase of the present invention can be effectively applied to medical diagnosis of diseases and research of recombinant DNA.

According to another preferred embodiment of the present invention, the PCR kit includes more than one matched primer, more than one mismatched primer, or contains more than one matched primer and more than one mismatched primer, the more than one The matched primer and one or more mismatched primers hybridize to the target sequence, and the mismatched primers contain non-canonical nucleotides from the 3′ end to the 7th base position for the hybridized target sequence .

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

The PCR kit of the present invention also includes (i) one or more buffers; (ii) quantitative reagents that bind to double-stranded DNA; (iii) polymerase blocking antibody; (iv) one or more control values or control sequences ; (V) One or more templates; as shown.

The present invention will be described in detail below through examples. These embodiments are exemplary illustrations of the present invention, and those with general knowledge in the art should understand that the scope of patent application of the present invention is not limited to the embodiments.

Example 1 Inducing Taq polymerase mutation

1-1. Fragment PCR

In this example, the Taq DNA polymerase (hereinafter referred to as "R536K") in which the arginine acid of the 536th amino acid residue in the amino acid sequence of SEQ ID NO: 1 was substituted with lysine was prepared according to the following method ), Taq DNA polymerase with the 660th amino acid residue of arginine replaced by valine (hereinafter referred to as "R660V"), and the 536th amino acid residue of arginine with lysine Taq DNA polymerase (hereinafter referred to as "R536K/R660V") in which the arginine of the 660th amino acid residue is substituted with valinate is substituted.

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

The PCR product was confirmed by electrophoresis. As shown in FIG. 1(b), the band of each fragment was confirmed to confirm that the target fragment had been amplified.

1-2. Overlap PCR

Using each fragment amplified in 1-1 as a template, the primers (Eco-F and Xba-R primers) were used to amplify the full length at both ends. The reaction conditions are shown in Table 3 and Table 4.

As a result, as shown in FIG. 1(c), it was confirmed that the "R536K", "R660V", and "R536K/R660V" Tag polymerases were amplified.

1-3. Ligation

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

For the insert, the overlapping PCR products of Examples 1-2 were purified and decomposed with the restriction enzyme EcoRI/XbaI at 37°C for 3 hours under the conditions shown in Table 7, and then combined with the prepared vector The gel extraction was performed together (Figure 2).

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

Example 2 Introduction of E507K mutation

2-1. Fragment PCR

The activity of the "R536K", "R660V" and "R536K/R660V" Taq DNA polymerase prepared in Example 1 was tested, and the results confirmed that the activity decreased (data not shown), respectively for "R536K" and "R660V" And "R536K/R660V" introduced the E507K mutation (in the amino acid sequence of SEQ ID NO: 1, the glutamic acid of the 507th amino acid residue was replaced by lysine), as a control group, in the wild type (WT ) Taq DNA polymerase also introduces E507K mutation. The preparation method of Taq DNA polymerase introducing the E507K mutation is the same as in Example 1.

Taq DNA polymerase fragments (F6 to F7) were amplified by PCR using the mutation-specific primers shown in Table 9, as shown in FIG. 3. The reaction conditions are shown in Table 10.

2-2. Overlap PCR

Using each fragment amplified in 2-1 as a template, the primers (Eco-F and Xba-R primers) were used to amplify the full length at both ends. The reaction conditions are shown in Table 11.

2-3. Ligation

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

For the insert, the overlapping PCR product of Example 2-2 was purified and decomposed with the restriction enzyme EcoRI/XbaI at 37°C for 3 hours under the conditions shown in Table 7, and then combined with the prepared vector The gel extraction was performed together (Figure 4).

Under the conditions shown in Table 8, after ligation at room temperature (RT) for 2 hours, E. coli E. coli DH5α or DH10β was transformed and selected on medium containing ampicillin. The plastids prepared from the obtained colonies were sequenced to obtain Taq DNA polymerase mutants ("E507K/R536K", "E507K/R660V" and "E507K/R536K/R660V") into which the desired mutations were introduced.

Example 3 qPCR using the DNA polymerase of the present invention

Using Taq polymerases each containing the "E507K/R536K", "E507K/R660V", and "E507K/R536K/R660V" mutations obtained in Example 2 above, it was confirmed whether the ability to extend mismatched primers for the SNP-containing template was reduced . As a control group, "E507K" Taq polymerase containing E507K mutation was used.

The SNP-containing templates used in the present invention are rs1408799, rs1015362 and rs4911414. The genotype of each template and the sequence information of its specific primers (IDT, USA) are shown in Table 12 and Table 13.

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

The probes are double labeled, as shown in Table 15 below.

The oral epithelial cell collection kit purchased from Noble Bio was used to collect oral epithelial cells, dissolved in 500 μl of lysis solution, and centrifuged at 12,000×g for 3 minutes. Transfer the supernatant to a new tube, using 1 μl each time (Figure 5).

The reaction conditions are shown in Table 16, and the composition of the reaction buffer is shown in Table 17.

Except for the specific primers shown in Table 13, other reaction solutions were prepared in two test tubes in the same manner, and qPCR was performed by adding each allele-specific primer. At this time, the fluorescent signals detected in each test tube are combined to calculate and analyze the difference in the cycle (Ct) value of the fluorescence value reaching the threshold on the AB 7500 software (v 2.0.6). The longer the delay of the Ct value in the amplification caused by the mismatched primer, the better the mutation specificity or allele specificity.

The results of AS-qPCR on rs1408799, rs1015362, and rs4911414, as shown in FIGS. 6 to 8, compared with the control group E507K, it can be confirmed that Taq polymerization containing E507K/R536K, E507K/R660V, or E507K/R536K/R660V mutations In the case of enzymes, the amplification delay caused by mismatched primers is most obvious in the E507K/R536K/R660V mutation.

This confirms that the Taq DNA polymerase of the present invention containing the E507K/R536K, E507K/R660V, or E507K/R536K/R660V mutation has better selectivity for mismatch extension than the Taq polymerase containing the E507K mutation. Therefore, it is expected that the Taq DNA polymerase of the present invention can be effectively applied to medical diagnosis of diseases and research of recombinant DNA.

Example 4 Optimization of KCl concentration in reaction buffer

In this embodiment, in order to find a high cation concentration that maximizes the delay in amplification caused by mismatches without reducing the amplification efficiency caused by matching, the KCl concentration in the PCR reaction buffer is adjusted to determine the optimal KCl concentration.

The Taq polymerase obtained in Example 2 containing the mutations "E507K/R536K", "E507K/R660V" or "E507K/R536K/R660V", respectively, was used, and the KCl concentration threshold was compared with the Taq polymerase including the E507K mutation.

The template containing SNP uses rs1408799, the template genotype is TT, and the primer uses the rs1408799 primer described in Table 12. The qPCR conditions (Applied Biosystems 7500 Fast) were performed according to the conditions in Table 14, the double-labeled probe used 1408799-FAM in Table 15, the reaction conditions are shown in Table 18, and the reaction buffer composition is shown in Table 19.

The results are shown in FIG. 9, the KCI concentration threshold of E507K/R536K/R660V Taq polymerase is the lowest, and the KCl concentration threshold of E507K/R536K and E507K/R660V is lower than that of E507K.

Based on the above results, in order to determine the appropriate KCl concentration, E507K/R536K/R660V Taq polymerase was used for further experiments, and the rs1408799-T specific primer described in Table 13 was used as the primer. The qPCR conditions (Applied Biosystems 7500 Fast) were carried out for 35 cycles under the conditions of Table 14, and the reaction conditions are shown in Table 20.

The composition of the reaction buffer in the control group is the same as that in Table 21. The composition of the reaction buffer in the experimental group is shown in Table 8. The (NH ₄ ) ₂ SO ₄ concentration was fixed at 2.5 mM, and the KCl concentration was variously changed.

The result of confirming the PCR product by electrophoresis after amplification under the above conditions, as shown in FIG. 10, it can be confirmed that the amplification caused by the mismatch can be delayed to the maximum while the amplification efficiency caused by the matching is not reduced. The appropriate KCl concentration is 75mM.

Example 5 (NH ₄ ) ₂ SO ₄ Concentration optimization

In this example, based on the results of Example 4, the KCl concentration in the reaction buffer was fixed at 75 mM, and the (NH ₄ ) ₂ SO ₄ concentration was changed variously to confirm the optimal (NH ₄ ) ₂ SO ₄ concentration. For the primers, use the rs1408799-T specific primer described in Table 13, and perform qPCR conditions (Applied Biosystems 7500 Fast) under the conditions of Table 14 for 35 cycles. The reaction conditions are shown in Table 20. The composition of the reaction buffer in the control group is shown in Table 21. the same.

As a result, as shown in FIG. 11, the appropriate (NH ₄ ) ₂ SO ₄ concentration is 5 mM.

Based on the above results, the KCl concentration in the reaction buffer was fixed at 75 mM, and the (NH ₄ ) ₂ SO ₄ concentration was set at about 5 mM (respectively 2.5 mM, 5 mM, and 10 mM) to further confirm the mismatch. Amplify the delay effect.

The rs1408799 primer described in Table 13 was used as the primer, and the 1408799-FAM of Table 15 was used as the double-labeled probe. The reaction conditions are shown in Table 22.

As a result, as shown in FIG. 12, the concentration of (NH ₄ ) ₂ SO _{4 had} the largest difference in Ct value at 10 mM. However, it was confirmed that the amplification Ct caused by the match was slightly delayed and the peak fell down, so the appropriate (NH ₄ ) The concentration of ₂ SO ₄ is 5mM.

Combining the results of Example 4 and Example 5, it was confirmed that the optimal reaction buffer combination contained 50 mM Tris. Cl, 2.5 mM MgCl ₂ , 75 mM KCl, 5 mM (NH ₄ ) ₂ SO ₄ , 0.1% Tween 20, and 0.01% BSA.

Example 6 Adding TMAC to the reaction buffer and optimizing its concentration

In this example, TMAC was added to the reaction buffer, and its optimal concentration was confirmed. Based on the results of Examples 4 and 5, the concentration of KCl in the reaction buffer was fixed at 75 mM, and the concentration of (NH ₄ ) ₂ SO _{4 was} fixed at 5 mM, and various changes were made to the concentration of TMAC to confirm the best The best TMAC concentration.

E507K/R536K or E507K/R536K/R660V Taq polymerase was used, the template containing SNP used rs1408799, the template genotype was TT, and the rs1408799 primer described in Table 12 was used for the primer. The qPCR conditions (Applied Biosystems 7500 Fast) were performed according to the conditions in Table 14. The double-labeled probe used 1408799-FAM in Table 15 and the reaction conditions are shown in Table 22.

As shown in FIG. 13, the experimental results confirmed that the appropriate TMAC concentration was 60 mM for E507K/R536K Taq polymerase and 25 mM for E507K/R536K/R660V Taq polymerase, and it was confirmed that if the TMAC concentration is too high, the amplification efficiency is reduced.

Example 7 KCl, (NH ₄ ) ₂ SO ₄ And TMAC concentration optimization

In this example, based on the results confirmed in Example 6, the optimal KCl, (NH ₄ ) ₂ SO ₄ and TMAC concentrations in the reaction buffer were confirmed using E507K/R536K/R660V Taq polymerase.

Specifically, the TMAC concentration in the reaction buffer was fixed at 25 mM, the (NH ₄ ) ₂ SO ₄ concentration was fixed at 2.5 mM, and the KCl concentration was adjusted to 20, 40, 60, and 80 mM, respectively. For the two SNPs, rs1015362 and rs4911414, the genotype of the template was as described in Table 12, the primers were the rs1015362 and rs4911414 primers described in Table 13, and the qPCR conditions (Applied Biosystems 7500 Fast) were carried out under the conditions of Table 14, double labeling The probe used 1408799-FAM in Table 15, and the reaction conditions are shown in Table 22.

Experimental results, as shown in FIG. 14, for two SNPs, when the appropriate KCl concentration is 60 mM and the concentration is 80 mM, a decrease in amplification efficiency can be observed.

From the above results, it was confirmed that the optimal KCl concentration in the reaction buffer was 60 mM, the (NH ₄ ) ₂ SO ₄ concentration was 2.5 mM, and the TMAC concentration was 25 mM. More specifically, the confirmation of E507K / R536K polymerase, the concentration of KCl 75mM, 5mM of _{(NH 4) 2 SO 4,} 60mM the most effective TMAC; for E507K / R536K / R660V polymerase, used at a concentration of 60mM KCl, 2.5 mM (NH ₄ ) ₂ SO ₄ concentration, and 25 mM TMAC are most effective.

<110> Gene Kester Co., Ltd.

<120> PCR buffer composition for enhancing DNA polymerase activity with increased gene mutation specificity

<130> 107E0252

<150> KR 10-2017-0088376

<151> 2017-07-12

<160> 30

<170> PatentIn version 3.5

<210> 1

<211> 832

<212> PRT

<213> Artificial sequence

<400> 1

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Claims (10)

A PCR buffer composition for enhancing DNA polymerase activity with increased gene mutation specificity, wherein the composition comprises: 60 to 90 mM KCl; and 2.5 to 8 mM (NH ₄ ) ₂ SO ₄ ; the final pH is 8.0 To 9.0, wherein, in the PCR buffer composition for enhancing DNA polymerase activity with increased gene mutation specificity, the DNA polymerase system with increased gene mutation specificity has an amine group represented by SEQ ID NO: 1 In the acid sequence, the 507th amino acid residue, glutamic acid (E), was replaced by lysine (K), and the 536th amino acid residue, arginine (R), was replaced by lysine (K). And the DNA polymerase of Taq polymerase consisting of the amino acid sequence of the 660th amino acid residue, that is, an amino acid sequence in which arginine (R) is replaced by valine (V). The PCR buffer composition for enhancing DNA polymerase activity with increased gene mutation specificity as described in item 1 of the patent application scope, wherein the KCl concentration is 70 to 80 mM, and the (NH ₄ ) ₂ SO ₄ concentration is 4 to 6 mM. A PCR buffer composition for enhancing DNA polymerase activity with increased gene mutation specificity, wherein the composition comprises: 50 to 80 mM KCl; 1.5 to 6 mM (NH ₄ ) ₂ SO ₄ ; and 5 to 30 mM Tetra methyl ammonium chloride (TMAC); the final pH is 8.0 to 9.0, wherein the PCR buffer composition for enhancing DNA polymerase activity with increased gene mutation specificity has an increased The gene mutation-specific DNA polymerase system has a 507th amino acid residue in the amino acid sequence of SEQ ID NO: 1, that is, glutamic acid (E) is replaced by lysine (K), and the 536th amine Amino acid sequence consisting of arginine (R) substituted by lysine (K) and arginine (R) substituted by valine (V) Taq polymerase DNA polymerase. The PCR buffer composition for enhancing DNA polymerase activity with increased gene mutation specificity as described in item 3 of the patent application scope, wherein the TMAC concentration is 25 mM, the KCl concentration is 60 mM, and the (NH ₄ ) ₂ The SO ₄ concentration is 2.5 mM. As described in the first or third paragraph of the scope of the patent application, the PCR buffer composition for enhancing DNA polymerase activity with increased gene mutation specificity, wherein the PCR buffer composition further comprises Tris. Cl and MgCl ₂ . A PCR kit for detecting gene mutations or SNPs comprising the PCR buffer composition described in item 1 or item 3 of the patent scope. The PCR kit for detecting gene mutations or SNPs as described in item 6 of the patent application scope, wherein the PCR kit further comprises a DNA polymerase, and the DNA polymerase has an amino acid represented by SEQ ID NO: 1 In the sequence, the 507th amino acid residue, glutamic acid (E), was replaced by lysine (K), and the 536th amino acid residue, arginine (R), was replaced by lysine (K). And the Taq polymerase consisting of the amino acid sequence of the 660th amino acid residue, that is, the amino acid sequence in which arginine (R) is replaced by valine (V). The PCR kit for detecting gene mutations or SNPs as described in item 6 of the patent application scope, wherein the PCR kit further comprises: (i) nucleoside triphosphate; (ii) quantitative reagents that bind to double-stranded DNA; (iii) polymerase blocking antibody; (iv) one or more control values or control sequences; and (v) one or more templates. A detection method for in vitro detection of at least one gene mutation or SNP from more than one template using the PCR kit described in item 6 of the patent application scope. The method as described in item 9 of the patent application scope, wherein the PCR kit further comprises a DNA polymerase, and the DNA polymerase has the 507th amino acid residue in the amino acid sequence of SEQ ID NO: 1 That is, glutamic acid (E) is substituted with lysine (K), the 536th amino acid residue, arginine (R), is substituted with lysine (K), and the 660th amino acid residue is, Taq polymerase consisting of an amino acid sequence in which arginine (R) is replaced by valine (V).

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