TWI733038B - DNA polymerase with improved gene mutation specific amplification efficiency - Google Patents

Abstract

The present invention relates to a DNA polymerase with improved gene mutation specific amplification efficiency and its use. Specifically, it is a DNA polymerase with a specific amino acid site that induces mutations and improves gene mutation specific amplification efficiency. The nucleic acid sequence of the polymerase includes a vector of the nucleic acid sequence and provides a transgene host cell to the vector. The present invention also provides a method for detecting more than one gene mutation or SNP in more than one template in vitro ( in vitro ) using a DNA polymerase with improved specific amplification efficiency of gene mutations, and a method comprising the DNA polymerase A composition for detecting genetic variation or SNP and a PCR kit containing the composition.

Description

DNA polymerase with improved gene mutation specific amplification efficiency

The present invention relates to a DNA polymerase with improved gene mutation-specific amplification efficiency and its use. Specifically, it is a DNA polymerase with increased efficiency of gene mutation-specific amplification by inducing mutations at amino acid sites, encoding said The nucleic acid sequence of the polymerase includes a vector of the nucleic acid sequence and provides a transgene host cell to the vector. The present invention also provides a method for detecting more than one gene mutation or SNP in more than one template in vitro ( in vitro ) using a DNA polymerase with improved specific amplification efficiency of gene mutations, and a method comprising the DNA polymerase A composition for detecting genetic variation or SNP and a PCR kit containing the composition.

Since the first human gene sequence was discovered, researchers have focused on discovering single nucleotide mutations (single nucleotide polymorphisms, SNPs) and other genetic differences between individuals. Single nucleotide variation in the genetic body is related to different drug resistance and pathogenic factors for various diseases. As this point becomes more and more clear, it becomes the object of attention. In the future, medical knowledge about nucleotide variations can be used in the treatment of individual genetic supply, and can prevent ineffective or side-effect drug treatments (Shi, Expert Rev. Mol. Diagn. 1.363-365 (2001)). The development of efficient nucleotide variation identification technology in terms of 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 variants of the target nucleic acid can be achieved by hybridizing the nucleic acid sample to be analyzed with hybridization primers specific to the 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 clinical needs, especially in terms of the sensitivity necessary for the determination. Therefore, PCR has been widely used in molecular biology, SNP and other allelic sequence variants and other mutation detection diagnostic detection methods (Saiki et al., Science 239, 487-490 (1988)), among which, considering the change In the presence of the body, the target nucleic acid to be detected is amplified by polymerase chain reaction (PCR) before hybridization. The hybridization primers used in this measurement usually use single-stranded oligonucleotides. 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 the determination of SNP and other sequence variations. (Gut, Hum. Mutat. 17, 475-492 (2001)).

The scheme of specific hybridization of sequence variation known in the corresponding technical field is provided by so-called gene variation specific amplification. In these detection methods, mutation-specific amplification primers have been used in the amplification process, which usually have so-called differential terminal nucleotide residues in the 3'-end of the primer, and the residues are only the target to be detected. A specific variation of nucleic acid is complementary. In this method, nucleotide variants are determined based on the presence or absence of DNA products amplified by PCR. The principle of gene variation specific amplification is based on the formation of canonical or non-standard primer-template complexes in the ends of gene variation specific amplification primers. In a precisely matched 3'primer end, DNA polymerase causes amplification, on the other hand, the extension of the mismatched primer end is inhibited.

For example, US Patent No. 5,595,890 discloses a method for specific amplification of gene variants and its application, as well as an application for detecting clinical-related 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 to detect point mutations that cause sickle cell anemia. However, gene variant-specific amplification or allele-specific amplification has the problem of low selectivity, and therefore requires more complicated and time- and cost-intensive optimization steps.

The methods described above for detecting sequence variation, polymorphism and focusing on point mutations, especially when the detected sequence variation is not sufficiently compared with the dominant mutation with the same nucleic acid segment (or the same gene), Allelic gene-specific amplification (or gene variant specific amplification) is required.

For example, this situation occurs when sporadic tumor cells are detected in body fluids such as blood, serum or plasma using gene mutation-specific amplification (US Patent No. 5,496,699). For this reason, DNA is first separated from body fluids such as blood, serum or plasma. DNA is composed of lack of sporadic tumor cells and excess non-proliferative cells. Therefore, in the presence of excess wild-type DNA, important mutations in the tumor DNA in the k-ras gene should be detected from multiple replications.

All the methods for specific amplification of gene variants disclosed in the traditional technology have the defect that 3'-end differential oligonucleotide residues must be used. In addition, there is a defect that although 3'-different nucleotide residues are used, and even if the target nucleic acid is not completely consistent with the sequence variant to be detected, primer extension occurs in the presence of a suitable DNA polymerase. Lower standard. Especially when specific sequence variants are detected with excessive background nucleic acid containing different sequence variants, false positive results will be produced. The main reason for the defects of these PCR-based methods is poor synthesis of the polymerase used in the method for sufficiently distinguishing mismatched bases. Therefore, it has not been possible to use PCR to directly obtain clear information on the presence or absence of mutations. So far, the definitive diagnosis of mutations requires more time and input cost of purification and analysis methods. Therefore, a new method that can improve gene variation specificity or the selectivity of opposite allele-specific PCR amplification will have a significant impact on the reliability and potency of direct gene variation or SNP analysis by PCR.

The inventors of the present invention are committed to developing a new type of DNA polymerase that can improve the selectivity of gene mutation-specific PCR amplification. The results confirmed that when mutations are induced to amino acid residues at specific sites of Taq polymerase, gene mutations The specific amplification efficiency is significantly improved, thus completing the present invention.

The present invention was developed to solve the above-mentioned problems and provides a DNA polymerase for detecting one or more gene variants or SNPs in a target sequence containing gene variants or SNPs.

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

Another object of the present invention is to provide a method for preparing DNA polymerase according to the present invention.

Another object of the present invention is to provide a kit for detecting one or more gene mutations or SNPs in more than one template in vitro using the DNA polymerase of the present invention.

Another object of the present invention is to provide a composition for detecting genetic variation or SNP containing the DNA polymerase of the present invention.

Another object of the present invention is to provide a kit for detecting gene variation or SNP comprising the composition for detecting gene variation or SNP according to the present invention.

In order to achieve the above objective, the present invention provides a DNA polymerase with Taq polymerase composed of the amino acid sequence of SEQ ID NO: 7 (the base sequence of SEQ ID NO: 7). The DNA polymerase includes: (a) The substitution of the 507th amino acid residue in the amino acid sequence of sequence number 1; and (b) the substitution of the 536th amino acid residue in the amino acid sequence of sequence number 1, and the Substitution of 660 amino acid residues or substitution of two amino acid residues of the 536th and 660th, or substitution of three amino acid residues of the 536th, 587th and 660th.

According to a preferred embodiment of the present invention, the replacement of the 507th amino acid residue is the substitution of lysine (K) for glutamine (E), and the 536th amino acid residue is replaced Is the substitution of lysine (K) for arginine (R), the 587th amino acid residue is replaced by isoleucine (I) for arginine (R), the 660th The replacement of amino acid residues is the substitution of valine (V) for arginine (R).

According to another preferred 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 3'end of the mismatched primer May include atypical nucleotides.

According to another preferred embodiment of the present invention, the DNA polymerase exhibits a lower Ct value (high amplification efficiency) improvement in the amplification of the target sequence containing the matched primer than the amplification of the target sequence containing the mismatched primer.

The present invention also provides a nucleic acid sequence encoding the DNA polymerase involved in the present invention, a vector including the nucleic acid sequence, and a host cell transgene using the vector.

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

The present invention also provides a detection method, comprising: a) one or more templates contacted with the DNA polymerase of the present invention; b) more than one matched primer, more than one mismatched primer, or more than one matched primer and one Both of the above mismatched primers; and c) nucleotide triphosphates; hybridizing the one or more matched primers and mismatched primers with the target sequence, and for the target sequence that hybridizes with the mismatched primer, from From the 3'end to the 7th base position, including unconventional (Non-canonical) Nucleotide, which detects more than one gene variation or SNP in vitro in more than one template.

According to a preferred embodiment of the present invention, the method may include using dual-strand specific dyes for melting point analysis.

According to another preferred embodiment of the present invention, the method can be performed by real-time PCR, analysis in agarose gel after standard PCR, gene variation-specific amplification or allele-specific amplification by real-time PCR, Four-primer amplification-hindered mutation system PCR or isothermal amplification to complete.

The present invention also provides a composition for detecting gene variation or SNP containing the DNA polymerase of the present invention.

The present invention also provides a kit containing a composition for detecting genetic variation or SNP.

According to an embodiment of the present invention, the PCR kit can be used for transfection PCR (competitive allele-specific TaqMan PCR), droplet digital PCR (Droplet digital PCR) or nucleic acid mass spectrometry system (MassARRAY).

According to a preferred embodiment of the present invention, the PCR kit further includes more than one matched primer, more than one mismatched primer, or both more than one matched primer and more than one mismatched primer, so The one or more matched primers and one or more mismatched primers hybridize to the target sequence, and the target primer that hybridizes to the mismatched primer may include unconventional primers starting from the 3'end to the 7th base position. (non-canonical) nucleotides.

According to another preferred embodiment of the present invention, the PCR kit may further include nucleotide thiophosphate.

According to another preferred embodiment of the present invention, the PCR kit may further include: a) one or more buffers; b) a quantification reagent that binds to double-stranded DNA; c) a polymerase blocking antibody; d ) More than one comparison value or comparison sequence; and e) one or more templates.

Compared with the prior art, the present invention has the following beneficial effects: the DNA polymerase with improved gene variant specific amplification efficiency according to the present invention has higher mismatch matching extension selectivity than conventional Taq polymerases. , Without any matrix variation, reliable gene variation-specific amplification can occur. In addition, the DNA polymerase involved in the present invention can be effectively applied to the medical diagnosis of diseases and the research of recombinant DNA.

By reading the detailed description of the non-limiting examples with reference to the following drawings, other features, purposes and advantages of the present invention will become more apparent: Figure 1 shows Taq DNA including R536K, R660V and R536K/R660V variants, respectively The polymerase preparation process, (a) shows a schematic diagram of fragment PCR and overlap PCR, (b) shows the identification of amplified products in fragment PCR by electrophoresis, and (c) shows the full length amplified by overlap PCR. Confirm the results of the amplified products by electrophoresis; Figure 2 is to extract the gel, decompose it with the restriction enzyme EcoRI/XbaI, and then use electrophoresis to identify the pUC19 vector treated with SAP and the purified Figure 1(c) overlap PCR product results; Figure 3 is a schematic diagram of fragment PCR and overlap PCR in the preparation process of Taq DNA polymerases that include E507K, E507K/R536K, E507K/R660V and E507K/R536K/R660V variants respectively; Figure 4 is to extract the gel with restriction enzymes After EcoRI/XbaI was decomposed, the SAP-treated pUC 19 vector was identified by electrophoresis and the results of the purified overlapping PCR products in Figure 3; Figure 5 is a schematic diagram of the process of collecting oral epithelial cells to prepare PCR templates; Figure 6 shows the use of the E507K/ of the present invention. The results of AS-qPCR on rs1408799 by R536K, E507K/R660V and E507K/R536K/R660V Taq polymerases, the comparison group used Taq polymerase containing E507K variants; Figure 7 shows the use of the present invention with E507K/R536K, E507K /R660V and E507K/R536K/R660V Taq polymerase, AS-qPCR detection results of rs1015362, the comparison group used Taq polymerase including E507K variants; Figure 8 shows the use of the present invention with E507K/R536K, E507K /R660V and E507K/R536K/R660V Taq polymerases performed AS-qPCR results on rs49114. The comparison group used Taq polymerase including E507K variants; Figure 9 shows Taq DNA including E507K/R536K/R587I/R660V variants The polymerase preparation process, (a) shows the graphical fragment PCR and overlap PCR, (b) shows the result of electrophoresis to identify the amplified products in the fragment PCR; Figure 10 shows the use of E507K/R536K/R587I/R660V Polymerase, the result of AS-qPCR on the template including the Q61H SNP in the KRAS gene, (a) and (b) are the results of using a primer with a length of 24mer, (c) and (d) are using a length of 18mer The results of the primers, the comparison group uses the variants containing E507K/R536K/R660V Figure 11 shows the results of AS-qPCR using the E507K/R536K/R587I/R660V polymerase on the template including the G13D SNP in the KRAS gene. The comparison group uses the E507K/R536K/R660V variant Figure 12 shows the results of AS-qPCR using E507K/R536K/R587I/R660V polymerases on templates including G12S SNPs in the KRAS gene. The comparison group uses E507K/R536K/R660V variants. Figure 13 shows the results of AS-qPCR using the E507K/R536K/R587I/R660V polymerase on the template including the L585R SNP in the EGFR gene. The comparison group uses the E507K/R536K/R660V variant Taq polymerase.

The present invention will be described in more detail below.

As mentioned above, in order to improve the shortcomings of gene mutation-specific amplification methods disclosed in traditional technologies, it is necessary to continue to develop methods that can increase the selectivity of gene mutation-specific amplification rates. The credibility and robustness of SNP analysis have a major impact. The inventors of the present invention are committed to developing a method that can improve the selectivity of gene mutation-specific PCR amplification, confirming that when the amino acid residue at a specific site of Taq polymerase is mutated, the gene mutation-specific amplification efficiency Significant improvement, thus completing the present invention.

Compared with the conventional Taq polymerase, the DNA polymerase with improved gene mutation specific amplification efficiency has higher mismatch matching extension selectivity, and can generate highly reliable gene mutation without any matrix mutation. Specific amplification. In addition, the DNA polymerase involved in the present invention can be effectively applied to the medical diagnosis of diseases and the research of recombinant DNA.

The terminology used in this manual will be explained below.

"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), asparagine ( Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamine (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), Phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

Amino acids are typical organic acids, including substituted or unsubstituted amine groups, substituted or unsubstituted carboxyl groups, and more than one sidechain or group, or any analog of these groups , Exemplary side chains include sulfhydryl, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazino, cyano, halo, hydrazine, alkenyl, alkyne Group, ether group, borate, boronic acid group, dioxophosphorus group, phosphine sulfonic acid group, phosphine group, heterocyclic ring, enone, imine, aldehyde group, ester group, thioacid, hydroxylamine or any of these groups Any combination.

Other exemplary amino acids include, but are not limited to, the following: amino acids containing photoactivated crosslinkers, metal bond amino acids, spin-labeled amino acids, fluorescent amino acids, metal-containing amino acids, Amino acids with novel functional groups, amino acids that interact covalently or non-covalently with other molecules, rasterized and/or photoisomerizable amino acids, radioactive amino acids, including biotin or Amino acids of biotin analogs, glycosylated amino acids, other carbonate-modified amino acids, amino acids including polyethylene glycol or polyether, heavy atom substituted amino acids, chemical degradability And/or photodegradable amino acids, amino acids containing carbon-linked sugars, redox-active amino acids, amino acids containing sulfur carboxylic acids, and amino acids containing more than one toxic moiety.

In the DNA polymerase of the present invention, the term "mutant" refers to a recombinant polypeptide that contains more than one amino acid substitution relative to a naturally-occurring or unmodified DNA polymerase.

The term "thermostable polymerase" (refers to a thermostable enzyme) means that it has heat resistance and maintains sufficient activity to achieve the subsequent polynucleotide extension reaction. The process is heated within the time required to achieve double-stranded nucleic acid denaturation. No irreversibility (inactivation) will occur. As used in the present invention, it is suitable for use at the circulating temperature in reactions such as PCR. Irreversibility in the present invention refers to the complete loss of permanent enzyme activity. Regarding thermostable polymerases, enzymatic activity refers to catalyzing the combination of nucleotides in an appropriate manner to form polynucleotide extension products that complement each other with the template nucleic acid strand. Examples of thermostable DNA polymerases derived from thermophilic bacteria are as follows: Thermotoga maritima , Thermus aquaticus , Thermus thermophilus , Thermus aquaticus Thermus flavus , Thermus filiformus , Thermus species Sps17, Thermus species Z05, Thermus caldophilus , Bacillus caldo tenax , New Apo Thermotoga neapolitana and DNA polymerase derived from Thermosipho africanus.

The term "thermal activity" refers to an enzyme that maintains catalytic performance during reverse transcription or adhesion in RT-PCR and/or PCR reactions/at the usual temperature in the extension stage (ie, 45-80°C). A thermostable enzyme refers to an enzyme that is irreversibly inactivated or not modified during the high temperature treatment required for nucleic acid modification. Thermoactive enzymes may or may not be thermostable. A thermoactive DNA polymerase may include the following DNA or RNA dependent on thermophilic or mesophilic species, but is not limited thereto.

The term "host cell" refers to single-celled prokaryotes and eukaryotic organisms (such as bacteria, yeast, and actinomycetes) and single cells of both higher plants or animals when cultured in a cell culture medium.

The term "vector" is a DNA molecule that can be cloned and can transfer genes and other foreign DNA to recipient cells, including plastids, bacteriophages, artificial chromosomes, and the like. In the present invention, "plastid", "carrier" or "plastid carrier" have the same meaning when used.

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

The term "nucleic acid" or "polynucleotide" refers to a DNA or RNA polymer, or a polymer that can correspond to its analogs. Nucleic acids may include, for example, chromosomes or chromosome segmentation, vectors (such as expression vectors), expression elements, naked DNA or RNA polymers, polymerase chain reaction (PCR) products, oligonucleotides, probes, and primers. The nucleic acid can be single-stranded, double-stranded, or triple-stranded, and is not limited to any specific length. Unless otherwise mentioned, certain nucleic acid sequences include complementary sequences or may encode them in addition to any sequences explicitly specified.

The term "primer" refers to a polynucleotide that can be used as a template-direction nucleic acid synthesis starting point when placed under the condition that the polynucleotide begins to extend. Primers can also be used in a variety of other oligonucleotide-mediated synthesis processes, including de novo RNA synthesis and as a promoter for in vitro transcription-related processes. A typical primer is a single-stranded oligonucleotide (e.g., oligodeoxynucleotide). The suitable 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, depending on the purpose of use. Short primer molecules usually require lower temperatures to form sufficiently stable hybrid complexes with the template. The primer does not need to reflect the exact sequence of the template, but it must have sufficient complementarity to hybridize with the template and extend the primer. In a specific implementation example, the term "primer pair" refers to a 5'-sense strand primer that hybridizes to the 5'-end of the amplified nucleic acid sequence and a 3'-antisense primer that hybridizes to the 3'-end of the amplified sequence. A set of primers for chain primers. If necessary, a label detected by spectroscopic, photochemical, biochemical, immunochemical or chemical means can be added to label the primer. For example, useful labels include the following: ³² P, fluorescent dyes, electron density reagents, enzymes (usually used in ELISA analysis), biotin or hapten, or proteins that can utilize antiserum or monoclonal antibodies.

The term "5'-nuclease probe" refers to an oligonucleotide that includes one or more luminescent labeling portions for performing a 5'-nuclease reaction for targeted nucleic acid detection. In some embodiments, for example, the 5'-nuclease probe includes only a single luminescent moiety (e.g., fluorescent dye, etc.). In some embodiments, the 5'-nuclease probe includes a self-complementary region, so that the probe can form a hairpin structure under selected conditions. In some embodiments, the 5'-nucleohydrolase probe includes two or more label parts, and after one label is separated or degraded from the nucleotide, the radiation intensity is increased and released. In some embodiments, the 5'-nuclease probe is labeled with two different fluorescent dyes, for example, a 5'-end reporter dye and a 3'-end quencher dye or partial label. In some embodiments, in addition to the end, the 5'-nuclease probe is also labeled at one or more positions other than the end position. Generally speaking, if the probe is intact, energy transfer takes place between the two fluorescent materials, so that the fluorescent part of the reporter dye is extinct. During the extension stage of the polymerase chain reaction, for example, the 5'-nucleohydrolase probe bound to the template nucleic acid is active, so that the fluorescent luminescence of the reporter dye is no longer quenched, such as by Taq polymerase or other polymerases. 5'to 3'-nucleic acid hydrolase activity for degradation. In some embodiments, the 5'-nuclease probe can be labeled with a variety of different reporter dyes and 3'-end quenching dyes or parts.

The term "FRET" or "fluorescence resonance energy transfer" or "Foster resonance energy transfer" refers to more than two chromophores, donor chromophores and acceptor chromophores (called quenchers) Energy transfer between. Generally speaking, when a donor is excited by emitting light of an appropriate wavelength, it transfers energy to the acceptor. The receptor usually re-emits the energy transferred in the form of light of a different wavelength. When the acceptor is a "dark" quencher, then it emits energy transferred in a form other than light. The role of certain fluorescent substances as donors or acceptors depends on the characteristics of FRET for other members. Commonly used donor-acceptor pairs include FAM-TAMRA pairs. Commonly used quenchers are DABCYL and TAMRA. Commonly used dark quenchers include the following: Black Hole Quencher ^TM (BHQ), (Biosearch Technologies, Inc., Novato, Cal.), Iowa Black ^TM (Integrated DNA Tech., Inc., Coralville, Iowa), and BlackBerry ^TM Quencher 650 (BBQ-650) (Berry & Assoc., Dexter, Mich.).

When referring to nucleobases, nucleoside triphosphates, or nucleotides, the term "conventional" or "natural" refers to those naturally occurring in the polynucleotide described (ie, for DNA they are dATP, dGTP, dCTP and dTTP ). In addition, dITP and 7-decazepine-dGTP are often used instead of dGTP, and can be used to replace dATP in in-tube DNA synthesis reactions such as sequencing.

When referring to nucleic acid bases, nucleosides, or nucleoside triphosphate triphosphates, the term "unconventional" or "modification" includes the modification or derivation of conventional bases, nucleosides, or nucleotides naturally occurring in certain polynucleotides.物 or the like. Compared with conventional dNTPs, certain unconventional nucleotides are modified at the 2'position of the ribose sugar. Therefore, even if the naturally occurring nucleotides for RNA are ribonucleosides (ie, ATP, GTP, CTP, UTP, collective rNTP), since the nucleotide has a hydroxyl group at the 2'position of the sugar, relatively speaking, dNTP is missing, and Like the nucleotide used in the present invention, ribonucleoside is an unconventional nucleotide as a substrate of DNA polymerase. As used herein, unconventional nucleotides include, but are not limited to, compounds used as terminator for nucleic acid sequencing. Exemplary terminator compounds include, but are not limited to, compounds with a 2',3'-dideoxy structure, known as dideoxynucleoside triphosphates. Dideoxynucleoside triphosphate ddATP, ddTTP, ddCTP and ddGTP are collectively referred to as ddNTP. Other examples of terminator compounds include 2'-PO ₄ analogs of ribonucleotides. Other unconventional nucleotides include but are not limited to thiodNTP ([[ α ]-S]dNTP), 5'-[ α ]-borano-dNTP, [ α ]-methyl-phosphonic acid dNTP, ribose Nucleoside triphosphate (rNTP). Unconventional bases can be radioisotopes such as ³² P, ³³ P or ³⁵ S; fluorescent labels; chemiluminescent labels; bioluminescent labels; hapten tags such as biotin; or enzyme tags such as streptavidin or antibiotics Vegetarian protein to label. Fluorescent labels may include negatively charged dyes such as luciferin family dyes, or neutrally charged dyes such as rhodamine family dyes, or positively charged dyes such as 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 pass Perkin-Elmer (Boston, Massachusetts), Applied Biosystems (Fox Special City, California), Invitrogen/Molecular Probes (Eugene, Oregon) market. The cyanine family dyes include Cy2, Cy3, Cy5, and Cy7, which are marketed by GE Healthcare UK Limited (Amersham Place, Little Chalfont, Buckinghamshire, England).

The term "discrimination of mismatches" refers to when one or more nucleotides are adhered (e.g., covalently) to the nucleic acid to extend the nucleic acid (e.g., primers or other oligonucleotides) in a template-dependent manner. From mismatches-include the ability to distinguish completely complementary sequences from biocatalysis (such as polymerases, ligases and other enzymes). The term "discrimination of mismatches" means that the extended nucleic acid (such as primers or other oligonucleotides) is compared with the template to which the nucleic acid hybridizes, from the 3'end of the nucleic acid that has mismatched mismatches-including (approximately complementary) sequence discrimination is complete The biocatalytic ability of complementary sequences. In some embodiments, the extended nucleic acid contains a mismatch at the 3'end of the fully complementary sequence. In some embodiments, the extended nucleic acid contains a mismatch at the second (N-1) 3'-position and/or N-2 position of the end to the fully complementary sequence.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as commonly understood by the parties concerned.

The present invention relates to a DNA polymerase, as a DNA polymerase with Taq polymerase composed of an amino acid sequence of sequence number 1 (the base sequence of SEQ ID NO: 7), including: (a) sequence number 1 The substitution of the 507th amino acid residue in the amino acid sequence; and (b) the substitution of the 536th amino acid residue and the 660th amino acid residue in the amino acid sequence of sequence number 1. Or the 536th and 660th two amino acid residues, or the 536th, 587th and 660th three amino acid residues.

The "Taq polymerase" is a heat-resistant DNA polymerase named after the name of the thermophilic bacterium Thermus aquaticus, which is first isolated from the bacteria. As a kind of bacteria inhabiting hot springs and hot water eruption holes, the thermophilic bacteria inhabiting confirmed that Taq polymerase can withstand the protein modification conditions (high temperature) required by the PCR process. The best activity temperature of Taq polymerase is 75~80℃, the half-life at 92.5℃ is more than 2 hours, the half-life at 95℃ is 40 minutes, the half-life at 97.5℃ is 9 minutes, and the half-life at 72℃ can be 10 seconds. 1,000 base pairs of DNA are cloned within. The correcting activity of its deletion 3'→5' nucleic acid terminal hydrolase (exonuclease) was deleted, and an error rate of about 1 in 9,000 nucleotides was determined. For example, heat-resistant Taq can be used to run PCR at high temperatures (above 60°C). The amino acid sequence shown in Sequence 1 of Taq polymerase is taken as the standard sequence.

According to a preferred embodiment of the present invention, the replacement of the 507th amino acid residue is the substitution of lysine (K) for glutamine (E), and the 536th amino acid residue is replaced Is the substitution of lysine (K) for arginine (R), the 587th amino acid residue is replaced by isoleucine (I) for arginine (R), the 660th The replacement of amino acid residues is the substitution of valine (V) for arginine (R).

In the present invention, the Taq polymerase in which the 507th amino acid residue in the amino acid sequence of sequence number 1 is replaced by lysine (K) for glutamate (E) is named "E507K" (SEQ ID NO: 2 , The base sequence is SEQ ID NO: 8); in the amino acid sequence of SEQ ID NO: 1, the 507th amino acid residue is replaced by lysine (K) for glutamate (E), and the 536th amino acid residue The Taq polymerase whose acid residue is replaced with arginine (R) by lysine (K) is named "E507 K/R536K" (SEQ ID NO: 3, base sequence is SEQ ID NO: 9); SEQ ID NO: 1 In the amino acid sequence, the 507th amino acid residue is replaced by lysine (K) for glutamine (E), and the 660th amino acid residue is replaced by valine (V) for arginine (R) Taq polymerase was named "E507K/R 660V" (Sequence No. 4, base sequence is SEQ ID NO: 10); finally, the 507th amino group in the amino acid sequence of SEQ ID NO. The acid residue is replaced by lysine (K) for glutamine (E), the 536 amino acid residue is replaced by lysine (K) for arginine (R), and the 660th amino acid residue is replaced by valerate. The Taq polymerase with amino acid (V) substituted for arginine (R) was named "E507 K/R536K/R660V" (SEQ ID NO: 5, base sequence is SEQ ID NO: 11). Finally, the 507th amino acid residue in the amino acid sequence of sequence number 1 was replaced by lysine (K) for glutamine (E), and the 536th amino acid residue was replaced by lysine (K). ) Replaced arginine (R), the 587th amino acid residue was replaced by isoleucine (I), the 660th amino acid residue was replaced by valine (V) The Taq polymerase of arginine (R) was named "E507K/R536K/R587I/R660V" (SEQ ID NO: 6, the base sequence is SEQ ID NO: 12).

According to a preferred embodiment of the present invention, the DNA polymerase distinguishes between matched primers and mismatched primers, the matched primers and mismatched primers hybridize to the target sequence, and the 3'end of the mismatched primers Contains atypical nucleotides.

The mismatched primer is an oligonucleotide and must be sufficiently complementary to hybridize with the target primer, but it cannot reflect the exact sequence of the target sequence.

The "canonical nucleotides" or "complementary nucleotides" refer to standard Watson Crick base pairs, A-U, A-T, and G-C.

The "non-canonical nucleotide" or "non-complementary nucleotide" refers to AC, AG, GU, GT, TC, TU, AA other than Watson Crick base pair , GG, TT, UU, CC, CU.

According to a preferred embodiment of the present invention, the Ct value displayed by the DNA polymerase shows that the amplification of the target sequence containing the matched primer is lower than the amplification of the target sequence containing the mismatched primer.

For example, by covalently linking one or more nucleotides to a primer, a DNA polymerase can extend a matched primer more efficiently than a primer that is mismatched in a target sequence-dependent manner. Here, the higher efficiency can be observed by the following example, such as in RT-PCR, the matched primer has a lower Ct value than 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, the matched forward primer and reverse primer in the first reaction, the mismatched forward primer and the matched reverse primer were used in the second reaction under the same experimental configuration, which means that the standard PCR The formed product has a greater impact on the first reaction than the second reaction.

The Ct (threshold crossing cycle) value represents the DNA quantification method of quantitative PCR and depends on drawing the fluorescence on the number of logarithmic phase cycles. The threshold for DNA-based fluorescence detection is set to be 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 MIQE criteria. The Ct value for a given reaction is defined as the number of cycles at which the fluorescence emission crosses a fixed limit value. For example, SYBR Green I and fluorescent probes can be used for real-time PCR for template DNA quantification. The fluorescence from the sample was collected at each 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-mentioned 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 a 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 sequences refer to DNA sequences required for the expression of operably linked coding sequences in a specific host organism. For example, suitable regulatory sequences for prokaryotes include promoters, any operating sequences, and alc ribosome binding sites. In addition, the vector may contain a "Positive Retroregulatory Element (PRE)" to enhance the half-life of the transcribed mRNA. Controlling transcription and translation of nucleic acid regions is generally applicable to 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 may include, for example, promoter sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or promoter sequences. In a typical embodiment, the regulatory sequence includes a promoter and transcription initiation and termination sequences. The vector usually also contains a multi-site adaptor region containing several restriction sites for insertion of foreign DNA. In certain embodiments, a "fusion tag" is used to facilitate purification, and if necessary, the tag/tag is subsequently removed (e.g., "His-tag"). However, these are generally unnecessary when purifying thermoactive and/or thermostable proteins from a mesophilic host (e.g. E. coli) that uses a "heating step". Standard recombinant DNA techniques are used to prepare suitable vectors containing coding DNA replication sequences, regulatory sequences, phenotypic selection genes, and mutant polymerases of interest. As known in the art, the isolated plastids, viral vectors, and DNA fragments are broken down and cut, and joined together in a specific order to produce the desired vector.

In a preferred embodiment of the present invention, the expression vector contains a selectable 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 in which these vectors are cultured 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 a cell alone or after being used in combination with a vector. Introduction or its equivalent expression means that the nucleic acid enters the cell in a suitable manner such as subsequent integration, amplification, and/or expression. Introduction methods include CaPO ₄ precipitation, liposome fusion, adiponectin, electrophoresis, virus infection and so on.

Prokaryotes are used as host cells in the early colonization stage of the present invention. For the rapid preparation of large amounts of DNA, the production of single-stranded DNA templates for generating positional mutations, the screening of a large number of mutants, and the resulting mutant DNA serialization is particularly useful. Suitable host cells for prokaryotic cells include E.coli K12 strain 94 (ATCC No.31,446), E.coli strain W3110 (ATCC No.27,325), E.coli K12 strain DG116 (ATCC No.53,606), E.coli X1776 (ATCC No.31,537) and E.coli B; E.coli and many other different strains, such as HB101, JM101, NM522, NM538, NM539, and various other types, including Bacillus subtilis , etc. Bacillus group, Salmonella typhimurium (Salmonella typhimurium) and other intestinal flora, or Serratia marcesans (Serratia marcesans) and other various Pseudomonas sp. (Pseudomonas sp.) prokaryotic organisms are used as the main body. A typical plastid used for E. coli transgene includes pBR 322, pUCI 8, pUCI 9, pUCI18, pUC119 and Bluescript M13. However, many other suitable carriers can also be used.

The present invention also provides a method for preparing DNA polymerase, including the step of culturing the host cell; and the 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 that is genetically transformed into an expression vector containing a nucleic acid sequence encoding the DNA polymerase under appropriate conditions that induce or cause the expression of the DNA polymerase. Methods for culturing transgenic host cells under conditions suitable for protein expression have been published in the industry. A host cell suitable for preparing polymerases derived from the lambda pL promoter-containing plastid vector includes E. coli strain DG116 (ATCC No. 53606). Depending on the expression, the polymerase can be harvested and isolated.

Once purified, the mismatch difference of the DNA polymerase of the present invention can be identified. For example, the mismatch discrimination activity is determined by comparing the amplification of a target sequence with a single base mismatch in the 3-terminus of the primer and the amplification of a target sequence that perfectly matches the primer. Amplification can be detected immediately by the use of ^{TaqMan TM, etc.} The ability of the polymerase to distinguish between the two target sequences can be estimated by comparing the Ct of the two reactions.

Therefore, the present invention provides a detection method that includes contacting DNA polymerase with: a) more than one template; b) nucleotide triphosphate; and c) more than one matched primer, more than one mismatched primer, or one Both the above matched primer and one or more mismatched primers; hybridize the one or more matched primers and the mismatched primer with the target sequence, and for the target sequence that hybridizes with the mismatched primer, start from its 3'end From the beginning to the 7th base position, non-canonical nucleotides are included, and more than one gene mutation or SNP is detected in vitro in more than one template.

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

In the in vitro gene mutation or SNP detection method of the present invention, the target sequence may be present in the test sample, and may include DNA, cDNA or RNA, of which genomic DNA is preferred. 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 animals, preferably in vertebrates, and preferably in human subjects. A target sequence may be contained in genomic DNA, preferably contained in the genomic DNA of an individual, more preferably contained in bacteria or vertebrates, and most preferably contained in the genomic DNA of a human subject.

The SNP detection method of the present invention may include the use of SYBR Green I and other double-strand specific dyes for deconstruction temperature analysis.

Deconstruction temperature curve analysis can be performed on real-time PCR equipment such as ABI 5700/7000 (96-well format) or ABI 7900 (384-well format) equipment including onboard software (SDS 2.1). Or, the analysis of the deconstruction temperature profile can be performed as an end point analysis.

The "double-stranded DNA-binding dye" or "double-stranded specific dye" can be used in the case of higher fluorescence when combined with double-stranded DNA than when it is not combined with 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 gene mutation or SNP detection method of the present invention can be performed by real-time PCR, agarose gel analysis after standard PCR, gene mutation-specific amplification or allele-specific amplification by real-time PCR, four primers Amplification hindered mutation system PCR or isothermal amplification is carried out, but it is not limited to this.

For example, the SNP detection method of the present invention can use sequencing, mini-sequencing, allele specific PCR (Allee specific PCR), dynamic allele-specific hybridization (DASH); PCR extended analysis (Such as single base extension (SBE), PCR-SSCP, PCR-RFLP analysis or TaqMan technology, SNPlex platform (Thermo Fisher, Applied Biosystems), nucleic acid mass spectrometry system (such as Sequenom's MassARRAY system) ), Bio-Plex system (Bio-Rad, Bio-Rad) and so on.

The "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 thermostable DNA polymerases such as Taq polymerase or Klen Taq. DNA polymerase assembles new DNA strands from nucleotidase by using single-stranded DNA as a template and oligonucleotides (primers). The amplicons generated by PCR can be analyzed in an agarose gel.

The "real-time PCR" is to monitor and monitor the process in real time during the PCR. Therefore, the data is collected during the entire PCR process rather than at the end of the PCR. In real-time PCR, the reaction is characterized by the time point at which amplification is first detected in the cycle, rather than the target amount accumulated after a fixed number of cycles. Two methods of dye-based detection and probe-based detection are mainly used for quantitative PCR.

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 "allele-specific amplification or gene mutation specific amplification" uses a very efficient method to detect gene mutations. Unlike most other methods for detecting genetic variation or SNP, pre-amplification of target genetic material is not required. ASA combines amplification and detection in one reaction on the basis of distinguishing between matched and mismatched primer/target sequence complexes. The increase in the amplified DNA during the reaction can be monitored in real time by the increase in the fluorescence signal caused by dyes such as SYBR Green I that emit light when combined with the double-stranded DNA. Allele-specific amplification or gene variation-specific amplification by real-time PCR shows a delay or loss of fluorescence signal in the case of mismatches. In genetic variation or SNP detection, it provides information about whether there is genetic variation or SNP.

The "four-primer amplification hindered mutation system PCR" simultaneously amplifies wild-type and mutant alleles together with the control fragment in a single-tube PCR reaction. The non-allele-specific control amplicon was amplified by two common (external) primers flanking the mutation region. Two allele-specific (internal) primers are designed in the opposite direction to the normal primers, and can simultaneously amplify wild-type and mutant amplicons together with the normal primers. As a result, the mutation positions of the two allele-specific amplicons are asymmetrical with the ordinary (external) primers, and therefore have different lengths, which are easy to separate by standard gel electrophoresis. The control amplicon not only fails to amplify, but also provides internal contrast for false negatives, so that at least one of the two allele-specific amplicons is present in the four-primer amplification hindered mutation system PCR.

The "isothermal amplification" means that the amplification of nucleic acid does not need to rely on a thermal cycler, but can be completed at a lower temperature. The ideal state is that the temperature does not need to be changed during the amplification process. The temperature used for isothermal amplification can be between room temperature (22-24°C) and about 65°C, or about 60-65°C, 45-50°C, 37-42°C, or 22-24°C. Isothermal amplification products can be passed through gel electrophoresis, ELISA, ELOSA (Enzyme linked oligosorbent assay), real-time PCR, ECL (improved chemiluminescence), analysis of RNA, DNA and protein or turbidity based Capillary electrophoresis device bioanalyzer (bioanalyzer) detection of wafers.

In an embodiment of the present invention, E507K/R536K, E507K/R660V or E507K/R536K/R660V Taq polymerase is used to confirm the ability of templates containing SNPs (rs1408799, rs1015362 and/or rs4911414) to extend mismatch primers Whether to reduce.

The results are shown in Figure 6 to Figure 8. Compared with E507K Taq polymerase, it can be confirmed that E507K/R536K, E507K/R660V or E507K/R536K/R660V Taq polymerase has delayed amplification caused by mismatched primers, and its effect is in E507K/R536K/R660V Taq polymerase is the most obvious.

This confirms that the three DNA polymerases have higher mismatch selectivity compared with the conventional Taq polymerase (E507K). Therefore, it is expected that the DNA polymerase of the present invention can be effectively applied to the medical diagnosis of diseases and the research of recombinant DNA.

In another embodiment of the present invention, using E507K/R536K/R/R660V Taq polymerase, it was confirmed that the KRAS gene contains Q61H, G13D or G12S and SNP templates and the EGFR gene contains L858R SNP templates. Whether the ability to extend mismatched primers is reduced.

The results are shown in Figures 10 to 13, confirming that the Taq DNA polymerase containing the E507K/R536K/R587I/R660V variant has better mismatch extension selectivity than the Taq polymerase containing the E507K/R536K/R660V variant. It can be seen that the Taq DNA polymerase containing E507K/R536K/R587I/R660V variants of the present invention can be applied to the medical diagnosis of diseases and the research of recombinant DNA.

The present invention also relates to a composition for detecting gene variation or SNP containing the DNA polymerase of the present invention and a PCR kit including the same.

According to an ideal embodiment of the present invention, the PCR kit is suitable for conventional PCR (1st generation PCR), real-time PCR (2nd generation PCR), digital PCR (3rd generation PCR) or nucleic acid mass spectrometry analysis system (MassARRAY) And use.

In the PCR kit of the present invention, the digital PCR may be a competitive allele-specific TaqMan PCR (Competitive allele-specific TaqMan PCR) or a droplet digital PCR (Droplet digital PCR; ddPCR), more specifically, it may be Allele-specific transfection PCR or allele-specific droplet digital PCR, but not limited to this.

The "transfection PCR" is a method to detect and quantify rare mutations in samples containing a large amount of normal wild-type gDNA, combining allele-specific TaqMan® qPCR with allele-specific MGB blockers Later, specificity better than traditional allele-specific PCR can be generated to suppress non-specific amplification from wild-type alleles.

The "microdroplet digital PCR" is a PCR reaction that divides and amplifies 20 μl of 20,000 droplets to calculate the number of target DNA. According to whether the target DNA in the droplet is amplified or not, it is like a digital signal. Receive and calculate the positive droplets (1) and negative droplets (0), calculate the copy number of the target DNA by Poisson distribution, and finally confirm the result value with the number of copies per μl sample. It can also be used for rare mutation detection and very few genes Amplification, detection of mutation types, etc.

The "MassARRAY" is a multiplex analysis method that uses MALDI-TOF mass spectrometry ((MALDI-TOF mass spectrometer) to perform genotyping analysis (genotyping) and other genetic studies, which can be used for low-cost and rapid analysis of multiple genetic bodies. Customized analysis of individual samples and targets or specific targets.

The PCR kit of the present invention may include any reagents or other elements known to those of ordinary skill to be used in the primer extension process.

According to a preferred embodiment of the present invention, the PCR kit includes more than one matched primer, more than one mismatch primer, or more than one matched primer and more than one mismatch primer at the same time. The one or more matched primers and the one or more mismatched primers hybridize with the target sequence, and the mismatched primers, relative to the hybridization target sequence, may include non-matches from its 3'end to the 7th base position. Standard (non-canonical) nucleotides.

The PCR kit of the present invention may also include nucleotide triphosphates.

The PCR kit of the present invention also includes: a) more than one buffer; b) quantification reagent for binding to double-stranded DNA; c) polymerase blocking antibody; d) more than one comparison value or control sequence; and e ) More than one template.

Hereinafter, the present invention will be explained in more detail through examples. These embodiments are merely examples of the present invention, and those with general knowledge in the industry should understand that the patent scope of the present invention is not limited to the embodiments.

Example 1 Induction of Taq polymerase mutation 1-1. Fragment PCR

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

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

The PCR products were confirmed by electrophoresis. As shown in Figure 1(b), the bands of each fragment were confirmed, thereby confirming that the target fragment was amplified.

1-2. Overlap PCR

Using each fragment amplified in 1-1 as a template, primers at both ends (Eco-F and Xba-R primers) were used to amplify the full length. 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 Tag polymerases of "R536K", "R660V", and "R536K/R660V" were amplified.

1-3. Ligation

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

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

Under the conditions shown in Table 8, at room temperature connection (RT) 2 h and transformed into E. coli E.coli DH5 α screening on a medium containing ampicillin. The plastids prepared from the obtained colonies were sequenced, and Taq DNA polymerase mutants ("R536K", "R660V" and "R536K/R660V") into which the desired mutations were introduced were obtained.

Example 2 Introduction of E507K mutation 2-1. Fragment PCR

The activity of the Taq polymerases of "R536K", "R660V" and "R536K/R660V" prepared in Example 1 was tested, and the results confirmed that the activity decreased (data not shown). "And "R536K/R660V" introduced the E507K variant (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 ) The E507K mutation has also been introduced in Taq DNA polymerase. The preparation method of the Taq DNA polymerase with the E507K mutation introduced is the same as in Example 1.

Using the mutation-specific primers shown in Table 9, each fragment (F6 to F7) of Taq DNA polymerase was amplified by PCR as shown in FIG. The reaction conditions are shown in Table 10.

2-2. Overlap PCR

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

2-3. Ligation

Under the conditions shown in Table 5, pUC19 was decomposed with restriction enzyme EcoRI/XbaI at 37°C for 4 hours and the DNA was purified. Under the conditions shown in Table 6, the purified DNA was treated with SAP at 37°C for 1 hour. To prepare the 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 Perform gel extraction together (Figure 4).

Under the conditions shown in Table 8, at room temperature connection (RT) 2 hours or E.coli DH5 α transformed into E. coli and selected on DH10β 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") introducing the desired mutations.

Example 3 Using the DNA polymerase of the present invention to perform qPCR

Using the Taq polymerases each containing the "E507K/R536K", "E507K/R660V" and "E507K/R536K/R660V" mutations obtained in Example 2, it was confirmed that for templates containing SNPs, they extended the mismatched primers. Whether the ability is reduced. As a control group, "E507K" Taq polymerase containing the E507K mutation was used.

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

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

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

The oral epithelial cells were collected using an oral epithelial cell collection kit purchased from Noble Bio, dissolved in 500 μl of lysis solution, and centrifuged at 12,000×g for 3 minutes. 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 fluorescence signals detected in each test tube are combined to calculate and analyze the difference in the cycle (Ct) value of the fluorescence value that reaches the threshold (threshold) on the AB 7500 software (v 2.0.6). It is judged that the longer the Ct value in amplification caused by mismatched primers is delayed, the better the gene variation specificity or allele specificity.

The results of AS-qPCR on rs1408799, rs1015362 and rs4911414 are shown in Figures 6 to 8. Compared with the control group E507K, it can be confirmed that Taq polymerase containing E507K/R536K, E507K/R660V or E507K/R536K/R660V variants is included. In the case of mismatched primers, the amplification delay is the most obvious in the E507K/R536K/R660V mutation.

It is confirmed that the Taq DNA polymerase containing the E507K/R536K, E507K/R660V or E507K/R536K/R660V mutation of the present invention has better mismatch extension selectivity than the Taq polymerase containing the E507K mutation. It can be seen that the three types of Taq DNA polymerases can be effectively used in medical diagnosis of diseases and recombinant DNA research.

Example 4 Introduction of R587I mutation 4-1. Fragment PCR

In order to additionally introduce the R587I mutation in the Taq selection of the "E507K/R536K/R660V" mutation prepared in Example 2 (the 587th amino acid residue in the amino acid sequence of SEQ ID NO: 1 was replaced with arginine) Leucine), the two fragments shown in Figure 9(a) were amplified to PCR using the primers recorded in Table 18 below. The reaction conditions are shown in Table 19.

The results of confirming PCR products on electrophoresis are shown in Fig. 9(b). The bands of each fragment are confirmed and the target fragments are amplified.

4-2. In-fusion cloning

The Taq plastid vector (E507K/R536K/R660V) was decomposed into restriction enzymes KpnI/XbaI under the conditions shown in Table 20 at 37°C for 4 hours, and purified (thawed: 25 μl) to prepare a decomposing linear vector. Then, under the conditions in Table 21, the In-Fusion colonization reaction was carried out at 37°C for 15 minutes and then biotransformed into E.coli DH5α or DH10β, which was selected from the ampicillin-containing medium. The plastids obtained for each selection were sequenced to obtain a Taq DNA polymerase mutant ("E507K/R536K/R587I/R660V") into which the R 587 I mutation was introduced.

Example 5 Using "E507K/R536K/R587I/R660V" Taq polymerase for qPCR 5-1. Differentiation of Q61H variants of KRAS gene

Using the Taq polymerase containing the "E507K/R536K/R587I/R660V" variant obtained in Example 4, it was confirmed whether the ability of the template containing the Q61H SNP in the KRAS gene to extend the mismatch primer was reduced. The comparison group used Taq polymerase containing "E507K/R536K/R660V" variants.

The SNP-containing template is gDNA (104copies, 33ng/rxn) obtained from the HepG2 liver cancer cell line, which is obtained by a conventional DNA extraction method. It was confirmed that the detection target parts were consistent with the NCBI reference sequence (NG_007524.1) and used as wild-type (WT).

The sequence information of the specific primers for the template is shown in Table 22 below.

The qPCR conditions (Applied Biosystems 7500 Fast) are the same as in Table 14 of Example 3.

The probes were labeled as shown in Table 23 below.

The reaction conditions are the same as in Table 16 of Example 3, and the composition of the reaction buffer is shown in Table 24 as follows.

The remaining reaction solutions except for the specific primers in Table 22 were put into two test tubes in the same amount, and each allele-specific primer was added to perform qPCR. At this time, the fluorescent signals measured in each test tube were combined and analyzed with AB7500 software (v2.0.6) to analyze the difference in cycle (Ct) value that reached the calculated and exported threshold fluorescent value. The more delayed the Ct value in the amplification caused by the mismatched primer, the better the gene variation specificity or allele specificity.

The AS-qPCR results are shown in Figure 10(a) and (b). It can be confirmed that compared with the comparison group E507K/R536K/R660V, the Taq polymerase △Ct containing the E507K/R536K/R587I/R660V mutation increased to 5. The amplification delay caused by the matching primer.

In addition, the inventors of the present invention also used the primers in Table 25 below to have a length of 18 mer instead of the primers of 24 mer in Table 22, and repeated the experiment again. Except for using the reaction buffer composition of Table 26 below, all conditions are the same as the experiments performed using the 24mer length primer.

As a result, as shown in Figures 10(c) and 10(d), it can be confirmed that the Taq polymerase containing E507K/R536K/R587I/R660V variants is amplified by mismatched primers compared with the comparison group E507K/R536K/R660V. Increase delay. In particular, the ΔCt of the polymerase introduced with R587I was significantly increased.

5-2. G13D variant differentiation of KRAS gene

Using the Taq polymerase containing the "E507K/R536K/R587I/R660V" variant obtained in Example 4, it was confirmed whether the ability of the template containing the G13D SNP in the KRAS gene to extend the mismatch primer was reduced. The comparison group used Taq polymerase containing "E507K/R536K/R660V" variants.

The SNP-containing template is gDNA (104 copies, 33ng/rxn) obtained from the HepG2 liver cancer cell line, which is obtained by a conventional DNA extraction method. It was confirmed that the detection target parts were consistent with the NCBI reference sequence (NG_007524.1) and used as wild-type (WT).

The sequence information of the specific primers for the template is shown in Table 27 below.

The qPCR conditions (Applied Biosystems 7500 Fast) are the same as in Table 14 of Example 3.

The probes are labeled as shown in Table 28 below.

The reaction conditions are the same as in Table 16 of Example 3, and the composition of the reaction buffer is the same as in Table 24 of Example 5-1.

The remaining reaction solutions except for the specific primers in Table 27 were put into two test tubes in the same amount, and each allele-specific primer was added to perform qPCR. At this time, the fluorescence signals measured in each test tube were combined and analyzed with the AB7500 software (v2.0.6) to analyze the difference in the cycle (Ct) value of the fluorescence value that reached the calculated and exported threshold (threshold). The more delayed the Ct value in the amplification caused by the mismatched primer, the better the gene variation specificity or allele specificity.

The AS-qPCR results are shown in Figure 11, which confirms that the Taq polymerase containing E507K/R536K/R587I/R660V variants has delayed amplification due to mismatched primers compared to the comparison group E507K/R536K/R660V.

5-3. G12S variant differentiation of KRAS gene

Using the Taq polymerase containing the "E507K/R536K/R587I/R660V" variant obtained in Example 4, it was confirmed whether the ability of the template containing the G13S SNP in the KRAS gene to extend the mismatch primer was reduced. The comparison group used Taq polymerase containing "E507K/R536K/R660V" variants.

The SNP-containing template is gDNA (104copies, 33ng/rxn) obtained from the HepG2 liver cancer cell line, which is obtained by a conventional DNA extraction method. It was confirmed that the detection target parts were consistent with the NCBI reference sequence (NG_007524.1) and used as wild-type (WT).

The sequence information of the specific primers for the template is shown in Table 29 below.

The qPCR conditions (Applied Biosystems 7500 Fast) are the same as in Table 14 of Example 3.

The probes were labeled as shown in Table 30 below.

The reaction conditions are the same as in Table 16 of Example 3, and the composition of the reaction buffer is the same as in Table 24 of Example 5-1.

The remaining reaction solutions except for the specific primers in Table 29 were put into two test tubes in the same amount, and each allele-specific primer was added to perform qPCR. At this time, the fluorescent signals measured in each test tube were combined and analyzed with AB7500 software (v2.0.6) to analyze the difference in cycle (Ct) value that reached the calculated and exported threshold fluorescent value. The more delayed the Ct value in the amplification caused by the mismatched primer, the better the gene variation specificity or allele specificity.

The AS-qPCR results are shown in Figure 12, and it can be confirmed that compared with the comparison group E507K/R536K/R660V, the Taq polymerase containing E507K/R536K/R587I/R660V variants has delayed amplification due to mismatched primers.

5-4. L858R variant differentiation of EGFR gene

Using the Taq polymerase containing the "E507K/R536K/R5871/R660V" variant obtained in Example 4, it was confirmed whether the ability of the template of the SNP containing L858R in the EGFR gene to extend the mismatched primer was reduced. The comparison group used Taq polymerase containing "E507K/R536K/R660V" variants.

Said template containing the SNP is obtained from the HepG2 hepatoma cell line ^{gDNA (10 4 copies, 33ng /} rxn), obtained by conventional DNA extraction methods. It was confirmed that the detection target parts were consistent with the NCBI reference sequence (NG_007726.3) and used as wild-type (WT).

The sequence information of the specific primers for the template is shown in Table 31 below.

The qPCR conditions (Applied Biosystems 7500 Fast) are the same as in Table 14 of Example 3.

The probes were double-labeled as shown in Table 32 below.

The reaction conditions are the same as in Table 16 of Example 3, and the composition of the reaction buffer is the same as in Table 24 of Example 5-1.

The remaining reaction solutions except for the specific primers in Table 31 were put into two test tubes in the same amount, and each allele-specific primer was added to perform qPCR. At this time, the fluorescent signals measured in each test tube were combined and analyzed with AB7500 software (v2.0.6) to analyze the difference in cycle (Ct) value that reached the threshold fluorescent value calculated and exported. The more delayed the Ct value in the amplification caused by the mismatched primer, the better the gene variation specificity or allele specificity.

The AS-qPCR results are shown in Figure 13, and it can be confirmed that compared with the comparison group E507K/R536K/R660V, the Taq polymerase containing E507K/R536K/R587I/R660V variants has delayed amplification due to mismatched primers.

As described above, it was confirmed that the Taq DNA polymerase containing the E507K/R536K/R587I/R660V variant of the present invention has superior mismatch extension selectivity compared to the Taq polymerase containing the E507K/R536K/R660V variant in some cases . It can be seen that the Taq DNA polymerase containing the E507K/R536K/R587I/R660V variant of the present invention can be effectively used in medical diagnosis of diseases and recombinant DNA research.

<110> GeneKester Co., Ltd.

<120> DNA polymerase with improved gene mutation specific amplification efficiency

<150> KR 10-2017-0088373

<151> 2017-07-12

<160> 55

<170> PatentIn version 3.5

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Claims (14)

A DNA polymerase with Taq polymerase consisting of an amino acid sequence with sequence identification number 1. This DNA polymerase includes: the 507th amino acid residue in the amino acid sequence with sequence identification number 1. The substitution of the 536th amino acid residue, and the 660th amino acid residue substitution of a Tag polymerase, wherein the 507th amino acid residue is replaced by lysine (K ) Substitute glutamine (E), the 536th amino acid residue is replaced by lysine (K) for arginine (R), and the 660th amino acid residue is replaced Is the substitution of valine (V) for arginine (R), where the DNA polymerase contains a matching primer for the amplification of the target sequence, which shows a higher Ct value than the amplification of the target sequence for the mismatched primer. The Ct value is low, and a difference between the Ct value displayed by the amplification of the target sequence containing the mismatched primer and the Ct value displayed by the amplification of the target sequence containing the matched primer is at least 16.7. The DNA polymerase according to item 1 of the patent application, wherein the DNA polymerase distinguishes between matched primers and mismatched primers, and the matched primers and mismatched primers hybridize with the target sequence, and the mismatched primers The 3'end includes atypical nucleotides. A nucleic acid sequence encoding the DNA polymerase described in item 1 of the scope of the patent application. A vector including the nucleic acid sequence described in item 3 of the scope of the patent application. A host cell containing the vector described in item 4 of the scope of the patent application. An in vitro method for detecting gene mutations or SNPs, including contacting the DNA polymerase described in item 1 of the scope of the patent application with the following: a) one or more templates; b) more than one matched primer, more than one Mismatched primers or both of one or more matched primers and more than one mismatched primers; and c) nucleotide triphosphates; wherein the one or more matched primers and mismatched primers are hybridized with the target sequence, Detect more than one gene variation or SNP in vitro in more than one template. The method described in item 6 of the scope of patent application includes the use of dual-strand specific dyes for deconstruction temperature analysis. The method as described in item 6 of the scope of the patent application, wherein the analysis in agarose gel after the standard PCR, the specific amplification of gene variation by the real-time PCR, the allele-specific amplification of the real-time PCR , Four-primer amplification-hindered mutation system PCR or isothermal amplification to complete. A composition for detecting genetic variation or SNP containing the DNA polymerase described in item 1 of the scope of patent application. A PCR kit containing the composition described in item 9 of the scope of the patent application. The PCR kit as described in item 10 of the scope of patent application, wherein the PCR kit can be used in a competitive allele-specific TaqMan PCR, droplet digital PCR or nucleic acid mass spectrometry analysis system. The PCR kit as described in item 10 of the scope of patent application, which includes more than one matching primer, more than one mismatch primer, or both more than one matching primer and more than one mismatch primer, the more than one matching primer Hybridize with more than one mismatch primer to the target sequence. As the PCR kit described in item 10 of the scope of patent application, it may also include nucleotide thiophosphate. The PCR kit described in item 10 of the scope of the patent application may further include: a) one or more buffers; b) a quantification reagent that binds to double-stranded DNA; c) polymerase blocking antibody; d) More than one comparison value or comparison sequence; and e) one or more templates.

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