KR102265417B1 - Primer for multiple analysis of single nucleotide polymorphism - Google Patents

Abstract

The present invention relates to a primer for multiple analyses of single nucleotide polymorphism and, more specifically, to: a primer for multiple analyses of single nucleotide polymorphism, which has a novel structure including a duplex amplification refractory mutation system (D-ARMS) structure capable of specifically amplifying different genotypes, a probe specific binding extension oligonucleotide (PBO) structure capable of genotype-specific binding to a probe in a microarray DNA chip without being involved in an amplification reaction, and a high-temperature activation primer (H-TAP) structure useful for a simultaneous amplification polymerase chain reaction (PCR) and microarray hybridization; and to utilization of the primer. According to the present invention, when a multiplex PCR and microarray DNA chip of a single nucleotide polymorphism gene are performed by using the primer of the novel structure, a genotyping result with high specificity can be acquired in one reaction.

Description

Primer for multiple analysis of single nucleotide polymorphism

The present invention relates to a primer for multiplex analysis of single nucleotide polymorphisms, and more particularly, a D-ARMS (Duplex Amplification Refractory Mutation System) structure capable of specifically amplifying different genotypes, a microarray without being involved in the amplification reaction A novel structure including a probe specific binding extension oligonucleotide (PBO) structure capable of genotype-specific binding to a probe in a DNA chip and a high-temperature activation primer (H-TAP) structure useful for simultaneous amplification PCR and microarray hybridization It relates to a primer for multiplex analysis of single nucleotide polymorphisms and their utilization.

Since 1990, the Human Genome Project has been officially launched by the National Institutes of Health and the U.S. Department of Energy to identify the complete DNA sequence of the human genome. It has become possible to provide a genetic epidemiologic method for the analysis and evaluation of the human genome by identifying the nucleotide sequence of the dog. In subsequent studies, interest in the type and frequency of genetic modification increased, and research to discover a large amount of SNPs began, and in the 2000s, a total of 1.8 million single nucleotide polymorphisms (SNPs) were discovered. After that, a study was conducted to compare genetic markers in the genomes of the normal group and the disease group by completing the genetic map between different ethnic groups and confirming SNPs across all genomes. It was confirmed that there would be a relationship between individual differences such as different appearances and external appearances, genetic diseases, susceptibility to diseases, and side effects to drugs.

A genetic change or mutation in which one nucleotide sequence differs in the DNA nucleotide sequence is called a single nucleotide polymorphism (SNP) and is called a snip. SNP is a genetic variant in which two alleles are combined with each other, and one base (wild-type) is replaced by another (mutant) at a specific base-pairing site. These SNPs can create genetic diversity in people. That is, some people have A (Adenine) in the protein synthesized according to a certain nucleotide sequence, while others have C (Cytosine). Due to these minute mutations, the function of the gene changes and the phenotype changes, making people with different looks and shapes, and even creating differences in susceptibility to diseases. SNP gene polymorphisms account for most of the 0.1% different genetic polymorphisms in human genes, and through analysis of these polymorphisms, it can have an important impact on personalized medicine such as disease prevention, diagnosis, and treatment.

SNPs are very useful for high-efficiency gene analysis due to their low mutation rate, high incidence in the human genome, and high intergenerational stability. In addition, it is a highly reliable genetic polymorphism that exists almost uniformly on the genome to search for genes related to susceptibility to diseases or side effects to drugs. For example, if a genotype is compared between a diseased group and a non-diseased control group, a specific genotype is correlated with the genotype of the diseased group. can be found

SNP genotype analysis techniques include SSCP (Single Strand Conformation Polymorphism), AFLP (Amplified Fragment Length Polymorphism), RFLP (Restriction Fragment Length Polymorphism), RAPD (Random Amplified Polymorphic DNAs), AS-PCR (Allele-Specific PCR), Tm-shift genotyping using GC-tail primer, DASH (Dynamic Allele-Specific Hybridization), TaqMan 5'-nuclease assay, Molecular Beacons, OLA (Oligonucleotide Ligase Assay), Invader Assay (Invasive Cleavage of Oligonucleotide Probes), Pyrosequencing, Single There are Base Extension, Fluorescence Polarization, and MALDI-TOF Mass Spectrometry technologies. Representative methods currently commercialized and used include ARMS PCR technology products, TaqMan probe technology products, and NGS technology analysis services.

ARMS (Amplification Refractory Mutation System) PCR technology is a technology to increase specificity in the PCR reaction process by making a mismatch oligo primer by changing the 2nd or 3rd base of the SNP and its neighboring genotype. to be. To identify different genotypes, after individual PCR for each genotype, each genotype-specific oligo primer is amplified through electrophoresis, respectively, in the normal group, the risk group, or both the normal group and the risk group to determine the genotype. The T-ARMS (Tetra primer Amplification Refractory Mutation System) PCR technology product, which has recently developed further general ARMS PCR, is an amplified product by dividing ARMS PCR oligo primers for the genotypes of normal and risk groups in the upstream and downstream directions of the SNP location. It is a product that can confirm the genotype by designing different sizes of the genotypes, and it is a product that can check one or two SNP genotypes with one amplification and electrophoresis.

TaqMan probe technology is a method using the 5' nuclease characteristics of Taq polymerase, and TaqMan fluorescent probes responding to each genotype and univesal PCR primers that can amplify them are used for the reaction. TaqMan probes do not show fluorescence when both reporter dye and quencher dye are bound, but TaqMan probes that are completely complementary to the SNP site are bound in the annealing step of PCR and 5' nuclease activity of Taq polymerase in the extension step. The reporter dye is separated and the fluorescence reaction is accumulated in proportion to the number of newly synthesized DNA strands. Accumulation of each fluorescence is checked in real time on a real-time PCR device, and the genotype is read by comparing the Ct values displayed by varying the fluorescence of TaqMan probes corresponding to the normal group and the risk group. When checking the results on Real-time PCR with TaqMan probe technology, when the genotype is normal, the Ct value of the normal group fluorescence is forward, the Ct value of the risk group fluorescence is backwards, and when the genotype is the risk group, it is reversed. Also, if the Ct values of the normal group and the risk group are similar, it can be confirmed as a heterotype having two genotypes at the same time.

The NGS technology analysis service can quickly analyze a large amount of nucleotide sequence using the NGS sequencing device and nucleotide sequence database, and is a method of analyzing SNPs using this. However, it is difficult to apply it in terms of cost and effort to use it to analyze 10 or less SNPs, which are not large in scale, and most of them have the disadvantage of having to request an analysis service from an analysis company with expensive equipment.

Although SNP analysis techniques such as the above methods have been introduced, most of the technologies used for products have limitations in the extent to which each genotype in SNPs or one SNP can be analyzed simultaneously, and multiple SNPs are simultaneously analyzed. It has disadvantages that cannot be analyzed. Although the NGS sequencing service can check the nucleotide sequences and SNPs for several genes at the same time, it has inefficient limitations in terms of cost and effort. There are registered patents related to microarray gene chips or DNA chips for SNP analysis, which introduces technologies that allow simultaneous amplification and simultaneous confirmation of SNPs, but commercialized products have not been identified. Although there are several limitations in the commercialization of technology, the main reason is that the probe for confirming SNP genotyping does not accurately distinguish a single base and shows a non-specific reaction, as can be seen in the example of confirming the result of the TaqMan probe technology. However, TaqMan probe fluorescence shows a difference in Ct value due to a difference in one nucleotide sequence and can be analyzed separately. However, it is difficult to distinguish between a probe of a gene chip or DNA chip of the same principle, and a non-specific reaction occurs according to minute changes in conditions. Since there is a high probability of appearing, it is difficult to manufacture by controlling the SNPs of several genes under the same conditions, and there is a limit to commercialization.

In the present invention, as a method for simultaneously performing genotyping on several SNPs, a specific oligonucleotide structure of a single nucleotide polymorphism microarray probe that is effective for simultaneous amplification and can be used for microarrays was developed.

As a result of repeated intensive studies to develop a primer and a kit including the same that can determine the genotype of a SNP simultaneously and in multiple ways, the present inventors have produced and utilized a primer set showing a special structure to achieve this object. It was found that the present invention was completed.

Accordingly, it is an object of the present invention to provide a primer represented by the following general formula (1):

[General formula 1]

5'-Ap-Bq-Cr-Ds-E-F-G-3'

(In the above formula,

A represents a site comprising a nucleotide having a nucleotide sequence complementary to the continuous nucleotide sequence at the 3' end of the primer,

B represents a site including a nucleotide having an arbitrary base sequence,

Wherein C represents deoxyuridine (Deoxyuridine),

D represents a region containing nucleotides having a nucleotide sequence complementary to a specific continuous nucleotide sequence of the template nucleic acid,

Wherein E and F are nucleotides complementary or non-complementary to the template nucleic acid, respectively, and only either E or F is complementary to the template nucleic acid,

wherein G represents a nucleotide complementary to a wild-type or mutant-type base of a single nucleotide polymorphism (SNP) site,

The p, q, r and s represent the number of nucleotides included in each site.)

Another object of the present invention is to provide a primer set comprising a first primer according to claim 1 and a second primer represented by the following general formula 2:

[General Formula 2]

5'-ap-bq-cr-ds-e-f-g-3'

(In the above formula,

wherein a represents a site comprising a nucleotide having a nucleotide sequence complementary to the continuous nucleotide sequence at the 3' end of the primer,

b represents a site including a nucleotide having an arbitrary base sequence,

Wherein c represents deoxyuridine (Deoxyuridine),

wherein d represents a region containing nucleotides having a nucleotide sequence complementary to a specific continuous nucleotide sequence of the template nucleic acid;

Wherein e and f are nucleotides complementary or non-complementary to the template nucleic acid, respectively, and only one of e or f is complementary to the template nucleic acid, and at least one of e and f is the corresponding E and It is a nucleotide of a base different from F,

wherein g is a nucleotide complementary to a wild-type or mutant-type base of a single nucleotide polymorphism (SNP) site of the template nucleic acid, and is a base different from G of the first primer;

The p, q, r and s represent the number of nucleotides included in each site.)

Another object of the present invention is a primer set according to the above; and a probe comprising a base sequence complementary to Bq of the first primer and a probe comprising a base sequence complementary to bq of the second primer, comprising a support immobilized on the surface, providing a single nucleotide polymorphism genotyping kit will do

Another object of the present invention is to provide a method for genotyping one or more single nucleotide polymorphisms in a nucleic acid sample, comprising the steps of:

(a) treating a mixture containing a template nucleic acid, a polymerase, and the primer set according to claim 11 with Uracil DNA Glycosylase (UDG) to remove the uracil base of deoxyuridine in the primer step;

(b) amplifying the template nucleic acid;

(c) hybridizing the amplification product with a probe including a nucleotide sequence complementary to Bq of the first primer and a probe including a nucleotide sequence complementary to bq of the second primer; and

(d) determining the genotype of the single nucleotide polymorphism in the nucleic acid sample through signal detection according to the hybridization.

In order to achieve the above object of the present invention, the present invention provides a primer represented by the following general formula 1:

[General formula 1]

5'-Ap-Bq-Cr-Ds-E-F-G-3'

(In the above formula,

A represents a site comprising a nucleotide having a nucleotide sequence complementary to the continuous nucleotide sequence at the 3' end of the primer,

B represents a site including a nucleotide having an arbitrary base sequence,

Wherein C represents deoxyuridine (Deoxyuridine),

D represents a region containing nucleotides having a nucleotide sequence complementary to a specific continuous nucleotide sequence of the template nucleic acid,

Wherein E and F are nucleotides complementary or non-complementary to the template nucleic acid, respectively, and only either E or F is complementary to the template nucleic acid,

wherein G represents a nucleotide complementary to a wild-type or mutant-type base of a single nucleotide polymorphism (SNP) site,

The p, q, r and s represent the number of nucleotides included in each site.)

In order to achieve the other object of the present invention described above, the present invention provides a primer set comprising a first primer according to claim 1 and a second primer represented by the following general formula 2:

[General Formula 2]

5'-ap-bq-cr-ds-e-f-g-3'

(In the above formula,

wherein a represents a site comprising a nucleotide having a nucleotide sequence complementary to the continuous nucleotide sequence at the 3' end of the primer,

b represents a site including a nucleotide having an arbitrary base sequence,

Wherein c represents deoxyuridine (Deoxyuridine),

wherein d represents a region containing nucleotides having a nucleotide sequence complementary to a specific continuous nucleotide sequence of the template nucleic acid;

Wherein e and f are nucleotides complementary or non-complementary to the template nucleic acid, respectively, and only one of e or f is complementary to the template nucleic acid, and at least one of e and f is the corresponding E and It is a nucleotide of a base different from F,

wherein g is a nucleotide complementary to a wild-type or mutant-type base of a single nucleotide polymorphism (SNP) site of the template nucleic acid, and is a base different from G of the first primer;

The p, q, r and s represent the number of nucleotides included in each site.)

In order to achieve another object of the present invention, the present invention provides a primer set according to the above; and a probe comprising a base sequence complementary to Bq of the first primer and a probe comprising a base sequence complementary to bq of the second primer, comprising a support immobilized on the surface, providing a single nucleotide polymorphism genotyping kit do.

In order to achieve another object of the present invention, the present invention provides a method for genotyping one or more single nucleotide polymorphisms in a nucleic acid sample, comprising the steps of:

(a) treating a mixture containing a template nucleic acid, a polymerase, and the primer set according to claim 11 with Uracil DNA Glycosylase (UDG) to remove the uracil base of deoxyuridine in the primer step;

(b) amplifying the template nucleic acid;

(c) hybridizing the amplification product with a probe including a nucleotide sequence complementary to Bq of the first primer and a probe including a nucleotide sequence complementary to bq of the second primer; and

(d) determining the genotype of the single nucleotide polymorphism in the nucleic acid sample through signal detection according to the hybridization.

Hereinafter, the present invention will be described in detail.

The present invention provides a primer represented by the following general formula (1):

[General formula 1]

5'-Ap-Bq-Cr-Ds-E-F-G-3'

(In the above formula,

A represents a site comprising a nucleotide having a nucleotide sequence complementary to the continuous nucleotide sequence at the 3' end of the primer,

B represents a site including a nucleotide having an arbitrary base sequence,

Wherein C represents deoxyuridine (Deoxyuridine),

D represents a region containing nucleotides having a nucleotide sequence complementary to a specific continuous nucleotide sequence of the template nucleic acid,

Wherein E and F are nucleotides complementary or non-complementary to the template nucleic acid, respectively, and only either E or F is complementary to the template nucleic acid,

wherein G represents a nucleotide complementary to a wild-type or mutant-type base of a single nucleotide polymorphism (SNP) site,

Wherein p, q, r and s represent the number of nucleotides.)

The primer provided by the present invention is characterized in that it provides three structural features and advantageous actions resulting therefrom.

The structural features of the primers provided by the present invention and advantageous effects resulting from each structural feature will be described.

1) H-TAP (High-temperature Activation Primer) structure

The H-TAP structure including the A site and the C site in the primer of Formula 1 provided by the present invention suppresses unintended PCR amplification at room temperature, and the nucleic acid amplification reaction is performed after reaching the desired reaction temperature. It is characterized in that the primers remaining unused in the PCR reaction form a self-binding structure in the hybridization reaction with the microarray DNA chip and do not affect it.

In the present invention, A is a site comprising nucleotides having a nucleotide sequence complementary to the continuous nucleotide sequence at the 3'-end of the primer according to Formula 1, the sequence of which is determined according to the 3'-terminal nucleotide sequence do. The p represents the number of nucleotides included in A, preferably, the p may be 3 to 8, most preferably 3 to 5.

The number of nucleotides included in p, that is, A may be determined from 3 to 8 depending on the desired Tm value of A, and in the present invention, the Tm of A is 10 to 30° C., preferably 10 to 25° C. , most preferably 15 to 25 ℃ can be set.

Since the Tm value of A in the primer of the present invention is 10 to 30 ° C., at room temperature conditions before proceeding with the polymerase chain reaction (PCR), the A is the 3'-terminal nucleotide sequence and It is bound to form a primer of a closed structure (ie, a dumbbell structure or a hairpin structure).

Therefore, because the primer exists in a closed structure at room temperature before the polymerase chain reaction, non-specific binding to the template nucleic acid does not occur, and the case of binding to the polymerase that recognizes the 3' end of the primer is reduced, resulting in an unwanted reaction product formation can be prevented.

After that, when the polymerase chain reaction is initiated and the appropriate reaction temperature is reached, the bond between A and the 3'-end nucleotide sequence, which shows a relatively low Tm value of 10 to 30°C, is broken, and the structure of the primer is in an unfolded form. It is modified to enable complementary binding to the template nucleic acid similar to the action of a conventional primer.

In the present invention, C represents deoxyuridine. In addition, r represents the number of deoxyuridine included in C. In the present invention, r may be 1 to 5, preferably 1 to 3, and most preferably 1 to 1.

In the primer of the present invention, C is a portion that does not affect the structure of the primer and does not complement the template nucleic acid, and is treated with UDG (Uracil DNA Glycosylase) before initiating the polymerase chain reaction to uracil. This is the part where the base is removed and the nucleotide sequence in the primer is induced.

The principle of operation of the H-TAP structure is expressed in time series as follows:

(i) At room temperature before the start of the polymerase chain reaction, the primer has a closed structure with its 5' end and 3' end complementarily bound by the A structure, so that it is non-specific binding with the template nucleic acid and polymerase binding is prevented.

(ii) Before the start of the polymerase chain reaction, the uracil base of the C part is removed by UDG (Uracil DNA Glycosylase), which is added to prevent cross-contamination, so that the base in the primer is cut off.

(iii) When the reaction temperature rises after the start of the polymerase chain reaction, the complementary bond between the 5' and 3' ends in the primer is broken, and the D region complementary to the template nucleic acid in the primer forms a bond with the template nucleic acid. Thereafter, nucleic acid amplification proceeds according to the same principle as a conventional primer.

(iv) Due to the cleavage of the uracil base at the C site in the primer, replication does not occur up to the A base sequence added to the 5' end in the nucleic acid amplification process after the start of the PCR reaction, so that a constant reaction efficiency is maintained without a change in the Tm value. .

(v) When the temperature is lowered after the PCR reaction is finished, the excess primers that do not participate in the amplification reaction form a closed structure again, so that in the process of hybridizing the PCR amplification product with the probe, the excess primers are separated from the probe Hybridization can be prevented.

The primer according to the present invention has the advantage that stable amplification is possible even if it is applied to multiplex PCR according to the structural features and operating principle.

In the case of multiplex PCR, a plurality of template nucleic acids and a plurality of primer pairs are mixed and a nucleic acid amplification reaction proceeds at the same time. Therefore, the most important of the conditions for obtaining a desired detection result through multiplex PCR is that all primers should exhibit similar Tm values.

On the other hand, in the case of the previously reported hairpin type primer or dumbbell type primer, there is a positive aspect in that it can prevent a non-specific reaction, but an additional nucleotide sequence capable of complementary binding to the 3' end of the primer is 5 'Because it is added at the end, there may be a problem that the Tm value increases as the nucleic acid amplification reaction proceeds. In this case, since the same increase in Tm value does not appear in all primer pairs, as the nucleic acid amplification reaction proceeds, the reaction activity of a specific primer pair increases while the activity of other primer pairs decreases, so that an objective detection result may not be obtained. have.

In contrast, in the case of the primer according to the present invention, a nucleotide cleavage site is generated by UDG (Uracil DNA Glycosylase) treatment before the start of the polymerase chain reaction, and as a result, a nucleotide sequence added to the 5' end for a closed structure Since it becomes impossible to act as a template for amplification, the Tm value of all primers can be kept constant from the beginning to the end of the reaction.

Therefore, when the primers according to the present invention are used for multiplex PCR, the activity of each primer optimized in the initial reaction conditions can be maintained the same until the end of the reaction.

2) PBO (Probe specific Binding extension Oligonucleotide) structure

The PBO structure including the B site in the primer of Formula 1 provided by the present invention is any nucleotide sequence capable of genotype-specific binding to the probe in the microarray DNA chip, and is an H-TAP structure, the A site and C located between the parts. Since the uracil base of the C part is removed through the UDG enzyme reaction, which is a decontamination step before the PCR amplification reaction, the amplification reaction does not occur to the A site and the B site in the primer.

Therefore, at one end of the amplification product generated using the primer of Formula 1 according to the present invention, the PBO structure site remains in the form of a single-strand, and as a result, the amplification product is denatured to form a single end. There is an advantage that hybridization is possible with a probe having a sequence complementary to the B site itself without forming a strand.

In the primer provided by the present invention, the PBO structure may include only the B site, or it may be understood that both A and B sites are included.

Specifically, when hybridizing a probe immobilized on a support and a PCR amplification product generated using the primer of the present invention, the probe has a sequence complementary to the B site sequence of the primer according to Formula 1, or the A Hybridization of the amplification product and the probe may be possible without a separate denaturation procedure by making it to have a sequence complementary to the region and the B region sequence.

In the primer provided by the present invention, the sequence of the B site is any sequence, and q means the number of nucleotides included in the B site, which may be 15 to 40, and Tm may be 55 to 70 ° C. have. Preferably, the Tm of the site B is 60 ~ 70 ℃, most preferably, the Tm of the region B may be configured such that the base sequence is 63 ~ 65 ℃.

The primer of the general formula 1 structure provided by the present invention can be detected by distinguishing a plurality of amplification products by differently preparing the sequence of the B region. For example, when preparing a primer for the purpose of discriminating and detecting wild type and mutant type in a template nucleic acid containing SNP, the sequence of the B site is prepared differently in the wild type and mutant type amplification primers, thereby hybridizing with the probe thereafter. It may be possible to distinguish and detect in the process.

That is, it is possible to simultaneously distinguish and detect various types of amplification products through the PBO structure including the B site.

In one embodiment of the present invention, it has been confirmed that the genotypes of SNPs present in six genes, that is, 12 different amplification products, can be simultaneously distinguished and detected through the primers provided by the present invention.

On the other hand, in the primer of Formula 1 provided by the present invention, the D region represents a portion including a nucleotide having a nucleotide sequence complementary to a specific continuous nucleotide sequence of the template nucleic acid. In addition, s represents the number of nucleotides included in D. In the present invention, s may be 15 to 30, and may be appropriately selected by those skilled in the art according to the Tm value of the desired primer. In the present invention, the Tm value of D may be 50 ~ 65 ℃. That is, the D region corresponds to a region of a sequence complementary to a template nucleic acid in a general primer.

3) D-ARMS (Duplex Amplification Refractory Mutation System) structure

The D-ARMS structure included in the primer of the present invention includes E, F and G in the primer of Formula 1, and refers to three nucleotides present at the 3' end of the primer of Formula 1 above. This is a PCR reaction by making mismatch oligo primers by changing the nucleotides of the single nucleotide polymorphism in the template nucleic acid and the first or second nucleotides adjacent thereto from those of the template so that different SNP genotypes can be distinguished and specifically amplified. It is a structure with increased specificity in the process.

This is a structure developed from the conventional oligo structure for ARMS PCR, and is a structure capable of amplifying SNP wild-type and mutant-type genes at the same time. That is, after the primary amplification of the template during PCR synthesis, even after the DNA nucleotide sequence of the secondary amplification product is replaced with the nucleotide sequence of the artificially manufactured primer, it can bind specifically without affecting it.

In the primer of Formula 1 provided by the present invention, E and F are nucleotides that are complementary or non-complementary to the template nucleic acid, respectively, and either one of E or F is complementary to the template nucleic acid,

G represents a nucleotide complementary to a wild-type or mutant-type base of a single nucleotide polymorphism (SNP) site.

The D-ARMS structure in the primers provided by the present invention is for the purpose of simultaneously detecting wild-type and mutant-type genes of SNPs included in the template nucleic acid, and its usefulness is demonstrated in a multiplex PCR reaction using primers specific for each. structure that can be

After the primer binds to the template nucleic acid, the sequence at the 3' end of the primer is very important for DNA synthetase to initiate replication. That is, if only one nucleotide at the 3' end of the primer of a sequence complementary to the template nucleic acid is not complementary to the template nucleic acid, the DNA synthetase can initiate replication, but three nucleotides present at the 3' end of the primer If two or more of the bases are not complementary to the template nucleic acid, replication is not initiated.

However, the sequence of the SNP site present in the template nucleic acid is different from the wild type and the mutant type, and when a primer that specifically binds to each SNP is prepared, the 3' end of the primer specific for each SNP wild type and mutant type is only one step. Only one sequence will show a difference. In this case, even if a primer specific to the wild type shows only one sequence difference between the mutant type template nucleic acid and the 3' end, there arises a problem of initiating replication.

To solve this problem, commercial ARMS primers change the first or second neighboring nucleotide in the 5' direction with respect to the SNP site of the template nucleic acid to a nucleotide of a base that is not complementary to the template nucleic acid. A specific primer shows a difference between the mutant type template nucleic acid and two nucleotide sequences at the 3' end, whereas a primer specific for the SNP mutant type shows a difference between the wild type template nucleic acid and two nucleotide sequences at the 3' end, They have been designed so that they do not cross each other to initiate replication.

However, such a conventional ARMS primer amplifies a template nucleic acid using only a primer specific for the SNP wild type, and then amplifies the template nucleic acid using only a primer specific for the SNP mutant type through a separate procedure. There is no particular problem when analyzing the genotype, but there is a limitation that the wild type and the mutant type are not distinguished in the final amplification product when the amplification reaction is performed simultaneously in a multiplex using primers specific for each SNP wild type and mutant type. That is, from the second amplification reaction after the template nucleic acid is amplified, the nucleic acid containing the primer sequence becomes the template and amplification starts, so the conventional D-ARMS primer specific for each SNP wild type and mutant type is specific to the SNP wild type. The 3' end of the primer shows only one nucleotide sequence difference from the primary amplified SNP mutant template nucleic acid (that is, the primary amplification product in which a primer specific for the SNP mutant type is bound to the 5' end), resulting in a cross-amplification reaction this will be initiated As a result, although the amplification reaction was performed using D-ARMS primers specific for each SNP wild type and mutant type, the final amplification product cannot be distinguished.

The D-ARMS structure included in the primer of the general formula 1 provided by the present invention solves the problems of the prior art, and even if wild-type and mutant SNPs are simultaneously amplified in a multiplex, each can be separately amplified. have.

The primer of the general formula 1 provided by the present invention exhibits functional usefulness by organically combining the structural features of 1) to 3) above, and when any one of the structures of 1) to 3) is not provided. The effect of simultaneously determining the genotype of the SNP as a multiplex may not be achieved.

The primer provided by the present invention may be characterized as a primer for multiplex polymerase chain reaction (SNP multiplex PCR) for detecting single nucleotide polymorphisms.

In addition, the primer provided by the present invention may be characterized as a primer for a microarray DNA chip.

The term “primer” used in the present invention is a nucleic acid sequence having a short free 3' hydroxyl group, which can form a complementary template and base pair and is used for copying the template strand. It refers to a short nucleic acid sequence that serves as a starting point. The primer can initiate DNA synthesis in the presence of a reagent for polymerization (DNA polymerase or reverse transcriptase) and four different deoxynucleoside triphospates (dNTPs) in an appropriate buffer and temperature.

The primer of the present invention can be chemically synthesized using a phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, "encapsulation", substitution of one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phospho poroamidates, carbamates, etc.) or charged linkages (eg phosphorothioates, phosphorodithioates, etc.). Nucleic acids may contain one or more additional covalently linked residues, e.g., proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalating agents (e.g., acridine, psoralen, etc.) ), chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents.

The present invention also provides a primer set comprising a first primer according to the general formula (1) and a second primer represented by the following general formula (2):

[General Formula 2]

5'-ap-bq-cr-ds-e-f-g-3'

(In the above formula,

wherein a represents a site comprising a nucleotide having a nucleotide sequence complementary to the continuous nucleotide sequence at the 3' end of the primer,

b represents a site including a nucleotide having an arbitrary base sequence,

Wherein c represents deoxyuridine (Deoxyuridine),

wherein d represents a region containing nucleotides having a nucleotide sequence complementary to a specific continuous nucleotide sequence of the template nucleic acid;

Wherein e and f are nucleotides complementary or non-complementary to the template nucleic acid, respectively, and only one of e or f is complementary to the template nucleic acid, and at least one of e and f is the corresponding E and It is a nucleotide of a base different from F,

wherein g is a nucleotide complementary to a wild-type or mutant-type base of a single nucleotide polymorphism (SNP) site of the template nucleic acid, and is a base different from G of the first primer;

The p, q, r and s represent the number of nucleotides included in each site.)

As for the general description of ap, bq, cr ming ds in the general formula 2, the description of the primer of the general formula 1 may be equally applied.

In Formula 2, p, q, r, and s may be the same as or different from p, q, r, and s in Formula 1, which can be easily changed by a person skilled in the art to produce an optimal primer You can choose.

The difference between the first primer and the second primer in the primer set provided by the present invention will be described in detail.

The function of the b site in the primer of Formula 2 is the same as the function of the B site in the first primer of Formula 1 above. That is, it is a single-stranded nucleic acid structure having a nucleotide sequence that can hybridize with the probe itself without denaturing the final amplification product.

In the primer set, the B of the first primer and the b of the second primer have different sequences, so that the SNP wild type and the mutant type can be easily distinguished by using a probe capable of complementary binding to each of them.

In the primer of Formula 2, e and f are nucleotides complementary or non-complementary to the template nucleic acid, respectively, and only one of e or f is complementary to the template nucleic acid, and at least one of e and f corresponds to each other Nucleotides of different bases from E and F of the first primer, and g is a nucleotide complementary to a wild-type or mutant base of a single nucleotide polymorphism (SNP) site of a template nucleic acid, and is a base different from G of the first primer .

The regions e, f and g are modified D-ARMS structures corresponding to E, F and G of the first primer, respectively, and are SNP wild-type structures by the modified D-ARMS structures of the first and second primers. Since the specific primer can selectively amplify only the wild type and the mutant type specific primer can selectively amplify only the mutant type, it is possible to simultaneously amplify the SNP wild type and the mutant type in a multiplex.

The sequence of the modified D-ARMS region of the first primer and the second primer will be described with specific examples:

In the template nucleic acid, assuming that the base sequence of three nucleotides in the 5' direction with respect to the SNP site is AAT (wild type) and AAC (mutant), the EFG site of the first primer specific for the SNP wild type is, for example, For example, it may be constructed with the sequence of ATT. In this case, e-f-g of the second primer specific for the SNP mutant type may be AGC, ACC, TAC, GAC or CAC.

Such an example shows any one of a number of possible combinations, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by the examples.

That is, "the e and f are nucleotides complementary or non-complementary to the template nucleic acid, respectively, and only one of e or f is complementary to the template nucleic acid, and at least one of e and f corresponds to the first primer, respectively. It is a nucleotide of a different base from E and F, and g is a nucleotide complementary to a wild-type or mutant-type base of a single nucleotide polymorphism (SNP) site of a template nucleic acid. A primer set including a multiplex can be used to differentiate and amplify the SNP wild type and the mutant type at the same time.

Accordingly, the primer set provided by the present invention may be characterized in that it is for genotyping analysis of single nucleotide polymorphisms in a gene.

The present invention also provides the primer set; and a probe comprising a base sequence complementary to Bq of the first primer and a probe comprising a base sequence complementary to bq of the second primer, comprising a support immobilized on the surface, providing a single nucleotide polymorphism genotyping kit do.

The "probe" of the present invention refers to a nucleic acid molecule corresponding to several to several tens of bases as a short nucleic acid sequence capable of specific detection of each SNP marker.

The probe may be a nucleic acid analog selected from traditional nucleic acids such as deoxynucleotides (DNA) and ribonucleotides (RNA), as well as peptide nucleotides (PNA), locked nucleotides (LNA) and di-hexitol nucleotides (HNA). . The nucleic acid analog has advantages of being stable to enzymes such as nucleases, structurally very specific binding to a nucleotide sequence, and stable to heat.

The probe may be characterized in that a detectable label is bound.

The assay kit may further include a reverse primer having a detectable label attached to the end thereof.

The detectable label may be a chemical label, an enzyme label, a radioactive label, a fluorescent label, a luminescent label, a chemiluminescent label, a fluorescence resonance energy transfer (PRET) label, or a metal label. For example, the detectable label is biotin, rhodamine, cyanine 3, pyrene, cyanine 5, Green Fluorescent Protein (GFP), calcein, fluorescein Isothiocyanate (FITC), Alexa488, 6-carboxy-fluorescein (FAM), 2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluoro Rescein (HEX), 2',7'-dichloro-6-carboxy-4,7-dichlorofluorescein (TET), Fluorescein Chlorotriazinyl, Fluorescein, Oregon Green ), Magnesium Green, Calcium Green, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE), Tetramethylrhodamine , tetramethyl-rhodamine isothiocyanate (TRITC), carboxytetramethyl rhodamine (TAMRA), rhodamine PHalloidin, Pyronin Y, Lissamine, ROX (X- rhodamine), Calcium Crimson, Texas Red (Texas Red), Nile Red (Nile Red), and may be selected from the group consisting of thiadicarbocyanine (Thiadicarbocyanine), but is not limited thereto.

In the kit of the present invention, the support is made of slide glass, plastic, membrane, semiconductor chip, silicon, gel, nano material, ceramic, metal material, optical fiber, or a combination thereof. characterized.

When the probe is immobilized on a support, a functional group such as an amino group, a thiol group, or biotin may be introduced, or a spacer may be additionally introduced between the functional group and the nucleotide. Although the kind of spacer used here is not specifically limited, For example, an alkane or ethylene glycol skeleton can be used.

The kit of the present invention may be a kit for a microarray, and a conventional pin microarray (Microarray printing technology, Don Rose, Ph.D., Cartesian Technologies, Inc., Anal Biochem, 320) known in the art. (2):281-91 (2003)), ink jet (Nat Biotech, 18;438-441 (2000), Bioconjug Chem, 13(1);97-103 (2002)), photolithography ) (Cur Opinion Chem Biol, 2;404-410 (1998), Nature genetics supplement, 21:20-24 (1999)), or an electric array (Ann Biomed Eng. 20(4):423-37) (1992), Psychiatric Genetics, 12;181-192 (2002)).

In addition to the primers and probes, the kit of the present invention may further include materials normally required for PCR reaction and nucleotide sequence analysis. For example, a DNA polymerase (such as a thermostable DNA polymerase obtained from Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), dNTP (dATP, dCTP, dGTP, dTTP), a reaction buffer, and a PCR reaction mixture consisting of distilled water, etc., may also include a color reagent capable of confirming whether a PCR product is detected or not. In addition, the kit of the present invention may be manufactured in a plurality of separate packaging or compartments containing the reagent components described above.

The present invention also provides a method for genotyping one or more single nucleotide polymorphisms in a nucleic acid sample, comprising the steps of:

(a) treating a mixture containing a template nucleic acid, a polymerase, and the primer set according to claim 11 with Uracil DNA Glycosylase (UDG) to remove the uracil base of deoxyuridine in the primer step;

(b) amplifying the template nucleic acid;

(c) hybridizing the amplification product with a probe including a nucleotide sequence complementary to Bq of the first primer and a probe including a nucleotide sequence complementary to bq of the second primer; and

(d) determining the genotype of the single nucleotide polymorphism in the nucleic acid sample through signal detection according to the hybridization.

The nucleic acid amplification method according to the present invention may be performed by the same method as the polymerase chain reaction (PCR) or multiplex PCR generally known in the art using the primer sets of the general formulas 1 and 2.

In the present invention, before initiating the polymerase chain reaction, UDG (Uracil DNA glycosylase) is treated in the reaction mixture containing the target nucleic acid (template nucleic acid), primer set, polymerase, etc. in step (a), and B of the primer A step of removing the uracil base present in the region is further performed.

In the present invention, each of the primers included in the primer set contains 1 to 5 uracil bases, and UDG (Uracil DNA Glycosylase) treatment causes cleavage of the bases in the primer.

After that, when the reaction temperature rises by initiating a conventional polymerase chain reaction, the closed primer is unfolded, the primer site having a sequence complementary to the template nucleic acid binds to the template nucleic acid, and the base extension reaction proceeds by the DNA polymerase. (1st amplification reaction).

When the second amplification reaction proceeds after the first amplification reaction is completed, the extension reaction of the base by the DNA polymerase is stopped at the cleavage site of the base from which the uracil base is removed. That is, the A and a sites added to the 5' end to form a closed primer; In addition, the base extension reaction cannot proceed to the base sequence of the B and b sites for hybridization with the probe, and as a result, the same Tm value can be maintained from the first amplification reaction to the end of the reaction.

On the other hand, as described above, cross-amplification does not occur in the first primer of Formula 1 and the second primer of Formula 2 provided by the present invention by each EFG and efg region, and the final amplification product is also accurately distinguished from each other. It is possible.

According to the method provided by the present invention, it is possible to simultaneously analyze the wild type and the mutant type of a SNP in one kind of gene, and also the wild type and the mutant type of the SNP in a plurality of genes can be simultaneously amplified and detected in a multiplex. There is this.

In the method provided by the present invention, the hybridization of the amplification product and the probe and the signal detection method according to the hybridization may be applied to the present invention without limitation as long as it is a method commonly used in the art.

According to another embodiment of the present invention, in order to confirm the specificity of the composition of the present invention, six SNPs related to skin care (OCA2, MC1R, AGER, NOTCH, SLC23A1(1), SLC23A1) (2)) Genotypes were confirmed for 4 samples using 6 SNP genotyping kits provided by ThermoFisher Scientific, which can select and confirm genes. After that, a total of 12 oligo primers for 6 normal group and risk group genotypes were subjected to multiplex PCR in one amplification reaction and then reacted to the microarray DNA chip. could

When multiplex PCR and microarray DNA chip of a single nucleotide polymorphism gene are performed using the primer of the novel structure according to the present invention, a genotyping result with high specificity can be obtained in one reaction.

1 is a SMPO primer structure of the present invention specific for SNP wild-type (A), a SMPO primer structure of the present invention specific for a SNP mutant type (B), and H-TAP, D-ARMS and PBO structures constituting the SMPO structure do.
Figure 2 shows the fluorescence reaction (A) after hybridizing the amplification product generated by using the primer provided by the present invention with the probe of the microarray DNA chip, and after PCR amplification with a primer having a general structure, it is unfavorable for hybridization with the probe After hybridization with the probe of the microarray DNA chip in the condition of double strand, check the fluorescence reaction (B), add the process of converting double strand into single strand by denaturation reaction, and microarray By confirming the fluorescence reaction (C) after hybridization with the probe of the DNA chip, it is the result of confirming that the different primer structures according to the present invention have superior hybridization efficiency with the probe.
3 shows multiplex PCR using 12 types of SMPO rescue primers specific for wild type and mutant type of 6 types of SNPs (OCA2, SLC23A1(1), SCL23A1(2), NOTCH, AGER, MC1R), and amplified products thereof Gold fluorescence reaction (A) and genotyping results (B) according to fluorescence reaction confirmed when hybridizing with the probe of the microarray DNA chip are shown.

Hereinafter, the present invention will be described in detail.

However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

Experiment preparation

1. Preparation of primer

The primers and probes used in the present invention are analyzed by using the DNAsis program of Hitachi Software for the Genebank sequence registered in the gene bank (www.ncbi.nlm.nih.gov) operated by NCBI under the National Institutes of Health. The nucleotide sequence was determined, and it was analyzed again by BLAST (www.ncbi.nlm.nih.gov/BLAST/) to confirm that it was a specific nucleotide sequence portion of six human single nucleotide polymorphism genes. The SMPO-structured primer can complementarily bind to the base sequence at the 3' end of the specific oligo site, and the binding temperature (Tm value) was set to 18 to 20 ° C. Each template DNA base sequence and the added base sequence Deoxyuridine was added in between. In addition, a total of 12 artificial oligo primers capable of discriminating 6 types of SNP wild type and mutant type were prepared in the deoxyuridine upstream direction to prepare a part capable of specifically binding to the probe on the microarray DNA chip. The single nucleotide polymorphism part and its neighboring 1st or 2nd base were changed to be different from the template to make a mismatch oligo, and the mismatch part was made so that all mismatch oligos of the template nucleic acid, wild-type genotype and mutant genotype were different.

Such a primer structure was named Single uncleotide polymorphim Microarray Probe-specific Oligonucleotide (SMPO).

2. Synthesis of primers

The primers analyzed in Experimental Preparation 1 were prepared using the same method as the oligonucleotide synthesis described in 10.42 of molecular cloning 3rd edition (sambrook and rusell, cold spring harborlaboratory press, New York, USA, 2001), using the same method as IDT (Integrated DNA Technologies, USA) was requested and synthesized.

Example 1: Evaluation of the efficiency of microarray DNA chip hybridization of general structural primers and SMPO structural primer amplification products

To check the hybridization reaction efficiency of the microarray DNA chip for the amplification product of the general structure and the SMPO structure primer, the primer and the SMPO structure of the general structure for the wild-type genotype of the four SNP genes (OCA2, MC1R, AGER, NOTCH) Primers to which were added were prepared, respectively. For the reverse primer, biotin capable of binding to Avidin and Streptavidin was prepared to be positioned at the 5' end of the primer for fluorescence reaction.

The probe to be attached to the microarray DNA chip was prepared by selecting a gene region capable of complementarily binding with a Tm value of 58°C to 68°C in the case of general structural primers, and in the case of SMPO structural primers, the PBO structure and complementary structure inside the structure were selected. Probes were prepared with nucleotide sequences.

Amplification products were generated by individual PCR amplification of 4 types of primers with general structures and 4 types of SMPO structures for 4 types of wild-type genotypes of single nucleotide polymorphism (SNP). As a template, wild-type and hetero genotype templates having the wild-type genotype of each SNP were selected and used, each of 2ul of template DNA, 0.5ul (10pmol/ul) of primer, 0.4ul (1 unit) of Solg ^TM h-Taq DNA Polymerase (Solgent co., Korea), 3 ul ( _{10X concentration with 15 mM MgCl 2} solution) of h-Taq reaction buffer (Solgent co., Korea), 0.1 ul (0.1 ul) to prevent contamination unit) of SolGent ^TM Uracil-DNA Glycosylase (Solgent co., Korea), 1ul (dATP, dCTP, dGTP, 2 mM and dUTP 6 mM, respectively) of dNTP (Solgent co., Korea), 1M betaine (Sigma, USA) in a mixture of DNase , RNase free water (Sigma-Aldrich co., USA) was added so that the total reaction solution was 30ul.

Each 10ul of the amplified reaction product was mixed with 40ul of the hybridization reaction solution and reacted on a microarray DNA chip at 63°C for 10 minutes. After that, a solution of 3ul of gold-streptavidin solution and 30ul of the hybridization reaction solution was mixed again on the membrane gene chip at 63°C. By reacting for 10 minutes, the efficiency of the hybridization reaction was confirmed by visually checking the fluorescence reaction displayed on the microarray DNA chip.

As shown in FIG. 2, the PCR amplification product by the SMPO structural primer according to the present invention showed a strong signal in all four SNP wild-type genotypes (FIG. 2A), but the PCR amplification product by the general structural primer showed no signal for the remaining three species except for the OCA2 SNP (Fig. 2B).

Since the PCR amplification product by the general structural primer is a double-stranded nucleic acid, it is disadvantageous to the hybridization reaction with the probe. After storage and changing to a single strand, it was reacted on a microarray DNA chip in the same manner. As a result, except for OCA2, which showed a signal even before denaturation, the signal of NOTCH SNP was newly detected, and the AGER SNP showed a faint response. However, despite the denaturation, the MC1R SNP still did not show a signal.

Through this, it was confirmed that the PCR amplification product by the primer having the SMPO structure provided by the present invention has a very good structure in the hybridization reaction with the microarray DNP chip.

Example 2: SNP co-detection specificity evaluation of SMPO rescue primers

Six SNPs (OCA2, MC1R, AGER, NOTCH, SLC23A1(1), SLC23A1(2)) genes related to skin beauty among single nucleotide polymorphisms (SNPs) to evaluate the SNP genotyping ability and specificity of SMPO structural primer Genotypes were confirmed for 4 samples with 6 SNP genotyping kits provided by ThermoFisher Scientific, which can select and confirm them. Thereafter, a total of 12 primers and each reverse primer for 6 wild-type and mutant genotypes were subjected to multiplex PCR in one amplification reaction.

The reaction solution of the multiplex PCR is 2ul of template DNA, 0.5ul (10~20pmol/ul) of each primer, 1.2ul (3 units) of Solg ^TM h-Taq DNA Polymerase (Solgent co., Korea), 3ul (15) h-Taq reaction buffer (Solgent co., Korea) of 10X concentration with mM MgCl ₂ ^{solution, 0.1ul (0.1 unit) of SolGent TM} Uracil-DNA Glycosylase (Solgent co., Korea), 1ul to prevent contamination DNase, RNase free water (Sigma-Aldrich co., USA) was added to a mixture of dNTP (Solgent co., Korea) and 1M betaine (Sigma, USA) of (dATP, dCTP, dGTP, respectively, 2 mM and dUTP 6 mM), and the total reaction solution This was made to be 30ul.

10ul of the amplified reaction product was mixed with 40ul of each hybridization reaction solution and reacted on a microarray DNA chip at 63°C for 10 minutes. After that, a solution of 6ul of gold-streptavidin solution and 30ul of hybridization reaction solution was added to the membrane gene chip again at 63°C. By reacting for 10 minutes, the specificity of the hybridization reaction was confirmed by visually checking the fluorescence reaction displayed on the microarray DNA chip.

The genotypes of 6 skin beauty related SNPs for 4 samples were measured as shown in FIG. 3, and the results of the confirmed genotypes were 100% with 6 SNP genotyping kits provided by ThermoFisher Scientific, which can be confirmed by real-time PCR. exactly matched.

As described above, it was confirmed that the primer of the SMPO structure provided by the present invention makes it possible to analyze a plurality of single nucleotide polymorphism genotypes with high specificity by a single amplification reaction and a simple method.

When multiplex PCR and microarray DNA chip of a single nucleotide polymorphism gene are performed using the primer of the novel structure according to the present invention, a genotype determination result with high specificity can be obtained in a single reaction, so the industrial applicability is very high.

Claims (19)

A primer set comprising a first primer represented by the following general formula (1) and a second primer represented by the following general formula (2):
[General formula 1]
5'-AP-BQ-CR-DS-EFG-3'
(In the above formula,
A represents a site including a nucleotide having a nucleotide sequence complementary to the continuous nucleotide sequence at the 3' end of the primer,
B represents a site including a nucleotide having an arbitrary base sequence,
Wherein C represents deoxyuridine (Deoxyuridine),
D represents a region containing nucleotides having a nucleotide sequence complementary to a specific continuous nucleotide sequence of the template nucleic acid,
Wherein E and F are nucleotides complementary or non-complementary to the template nucleic acid, respectively, and only either E or F is complementary to the template nucleic acid,
wherein G represents a nucleotide complementary to a wild-type or mutant-type base of a single nucleotide polymorphism (SNP) site,
Wherein P, Q, R and S represent the number of nucleotides included in each site.)
[General Formula 2]
5'-ap-bq-cr-ds-efg-3'
(In the above formula,
wherein a represents a site including a nucleotide having a nucleotide sequence complementary to the continuous nucleotide sequence at the 3' end of the primer,
Wherein b is a region including a nucleotide having an arbitrary nucleotide sequence, the nucleotide sequence is different from that of the B region of the first primer,
Wherein c represents deoxyuridine (Deoxyuridine),
wherein d represents a region containing nucleotides having a nucleotide sequence complementary to a specific continuous nucleotide sequence of the template nucleic acid;
Wherein e and f are nucleotides complementary or non-complementary to the template nucleic acid, respectively, and only one of e or f is complementary to the template nucleic acid, and at least one of e and f is the corresponding E and is a nucleotide of a base different from F,
Wherein g is a nucleotide complementary to the wild-type or mutant-type base of the single nucleotide polymorphism (SNP) site of the template nucleic acid, and is a base different from G of the first primer,
The p, q, r and s represent the number of nucleotides included in each site.)
The primer set according to claim 1, wherein P and p are each independently 3 to 8.
The primer set according to claim 1, wherein Q and q are each independently 15 to 40.
The primer set according to claim 1, wherein R and r are each independently 1-5.
The primer set according to claim 1, wherein S and s are each independently 15 to 40.
The primer set according to claim 1, wherein the Tm of A and a are each independently 10 to 30°C.
The primer set according to claim 1, wherein the Tm of B and b are each independently 55 to 70°C.
The primer set according to claim 1, wherein the Tm of D and d are each independently 50 to 65°C.
The primer set according to any one of claims 1 to 8, wherein the primer set is a primer set for multiplex polymerase chain reaction (SNP multiplex PCR) for detecting single nucleotide polymorphisms.
The primer set according to any one of claims 1 to 8, wherein the primer set is a primer set for a microarray DNA chip.
delete delete A primer set according to claim 1; And A probe comprising a nucleotide sequence complementary to BQ of the first primer and a probe comprising a nucleotide sequence complementary to bq of the second primer, comprising a support immobilized on the surface. A single nucleotide polymorphism genotyping kit.
The genotyping kit of claim 13, wherein the kit can simultaneously determine wild-type and mutant sequences of single-nucleotide polymorphisms.
The genotyping kit of claim 13, wherein the probe is bound to a detectable label.
The genotyping kit of claim 13, wherein the assay kit further comprises a reverse primer having a detectable label attached to the end thereof.
The method according to claim 15 or 16, wherein the detectable label is a chemical label, an enzymatic label, a radioactive label, a fluorescent label, a luminescent label, a chemiluminescent label, a fluorescence resonance energy transfer (PRET) label, or a metal label. , single nucleotide polymorphism genotyping kit.
The method of claim 15 or 16, wherein the detectable label is biotin, rhodamine, cyanine 3, pyrene, cyanine 5, Green Fluorescent Protein (GFP), calcein ( Calcein), fluorescein isothiocyanate (FITC), Alexa 488, 6-carboxy-fluorescein (FAM), 2',4',5',7'-tetrachloro-6-carboxy- 4,7-dichlorofluorescein (HEX), 2',7'-dichloro-6-carboxy-4,7-dichlorofluorescein (TET), fluorescein chlorotriazinyl, fluorescein , Oregon Green, Magnesium Green, Calcium Green, 6-Carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE), Tetra Tetramethylrhodamine, Tetramethyl-Rhodamine Isothiocyanate (TRITC), Carboxytetramethyl Rhodamine (TAMRA), Phodamine PHalloidin, Pyronin Y, Lissamine ), ROX (X-rhodamine), Calcium Crimson, Texas Red (Texas Red), Nile Red (Nile Red) and thiadicarbocyanine (Thiadicarbocyanine) characterized in that at least one selected from the group consisting of , single nucleotide polymorphism genotyping kit.
A method for genotyping one or more single nucleotide polymorphisms in a nucleic acid sample, comprising the steps of:
(a) treating a mixture containing a template nucleic acid, a polymerase, and the primer set according to claim 1 with Uracil DNA Glycosylase (UDG) to remove the uracil base of deoxyuridine in the primer step;
(b) amplifying the template nucleic acid;
(c) hybridizing the amplification product with a probe comprising a base sequence complementary to BQ of the first primer and a probe comprising a base sequence complementary to bq of the second primer; and
(d) determining the genotype of the single nucleotide polymorphism in the nucleic acid sample through signal detection according to the hybridization.

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