KR101357237B1 - SNP genotyping method using the melting analysis comprising a dilution step of PCR amplicon - Google Patents

Abstract

The present invention relates to a SNP genotyping method using a DNA melting assay including a dilution step of a PCR amplicon, wherein the unlabeled probe by dilution of a PCR amplicon in a DNA melting assay for SNP genotyping (unlabeled probe) has the advantage of improving the binding efficiency (dislicmination) of alleles (allelic identities) and eliminating the need for 3'-end modification of the probe for SNP discrimination.

Description

SNP genotyping method using the melting analysis comprising a dilution step of PCR amplicon}

The present invention relates to a SNP genotyping method using a DNA melting analysis including a dilution step of a PCR amplicon, and by diluting a PCR amplicon in a DNA melting assay for SNP genotyping. There is an advantage that it is possible to increase the binding efficiency of the unlabeled probe and the discrimination of alleles and eliminate the need for 3'-end modification of the probe for SNP determination.

Polymorphic single nucleotides (SNPs), which account for more than 0.1% of the human genome sequence, have been the subject of linking human phenotypic variations, including complex diseases (The International HapMap Consortium, 2005). Various high throughput platforms have been developed for genotyping multiple SNPs in a relatively short time and have been successfully applied for large-scale analysis such as genome-wide association analysis (GWAS) (Ragoussis, 2009). ). These platforms typically have a limited number of oligonucleotide probes arranged densely on a small chip that can identify up to one million sequence variations through target DNA hybridization and in some cases biochemical reactions, resulting in very complete analysis of the entire genome. It can be done quickly (Kennedy et al., 2003; Ohnishi et al., 2001; Steemers et al., 2006).

The success of the GWAS has resulted in the identification of numerous SNPs associated with complex traits including asthma, diabetes and coronary heart disease (http://www.genome.gov/gwastudies /). These studies generally involve repeated studies to identify SNPs associated with specific trait (s) in a given population in other populations using a platform suitable for genotyping of small SNPs (Chanock et al., 2007). In addition to the biochemical reactions used in the GWAS platform, ligation mediated reactions, endonuclease cleavage, pyrosequencing, mass spectrophotometry, and high resolution DNA Various approaches have been applied for this purpose, including high resolution DNA melting (Fakhrai-Rad et al., 2002; Hardenbol et al., 2003; Jurinke et al., 2002; Kutyavin et al., 2006; Wittwer et al., 2003).

SNP genotyping using melt analysis of unlabeled oligonucleotide probes (UOP) applied to asymmetric amplification amplicons or small size amplicons has been successful in identifying SNP alleles as a relatively simpler and more affordable alternative. (Erali et al., 2008; Liew et al., 2007). Concentrations of PCR amplicons in the late PCR cycle, the plateau phase, show severe self re-annealing, depriving primers of their chance to attach to their target sequences. Ganda (Gevertz et al., 2005). In these methods, in order for the UOP to anneal to its target sequence enough to obtain sufficient signal from melting with a fluorescent DNA binding dye (dye), a selective enrichment or weak re-annealing rate of the target sequence is required. There was no choice but to use a small amplicon.

In the present invention, the inventors have found that amplicon dilution can lead to other conditions that can increase the target annealing opportunity of UOP. The inventors tested whether melt analysis of UOPs applied to diluted PCR amplicons could be used for genotyping purposes. The result demonstrates that the application of amplicon dilution followed by UOPs can provide a simpler, faster and more cost-effective SNP genotyping method and completes the present invention.

Therefore, the main object of the present invention is to provide a SNP genotyping method using simpler, faster and more cost-effective DNA melting analysis.

Another object of the present invention is to provide a SNP genotyping method using DNA melting analysis, which can eliminate the need for 3′-end modification through inactivation of polymerase.

According to one aspect of the invention, the invention provides a SNP genotyping method using DNA fusion analysis comprising the following steps: 1) PCR amplifying a target sequence comprising an SNP site; 2) dilution of the amplified PCR amplicons; 3) binding the diluted PCR amplicon with an unlabeled oligonucleotide probe in the presence of a fluorescent DNA binding dye; 4) measuring the fluorescence intensity that changes as the probe melts while gradually raising the temperature of the combined product (DNA melting analysis), and 5) obtaining a differential curve of the change curve of fluorescence intensity according to the temperature change. Thereafter, typing the SNP according to the presence or absence of a peak representing each type of SNP.

In the present invention, the SNP genotyping method is characterized in that the PCR amplification step of 1) does not put together the unlabeled oligonucleotide probes together after PCR amplification. In the conventional SNP genotyping method using DNA melting analysis, since the probe was put together in the PCR amplification step, the 3'-end modification of the probe was required so that the probe did not participate in the PCR amplification process, but in the present invention, PCR amplification was performed. After the probe is put separately, the inactivation of the polymerase (polymerase) by the dilution of the PCR amplicon can eliminate the need for 3'-end modification of the probe. In addition, in the conventional method, it was necessary to design the sequence of the probe so that the probe does not participate in the PCR amplification process so that the probe does not overlap with the primer sequence, but in the present invention, the probe sequence is not likely to be involved in the PCR amplification process. Can be designed freely regardless of primer sequence.

In the present invention, the PCR amplicon in step 2) provides a SNP genotyping method, characterized in that diluting 2 to 10 times in distilled water or PCR reaction buffer. Preferably, the present invention finally provides a SNP genotyping method, characterized in that the dilution factor of the final PCR product after mixing with the probe solution in step 3) is 6 to 96 times, more preferably 20 to 50 times. do. In Figure 1 of the present invention showed that the genotype (genotype) can be determined at the dilution level of 6 to 96 times, it was able to determine the genotype (genotype) most clearly at the dilution level of 24 and 48 times.

In the present invention, it is characterized in that in step 2) by diluting the PCR amplicon to increase the binding efficiency of the non-labeled probe and the discrimination between SNPs. Specifically, in step 2), the dilution of the PCR amplicon can increase the binding efficiency of the non-labeled probe physically / chemically fixed to the solid medium and increase the discrimination between SNPs.

In the present invention, the non-labeled probe in step 3) provides a SNP genotyping method, characterized in that it is physically / chemically fixed to a solid medium such as chips, beads, tubes, plates, and the like. The unlabeled probe is subjected to binding and denaturation with the diluted PCR product in a fixed state in a solid medium.

In the present invention, the PCR amplification in step 1) provides a SNP genotyping method, characterized in that the multiplexing (multiplexing) PCR to amplify multiple SNPs at once. The present invention is characterized in that step 2) eliminates the need for 3′-end modification of the probe through inactivation of the polymerase by dilution of the PCR amplicon.

In the present invention, in step 3), the diluted PCR amplicon is mixed with a fluorescent DNA binding dye and an unlabeled oligonucleotide probe solution having a sequence complementary to the SNP, and then the mixture is heated to a double helix PCR product. It provides a SNP genotyping method characterized in that the probe is bound to a target SNP site by inducing denaturation of the probe and leaving it at a temperature lower than the TM of the probe.

In the present invention, DNA melting analysis in step 4) provides a SNP genotyping method, characterized in that performed using a realtime thermal cycler (Realtime thermal cycler).

Hereinafter, a preferred example of the SNP genotyping method of the present invention will be described in more detail step by step.

1) Design stage of amplicon and probe

  -Primer TM value should be designed between 58-60 ℃.

  -Design the amplicon TM value between 75-85 ℃ if possible.

  The TM value of the probe should be designed between 62-72 ℃ as possible.

  -Design the probe so that TM value of probe is spread over 3 ℃ if possible.

2) PCR amplifying the target chain,

  Amplify the synthesis of the amplicon until it reaches the plateau in the PCR amplification step.

  Multiplexing PCR can be performed if necessary.

3) diluting the PCR amplicon to some extent,

  -Dilute 2 to 10 times in distilled water,

  Dilute 2 to 10 times in a composition similar to the PCR reaction product.

4) binding the diluted PCR amplicon with an unlabeled oligonucleotide probe in the presence of a fluorescent DNA binding dye,

   The dilution is mixed with a probe solution containing a DNA binding fluorescent dye (dye) and an unlabeled oligonucleotide probe for a specific SNP. At this time, the final dilution level of the PCR product is 20-50 times.

5) melting analysis,

   The mixture was heated to 95 ° C. using a realtime thermal cycler to induce denaturation of the double helix PCR product and allowed to stand at a temperature about 5 ° C. below the TM of the probe for 30 to 300 seconds. allow the probe to bind to the target DNA.

   Gradually raise the temperature (0.1 ° C / sec) to obtain fluorescence levels that change as the probe melts.

6) SNP typing step.

After obtaining the differential curve of the fluorescence level change curve according to the temperature change obtained above, the SNP is typed according to the presence or absence of a peak representing each type of SNP.

Applying DNA melting analysis for genotyping polymorphic single nucleotides (SNPs) using unlabeled oligonucleotide probes for polymorphic DNAs in the presence of fluorescent DNA binding dyes. There is a need for reaction conditions to efficiently associate with PCR amplified target sequences. The inventors have experimentally demonstrated that applying unlabeled probes to diluted PCR amplicons provides sufficient conditions for identifying allelic identities by fluorescence signals obtained by subsequent probe melting. The method of the present invention can be used most effectively in multiplexing PCR techniques that cover multiple SNPs in a given sample. That is, since the amplicon dilution step can inactivate polymerase through divalent cation chelation, the 3′-terminal modification of the probe, which was essential for conventional multiplexing PCR Eliminate the need for Thus, the present invention can provide a faster, simpler and more cost effective SNP genotyping method using inexpensive reagents and conventional laboratory equipment.

Figure 1 shows the sequence structure and melting curve analysis of the SNPs tested.
2 shows the effect of Amplicon Tm on melting analysis.
3 shows a melting analysis using amplicons generated by multiplexing PCR.
4 shows denaturation analysis of amplicons generated through multiplex PCR on 20 SNP sites.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example  One: PCR

PCR amplification was performed using 10-50 ng of genomic DNA as a template for a small fragment on the genome containing the SNP to be assayed. The simple PCR amplification reaction consists of 10 repetition cycles, and based on the first 94 ℃ 20 seconds, 64 ℃ 15 seconds and 72 ℃, the annealing reaction is lowered by 0.5 ℃ during the annealing reaction every 57 times. 40 additional cycles were performed for 20 seconds. Multiplex PCR amplification followed the method recommended by Henegariu et al. (Henegariu et al., 1997). The reaction sample was left at 95'C for 10 minutes to activate the enzyme, and the basic reaction of 95 ° C. 20 seconds, 55 ° C. 25 seconds, and 65 ° C. for 2 minutes was repeated 40 times. For each SNP, a primer was prepared such that the Tm value of the PCR reaction product was in the range of 75 to 85 ° C by the TM Utility v1.5 program distributed by Idaho Technology.

Example  2: Zino Typing Genotyping )

The PCR reaction product and its distilled water dilutions were mixed with a probe solution containing 5 volumes of 5 nM Syto® 9 (Invitrogen ^TM ) and 12.5 mM EDTA and 5 mM Tris (pH 8.0) to give a dilution factor of 6X to After 96X, temperature reaction was performed using a Lightcycler® 2.0 instrument (Roche Diagnostics). The reaction was denatured for 5 seconds at 95 ° C and induced complementary helix annealing at 60 ° C for 1 minute, and then fluorescence was measured while gradually raising the temperature to 0.1 ° C / sec. Genotype was determined based on the temperature at which each UOP was denatured

Experiment result

Result 1 . Dilution Probe ( probe ) Impact on bonding

Experiments were performed using three different PCR amplicons to verify that dilution of the PCR amplicon was effective for given UOP binding to target helices to distinguish polymorphic bases through its denaturation. Small DNA fragments corresponding to 113, 71, and 95 bp of three chromosomal sites, each containing known SNPs rs748514, rs2001599 or rs17170583, were PCR amplified. The UOP for each product was constructed to be 32-44 bases in size and complementary to one helix, with a denaturation temperature (Tm) of 68-71 ° C. for fully complementary binding (see Table 1).

Six genomic DNAs were selected for each SNP so that three genotypes appeared twice for the SNP, and the corresponding DNA fragments were amplified by PCR. Two-fold serial dilutions were made using a portion of the PCR product and mixed with a six-fold volume of fluorescent dye SYTO9 and a UOP solution containing the corresponding UOP. The double-stranded DNA of this mixture was initially denatured at 95 ° C. and allowed UOPs to bind to the target helix in glass microtubules. Gradually raising the temperature to obtain the level of fluorescence emitted and the results are shown in Figure 1b. At each dilution level, the SNP rs748514 showed a degeneration curve and a negative differential curve. For the other two SNPs, rs2001599 and rs17170583, only negative derivative curves were shown. Degeneration curves alone were insufficient to identify each genotype, but from the negative differential curve, genotypes were clearly identified, especially at 24 and 48-fold dilution levels. The relative ratio of UOP double helix to amplicon double helix was increased with increasing dilution level.

For better sensitivity, it is important that the UOP binds well to the target helix. Dilution of the amplicon can increase the fraction of target helix that binds to the UOP while reducing the absolute amount of the target helix, which can interfere with high signal levels. Our data show the highest sensitivity at dilution levels of 24-48 fold.

Results 2. Amplicon  Size Of genotyping  Impact on sensitivity

Next, the effect of dilution of amplicon on the sensitivity of this method was investigated. Amplicon chemistry, such as base composition and size, was also the first consideration because it would affect the binding rate of the UOP to the target helix by affecting the recombination of the amplicon. In order to examine this problem, several primers were prepared for genome sequences containing SNP rs7518199, and a combination thereof was used to produce amplicons having various sizes and Tm values including the SNPs.

Six genome samples were selected so that all genotypes of a given SNP appeared twice, and amplified five DNA fragments with different Tm values. In the 2-fold and 48-fold dilutions of Amplicon, negative differential curves were obtained through the same thermal reactions as shown above, and are shown in FIG. 2C. The only distinct genotypes at the 2-fold dilution were the lowest Tm and the smallest A amplicon. Other amplicons showed weak or indistinguishable UOP peaks, clearly indicating that amplicons with small size and low Tm values bind better to UOP. On the other hand, when all amplicons were analyzed at 48-fold dilutions, each genotype was clearly distinguishable. This observation suggests that dilution of the amplicon provides an environment in which the UOP can bind well to the target helix.

Results 3. Multiplexing ( 멀티플링 ) PCR Combination with

Our genotyping method consists of two distinct processes: DNA amplification, which involves PCR amplification and dilution of the amplicon in the UOP solution. The latter can be done very quickly in less than 10 minutes, so a PCR reaction that takes more than an hour will limit the speed of this assay. In this regard, the introduction of multiplexing PCR reactions can greatly improve the treatment efficiency of this method. To verify the success of this trial, 10 different sequences containing known SNPs were simultaneously amplified in one tube with 16 dielectric samples as template (see Table 1 below for base sequence information).

[Table 1]

Structure of amplicons and probes used in the analysis. Primers for generating each amplicon are shown in blue. The base sequence of the probe is indicated by underscore letters. The polymorphic bases are shown in red in the amplicon's surrounding nucleotide sequence, and R, Y, S, W and M represent G / A, C / T, C / G, T / A and C / A base polymorphisms, respectively. . The base corresponding to the orientation (F, forward; R, reverse) hyperplasia of the probe is indicated. All amplicons listed are amplified simultaneously by multiple PCR. Simple PCR analysis was performed on rs748514, rs2001599 and rs17170583.

** Primers shown for SNP rs7518199 were also used to amplify amplicon A in FIG. 2. Other amplicons of Figure 2 were generated by the combination of several primers as follows. F1 (5'-GCTGGGATCTCAGCCTACA-3 ') and R1 (5'-AGTGAGCATGACTCCTTTGC-3') for amplicon B; F1 and R2 for amplicon C (5'-GATCAGGCTGCACTGTGG-3 '); F2 (5'-GAGTGAACGGGGCTGGGT-3 ') and R2 for amplicon D; F3 (5'-CAGTAGTGTCGGGAGCAA-3 ') and R3 (5'-CCAGGTGACACTGAGCCA-3') for amplicon E.

Genotyping of 10 SNPs at 40-fold amplicon dilution was performed sequentially by binding and denaturation of the corresponding UOP. The negative differential curve of the fluorescence profile for each SNP is shown in two forms in FIG. 3. One is the amplicon and UOP denaturation at the same time (left) and the other is the UOP only degeneration (right). Regardless of which SNP, all samples were genotyping. In addition, a narrow temperature range fluorescence profile showing only UOP denaturation contained sufficient information for typing SNPs.

Next, an experiment was performed to simultaneously amplify 20 genome fragments each containing 20 known SNPs, which are also planted in each known Affymetrics GeneChip Human Mapping 500K set. GeneChip analysis was carried out to select 10 genomes that know the genotype of each SNP and can represent all genotypes, and used as PCR templates. There are various aspects of metamorphic curves for each of the 20 UOPs. 18 amplicons were amplified at various levels of efficiency in multiplex PCR reactions (Supplementary Figure 1). The genotype could be clearly determined from the degeneration curves of the 18 UOPs, but not the rest. As compared with GeneChip analysis, there was no discrepancy between the two genotyping methods. These results demonstrate that genotyping is effective for complex mixtures of more than 18 amplicons using amplicon specific UOPs.

Figure 1 shows the nucleotide sequence structure and denaturation curve analysis of the target SNP. A. Shows the nucleotide sequence and primer (blue) information of the amplicon containing the given SNPs. The registration number is indicated above the base sequence. Each SNP is indicated by red letters indicating polymorphism between background sequences (R, A / G; Y, C / T). The UOP sequence was indicated by the arrow line to indicate the base sequence and the orientation. The base corresponding to the polymorphic site is shown in red. B. As indicated, at different dilution levels between 6 and 96-fold, the amplicon denaturation curve (Rs748514 only, left) corresponding to a given SNP and the negative derivative curve of the denaturation curve were obtained. The negative denaturation curves of UOP corresponding to full complement and single mismatched base complementarity are shown as arrowheads in blue and red, respectively, at 6-fold dilutions.

2 shows the effect of Tm of Amplicon on denaturation analysis. A. As shown in parentheses, amplicons (A-E) containing SNP rs751819 with various sizes and Tm are shown relative to SNPs on the genome DNA (coil). Commonly used UOP probes are shown below the genomic DNA in dotted lines. B. PCR amplicons were isolated on 2% agarose gel. The size of the marker DNAs is indicated on the left side of the figure. Negative differential curves corresponding to dilution levels of 2 and 48 times each amplicon are indicated.

Figure 3 shows the denaturation analysis of amplicon generated by multiplexing PCR reaction. For a given SNP, a negative differential curve was obtained at two temperature ranges, one showing the deforming pattern of all the double helixes and one showing the UOP double helix.

4 shows denaturation analysis of amplicons generated through multiplexing PCR on 20 SNP sites. Negative differential curves for a given SNP were obtained over a range of temperatures showing only the denaturation patterns of the UOP double helix. Genotype was clearly determined for 18 SNPs except for 2 SNPs.

Interpret the results

The principle of the genotyping method used here is that the helixes of denatured PCR amplicons present in high concentrations, such as the plateau phase phase of the PCR amplification cycle, are exclusively small in size and complementary to oligonucleotides in the target helix due to their exclusive recombination. Based on the observation that it is difficult to combine. This exclusive recombination of amplicon DNA appears to explain the formation of a PCR plateau phase with little template amplification (Gevertz et al., 2005). The intensity of amplicon recombination may be weakened by lowering its concentration through dilution. This may reveal the activity of complementary molecules of small size, such as the UOP used in our analysis. Depending on the polymorphic base, the corresponding UOP will be complementary to the target helix completely or in a single base mismatch, which will be different in denaturation, which is easily observed in the presence of DNA-binding fluorescent dyes to distinguish polymorphic bases of a given SNP. It provides a way to do it.

UOP was also used in other genotyping methods, where the PCR reaction was set up either by amplifying small amplicons or by predominantly amplifying target helices. This applies a procedure that mitigates or avoids the intensity of exclusive recombination of amplicons for the purpose of typing a single SNP in a closed tube. The former uses asymmetric accumulation of target helix and the latter uses a smaller amplicon than the larger recombination strength. Both assays provide a simple and cost-effective method, but there is a need to address multiple PCR methods and their applicability to various sequencing backgrounds (especially the latter).

Benefits can be obtained by separating the genotyping step, including the PCR amplification step and the dilution step. The genotyping step takes only a few minutes due to its nature, and thus the yield can be greatly increased through multiple PCR amplification reactions. In addition, the PCR amplicon can be inactivated by thermal dilution of DNA polymerase in a UOP solution containing EDTA, which is a divalent cation chelator. Therefore, it is necessary to introduce UOP as a substrate of polymerase in a closed tube reaction. This reduces the need for a 3 'end modification of the UOP. In addition, since minor contamination of the reaction tube or plate does not significantly affect the genotyping reaction, genotyping costs can be further reduced in that they can be recycled.

Our method is very simple in understanding the analytical procedure or mechanism. Therefore, it is easy to optimize the procedure and analysis parameters for a given SNP. Nevertheless, we will consider some procedural requirements to better apply this method. Consideration should first be given to obtaining a high and distinct signal by the UOP. As the number of base pairs increases, more fluorescent dyes bind to the DNA double helix and thus a larger fluorescent signal can be obtained. Therefore, it is desirable to maximize the number of bases of the UOP at a level where the analysis is not disturbed by chemical properties. For example, it is desirable to design the Tm of the amplicon at a low level (see below), so increasing the length of the UOP makes the TOP of the UOP similar to the Tm of the amplicon, resulting in overlapping of the denaturation peak of the amplicon and the denaturation peak of the UOP. Can be caused and eventually make genotyping difficult.

Our results demonstrate that dilution of amplicons favors the recombination reaction of UOP double helix formation rather than amplcon double helix formation. Dilution of amplicons, however, keeps the absolute amount of UOP double helix to be formed by reducing the amount of target helix to which the simultaneously available UOP binds. Our results showed that the best UOP signal was obtained when the PCR amplicon was diluted to 24-48 fold.

The Tm of the amplicon double helix (and the size of the accompanying amplicon) is also an important consideration. Amplicons with high Tm have a higher recombination rate than those with low values. Amplicons having a reciprocal sequence for one UOP but having different Tm values show that the rate of formation of a double helix after denaturation is determined by the Tm value. The amplicon with the lowest Tm value showed the lowest recombination rate and the amplicon with the highest Tm value showed the highest recombination rate. Therefore, it is required to limit the Tm values of PCR amplicons in a range that is distinct from the Tm values of the UOP double helix. In this regard, it should be noted that the partial overlap between the UOP sequence and the primer sequence for amplicon amplification does not interfere with the analysis. This example can be seen in the analysis of rs17170538, rs7518199 and other SNPs, which helps to design low Tm value of PCR amplicon.

Based on the analytical method of the present invention, it is possible to propose a preferred design guide for PCR amplicon and UOP.

-For optimal performance, Tm value of primer should be between 58 ~ 62 ℃.

-Amplicon's Tm value should be in the range of 75 ~ 85 ℃ but lower value is better.

The Tm value of the UOP should be between 62 ° C and 72 ° C, regardless of whether it is incomplete or incomplete complementary binding to the target helix, so that the difference between the Tm values of the UOPs formed under the two binding conditions is at least 3 ° C. do.

In conclusion, UOP probe can be effectively used for SNP genotyping through simple denaturation analysis under certain conditions. These conditions include the design of small PCR amplicons containing SNPs and dilution of PCR amplicons. Our analytical method does not require special equipment other than the real-time thermoCycler equipment, which is a general laboratory equipment. The analytical procedure is simple, fast and the cost of reagents required is low. Due to the advantages it offers, our method offers a practical and economical alternative to SNP genotyping.

As described above, according to the present invention, the binding efficiency of the non-labeled probe and the discrimination of alleles are determined by dilution of the PCR amplicon in the DNA fusion analysis for SNP genotyping. There is an advantage in that it can increase and eliminate the need for 3′-terminal modification in multiplexing PCR.

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Claims (10)

SNP genotyping method using DNA fusion analysis comprising the following steps, characterized in that the PCR amplification step of the following step 1) separately put the PCR amplification without putting together the unlabeled oligonucleotide probe:
1) PCR amplifying the target sequence comprising the SNP site;
2) dilution of the amplified PCR amplicons;
3) binding the diluted PCR amplicon with an unlabeled oligonucleotide probe in the presence of a fluorescent DNA binding dye (dye);
4) measuring the fluorescence intensity changing as the probe melts while gradually raising the temperature of the bound product (DNA melting analysis);
5) After obtaining a differential curve of the change curve of the fluorescence intensity according to the temperature change, typing the SNP according to the presence or absence of a peak representing each type of SNP. delete The SNP genotyping method according to claim 1, wherein the PCR amplicon in step 2) is diluted 2 to 10 times in distilled water or a PCR reaction buffer. The SNP genotyping method according to claim 1, wherein the dilution factor of the final PCR product is 6 to 96 times after mixing with the probe solution in step 3). According to claim 1, SNP genotyping method characterized in that the dilution of the PCR amplicon in step 2) to increase the binding efficiency of the non-labeled probe and the discrimination between SNPs The SNP genotyping method according to claim 1, wherein the probe in step 3) is physically or chemically fixed to a specific solid medium. 2. The SNP genotyping method according to claim 1, wherein the PCR amplification in step 1) is a multiplexing PCR that amplifies multiple SNPs at once. The SNP genotyping method according to claim 1, wherein the step of 2) eliminates the need for 3′-end modification of the probe through inactivation of the polymerase by dilution of the PCR amplicon. The double-stranded PCR of claim 1, wherein in step 3), the diluted PCR amplicon is mixed with a fluorescent DNA binding dye and an unlabeled oligonucleotide probe solution having a sequence complementary to the SNP, and then the mixture is heated. SNP genotyping method, characterized in that the probe is bound to a target SNP site by inducing the denaturation of the product and then left at a temperature lower than the TM of the probe. The SNP genotyping method according to claim 1, wherein the DNA melting analysis in step 4) is performed using a realtime thermal cycler.

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