KR20210080220A - Primer for detecting the minimum rare single nucleotide variant and method for specific and sensitive detecting of the minimum rare single nucleotide variant using the same - Google Patents

Abstract

The present invention relates to a primer set for detecting a minimum rare single nucleotide variant (SNV) to be significantly effective in terms of time, labor, and cost, and a method for specifically and sensitively detecting a minimum rare SNV using the same. Specifically, the present invention relates to a primer composition for detecting an SNV, which includes a nucleotide sequence complementary to a DNA fragment to be detected with respect to the DNA fragment to be detected containing an SNV to be detected and includes a first primer containing 2 to 6 nucleotide sequences non-complementary to the DNA fragment to be detected at the 3' end of the primer, and a method for detecting a minimum rare SNV comprising a step of performing a polymerase chain reaction (PCR) using the composition.

Description

Primer for detecting the minimum rare single nucleotide variant and method for specific and sensitive detecting of the minimum rare single nucleotide variant using the same}

The present invention relates to a primer for detecting a very small amount of a rare single nucleotide variant and a method for specifically and sensitively detecting a very small amount of a rare single nucleotide variant using the same.

A single nucleotide mutation (SNV) refers to a mutation showing a difference in a single nucleotide, and includes single nucleotide polymorphism (SNP) and point mutation.

Single-base variations or differences in DNA or RNA sequences can have important biological consequences, and these single-base variations can be considered as important biomarkers for biomedical research and clinical applications, so the development of technologies to detect them has been a long time coming. Nucleic acid has been the focus of biotechnology. Conventional methods for single-base variant (SNV) detection include MALDI mass spectrometry, DNA sequencing, polymerase chain reaction, microarrays, enzyme-assisted methods, and hybridization-based methods have been developed.

However, these conventionally developed methods have problems with low specificity for detecting a very small amount of SNV, have problems with low detection efficiency due to poor sensitivity, and have limitations in that they take a lot of time and high cost.

On the other hand, due to the development of biotechnology, single nucleotide mutations related to diseases have been revealed, and thus, interest in technologies for diagnosing and treating diseases at an early stage by rapidly and accurately detecting them is increasing.

However, with the technologies developed so far, a technology capable of analyzing a very small amount of a rare single nucleotide variant with high sensitivity and high specificity has not been developed.

Republic of Korea Patent No. 10-1600039

Accordingly, the present inventors completed the present invention by developing a new primer capable of analyzing a very small amount of a rare single nucleotide variant with high sensitivity and high specificity, and a new PCR method capable of detecting a very small amount of a rare single nucleotide variant using the same.

Accordingly, it is an object of the present invention to provide a primer composition for detecting a single base variant capable of detecting a very small amount of a rare single base variant with high sensitivity and high specificity.

Another object of the present invention is to provide a kit for detecting a single base variant comprising the primer composition for detecting a single base variant of the present invention.

Another object of the present invention is to provide a method for detecting a very small amount of a rare single nucleotide variant (SNV).

Therefore, the present invention is a primer composition for detecting a single nucleotide variant, comprising a nucleotide sequence complementary to the detection target DNA fragment with respect to a detection target DNA fragment containing a single nucleotide variation (SNV) to be detected, It provides a primer composition for detecting a single nucleotide variant, comprising a first primer comprising 2 to 6 nucleotide sequences non-complementary to the DNA fragment to be detected at the 3' end of the primer.

In one embodiment of the present invention, the composition comprises a nucleotide sequence complementary to a DNA fragment to be detected, wherein the DNA fragment to be detected is a wild-type DNA fragment that does not contain a single nucleotide mutation, and the 3' end of the primer has the A second primer comprising 2 to 6 nucleotide sequences non-complementary to the wild-type DNA fragment and phosphorylated at the 3' end may be further included.

In one embodiment of the present invention, the primer composition for detecting single base variants may be for polymerase chain reaction (PCR).

The present invention also provides a kit for detecting single base variants, comprising the primer composition for detecting single base variants of the present invention.

In addition, the present invention is a step of performing a polymerase chain reaction (PCR) using the detection target DNA fragment containing a single nucleotide variant (SNV) as a template and using the primer composition for detecting single nucleotide variants of the present invention It provides a method for detecting a very small amount of a rare single nucleotide variation (SNV, Single Nucleotide Variant), including a.

In one embodiment of the present invention, the polymerase chain reaction (PCR) may be performed using a DNA polymerase having no 3'->5' exonuclease activity.

In one embodiment of the present invention, when a single base mutation is present in the DNA fragment to be detected, an amplification product by polymerase chain reaction (PCR) is generated, and when there is no single base mutation in the DNA fragment to be detected may not produce amplification products by polymerase chain reaction (PCR).

The present invention relates to a detection primer capable of very specific and sensitively detecting a very small amount of a rare single base variant, and a method for detecting a rare single base variant present in a very small amount using the same, and for detecting a single base variant according to the present invention The addition of non-complementary nucleotides to the 3' end of the primer has the effect of significantly increasing detection specificity. In particular, when the primer designed in the present invention is completely sequence-complementarily bound to a single base variant, the length of the non-complementary nucleotide at the 3' end is maintained, whereas when it binds to the template strand with a single nucleotide mismatch, the 3' Since the length of the non-complementary nucleotide at the end is increased, the primer provided in the present invention has the effect of very specifically amplifying only a single base variant (SNV). In addition, when a wild-type primer having a non-complementary nucleotide and a phosphate group at the 3' end is used together, detection specificity can be further increased, so that the single-nucleotide variant detection method provided in the present invention does not require sophisticated and complicated primer design. It is very effective in terms of time, labor and cost. Therefore, the primers provided in the present invention can very specifically and sensitively detect rare single nucleotide variants (SNVs) present in very small amounts.

1 is a schematic diagram showing a process for detecting a very small amount of a rare single nucleotide variant (SNV, Single Nucleotide Variant) by a PCR method using the primers designed in the present invention.
2 is a schematic diagram showing a process of analyzing the detection sensitivity (seneitivity) of a single nucleotide variation (SNV, Single Nucleotide Variant) by a PCR method using primers designed in an embodiment of the present invention.
Figure 3 shows the result of confirming the PCR sensitivity of the MT primer to various copies of the MT ultramer template synthesized in an embodiment of the present invention gel electrophoresis.
4 is a schematic diagram showing a process of analyzing the detection specificity of a single nucleotide variant (SNV, Single Nucleotide Variant) by a PCR method using primers designed in an embodiment of the present invention.
5 is a PCR method using primers designed in the present invention for a mixture of various ratios of MT ultramer template and WT ultramer template synthesized by adding nucleotide (T) 30 mer in one embodiment of the present invention to single base mutation (SNV, A schematic diagram of the process of analyzing the detection sensitivity and specificity of Single Nucleotide Variant) is shown.
Figure 6 shows a mixture of MT ultramer template and WT ultramer template in various ratios synthesized by adding ucleotide (T) 30 mer in one embodiment of the present invention using the primers designed in the present invention to perform PCR, gel electrophoresis The results of analysis of the sensitivity and specificity of detection through electrophoresis are shown.

The present invention is characterized by providing a primer for detecting a very small amount of a rare single nucleotide variant and a method for specifically and sensitively detecting a very small amount of a rare single nucleotide variant using the same.

A single nucleotide variant associated with a disease exists in a very small amount in the body, and thus, although it is an important marker for disease diagnosis, there is a problem in that it cannot be accurately and quickly detected.

Accordingly, the present inventors devised a primer composition for detecting a very small amount of a rare single base variant while researching a method for specifically sensitive and rapid detection of a very small amount of a rare single base variant, and a simple analysis method of the polymerase chain reaction It was also confirmed that rare single nucleotide variants can be rapidly and accurately detected with high specificity and sensitivity.

Accordingly, the present invention can provide a primer composition for detecting a single nucleotide variant, wherein the composition is complementary to the detection target DNA fragment with respect to the detection target DNA fragment containing the single nucleotide variation (SNV) to be detected. a nucleotide sequence, but at the 3' end of the primer, a first primer comprising 2 to 6 nucleotide sequences non-complementary to the detection target DNA fragment.

In addition, the primer composition for detecting single nucleotide variants of the present invention includes a nucleotide sequence complementary to a DNA fragment to be detected, wherein the DNA fragment to be detected is a wild-type DNA fragment that does not contain a single nucleotide mutation, and the 3' end of the primer to the wild-type DNA fragment and the non-complementary 2 to 6 nucleotide sequences, and may further include a second primer having a phosphorylated 3' end.

That is, the primer composition for detecting a single base of the present invention includes an SNV-specific primer (a first primer), wherein the first primer is complementary to a DNA fragment to be detected containing a single nucleotide variant (SNV). and a nucleotide sequence that is not complementary to the DNA fragment to be detected at the 3' end of the first primer. In this case, the non-complementary nucleotide sequence may consist of 2 to 10 nucleotide sequences, preferably 2 to 6 nucleotide sequences, more preferably 2 to 3 nucleotide sequences.

In addition, the primer composition for detecting a single base of the present invention may further include a WT-specific primer (a second primer), wherein the second primer is a base sequence complementary to a wild-type DNA fragment that does not contain a single base mutation. and a nucleotide sequence non-complementary to the wild-type DNA fragment is designed at the 3' end of the second primer. In this case, the non-complementary nucleotide sequence may consist of 2 to 10 nucleotide sequences, preferably 2 to 6 nucleotide sequences, more preferably 2 to 3 nucleotide sequences.

Here, the non-complementary nucleotide sequence that may be included in the first primer and the second primer may be any nucleotide sequence that is non-complementary to the nucleotide sequence of the DNA fragment to be detected. In addition, the non-complementary nucleotide sequences of the first primer and the second primer are the same.

In addition, the 3' end of the second primer is designed to be phosphorylated.

In the present invention, if the 3' end phosphorylated second primer is used, the PCR amplification reaction for the wild-type DNA fragment not containing a single base mutation can be blocked, so that the target single base variant by the first primer is more specific. can be detected sensitively.

A schematic diagram of the detection process of a single nucleotide variant using the primer composition for single nucleotide detection devised in the present invention is shown in FIG. 1 , and the present invention is very specific and sensitive to a very small amount of single-nucleotide variant (SNV). For detection, a polymerase chain reaction (PCR) may be performed using a competitive primer composition including the first primer and the second primer. In this case, the SNV-specific primer has a structure having a non-complementary base at the 3' end, and the WT-specific primer has a structure including a non-complementary nucleotide at the 3' end and a phosphate group for amplification blocking. The difference between the two primers lies in the single-nucleotide sequence on the 3' end region (blue area in Fig. 1). These SNV-specific primers and WT-specific primers mainly bind to a perfect match template. Therefore, with this combination, the two primers have a structure in which a short single strand exists due to the non-complementary nucleotide sequence at the 3' end, but the amplification reaction is performed using a DNA polymerase that does not have 3'->5' exonuclease activity. do. Therefore, the WT-specific primer cannot be amplified by PCR reaction because phosphoric acid is present at the 3' end. In addition, the SNV-specific primer and the WT-specific primer can bind to a mismatched template in a very small ratio. As a result, the mismatch binding generates noise, so the two primers are 3 ` It forms a relatively long single-stranded structure at the end. For this reason, DNA polymerase without 3`->5` exonuclease activity does not recognize the 3` end of the primer, so the amplification reaction does not proceed.

Therefore, since the PCR amplification reaction can proceed only when the SNV-specific primer binds to the SNV template strand, non-specific PCR reaction products are not generated and only a very small amount of the target SNV can be amplified. can detect single nucleotide mutations with high sensitivity and specificity.

In this regard, in one embodiment of the present invention, a G12D mutant DNA fragment of the KRAS gene, known to be highly related to cancer development, was prepared and used as a template for PCR analysis, in which case a single strand containing the G12D mutation of the KRAS gene was prepared. The template DNA sequence of SEQ ID NO: 1 is shown, and the single-stranded template DNA sequence including the G12D wild-type sequence of the KRAS gene is shown in SEQ ID NO: 2.

In addition, a reverse primer having a sequence completely complementary to the templates of SEQ ID NO: 1 and SEQ ID NO: 2 was designed and shown in SEQ ID NO: 5.

In one embodiment of the present invention, as the first primer, it includes a nucleotide sequence complementary to the template DNA sequence of SEQ ID NO: 1, which is a single strand containing the G12D mutation of the RAS gene, but is non-complementary to the 3' end of two bases The forward primer sequence of SEQ ID NO: 6 including the sequence 'TT' was prepared.

In addition, in one embodiment of the present invention, as a second primer, a nucleotide sequence complementary to the sequence of SEQ ID NO: 2 with respect to the single-stranded template DNA sequence of SEQ ID NO: 2 including the G12D wild-type sequence of the KRAS gene. However, the forward primer sequence of SEQ ID NO: 7 including 'TT', which is two non-complementary base sequences at the 3' end, was prepared, wherein the primer was phosphorylated at the 3' end.

As a result of performing PCR on the first primer and/or the second primer together with the reverse primer of SEQ ID NO: 5, it was found that the G12D single base mutation of the KRAS gene could be detected with high specificity and sensitivity, and the first primer It could be seen that detection was possible only with the first primer and the second primer was used together, indicating that the detection was possible with the highest specificity and sensitivity.

Accordingly, the present invention may provide a primer composition for detecting a single base variant, and a kit for detecting a single base variant comprising the primer for detecting a single base variant.

In the present invention, the term 'primer' is a short single-stranded oligonucleotide that serves as a starting point of DNA synthesis. A primer binds specifically to a polynucleotide as a template under suitable buffer and temperature conditions, and DNA polymerase adds a nucleoside triphosphate having a base complementary to the template DNA to the primer to link it. is synthesized The temperature (melting temperature, Tm) at which the primer binds to the template strand may vary depending on the base composition and length.

In addition, the sequence of the primer does not need to have a sequence that is completely complementary to a part of the base sequence of the template, and it is sufficient as long as it has sufficient complementarity within a range capable of hybridizing with the template to perform the intrinsic function of the primer.

In addition, the kit for detecting a single base variant of the present invention may include the primer composition for detecting a single base variant of the present invention described above, and the kit may include a test tube or other suitable container, reaction buffer, dNTPs, DNA polymerase ( DNA polymerase) and distilled water.

Furthermore, the present invention can provide a method for detecting a very small amount of a rare single nucleotide variant (SNV), wherein the method uses a detection target DNA fragment containing a single nucleotide variant (SNV) as a template, and performing a polymerase chain reaction (PCR) using the primer composition of the present invention.

In this case, the polymerase chain reaction (PCR) is performed using a DNA polymerase having no 3'->5' exonuclease activity.

In addition, in the case of the method of the present invention, when a single nucleotide mutation is present in the DNA fragment to be detected, an amplification product by polymerase chain reaction (PCR) is generated, and when there is no single nucleotide mutation in the DNA fragment to be detected, It has a characteristic that no amplification product is generated by the polymerase chain reaction (PCR).

Therefore, when the method of the present invention is applied, there is an effect that only a target DNA having a single nucleotide mutation of interest can be detected with high sensitivity and specificity, so that a single nucleotide mutation present in a very small amount related to a disease can be easily detected. and analysis.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these examples.

<Experiment method and preparation example>

All synthesized nucleotides used in the examples of the present invention were obtained from Integrated DNA Technologies, Inc. (IDT) was ordered and used. The synthesized nucleotide was received in a freeze-dried state, diluted with TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0), and then used for the experiment after incubation at 50 ° C. in a mixing block (MB102; Bioer) for 1 hour. In addition, the synthesized nucleotides used in the real-time PCR of the present invention are as shown in Table 1 below.

In Table 1, the templates of 1-1 & 1-2 are composed of a single stranded Ultramer (195 nt) containing the KRAS gene G12D mutant type sequence and a single stranded Ultramer (195 nt) containing the KRAS gene G12D wild type sequence. , each single-stranded ultramer has a phosphorylation modification at the 3' end.

In Table 1, the mutant type T30 Ultramer Template is composed of a single-stranded Ultramer (195 nt) containing the KRAS gene G12D mutant type sequence and nucleotide T 30 mer, and has a phosphorylation modification at the 3' end.

In Table 1, Forward Primer (FP) has a completely complementary sequence to the KRAS gene G12D mutant type Ultramer (template).

Reverse Primer (RP) has a completely complementary sequence in common to KRAS gene G12D mutant type and KRAS gene G12D wild type Ultramer (template).

In Table 1, Mutant-specific Forward Primer (FP) only has a sequence complementary to the KRAS gene G12D mutant type template, and has non-complementary nucleotides in the 3' end region.

In Table 1, Wild-specific Forward Primer (FP) only has a sequence complementary to the KRAS gene G12D wild type template, and has non-complementary nucleotides in the 3' end region. In addition, phosphorylation modification is performed at the 3' end.

In addition, a Taqman Probe was used as a signal detection method for real-time PCR amplification products. Real-time PCR was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems) equipment, and AmpliTaq Gold™ 360 Master Mix (Applied Biosystems™) was used as the DNA polymerase used for real-time PCR.

In addition, in the experiments of the present invention, Thermo Fisher Scientific products were used for electrophoresis, otherwise the products performed in each example were separately indicated.

For polyacrylamide gel electrophoresis (PAGE: Polyacrylamide gel electrophoresis), add 2 μl of Novex TBE-Sample Buffer (5X) to each sample, mix well, dispense 10 μl on Invitrogen 10% TBE gel, and electrophoresis at 180 V for 35 minutes. did. After electrophoresis, the gel was well separated, diluted with 5 μl of SYBR Gold Nucleic Acid gel stain in 100 ml of distilled water, and reacted in ROCKER (RF200; BLE) for 15 minutes.

<Example 1>

SNV-PCR performance and sensitivity confirmation using the primer set according to the present invention

In the preparation example, the SNV detection sensitivity for the primers designed in the present invention was analyzed by measuring the Ct value using the template DNA of Table 1 and each primer designed by the present inventors. Specifically, real-time PCR was performed using Mutant-specific Forward primers and Wild-specific Forward primers (non-complementary nucleotides: 0, 2nt) targeting Mutant type Ultramer (template). The conditions of real-time PCR and the composition of each experimental group are as described in Tables 2 and 3 below, and each experimental group consisted of samples according to the template concentration. A schematic diagram of SNV-PCR performance for sensitivity analysis according to the present invention described above is shown in FIG. 2 .

The results of the experiment are shown in Table 4 and FIG. 2 below, and these results are the results of confirming the PCR sensitivity of the MT primer to various copies of the synthesized mutant type (MT) ultramer template through real-time PCR. As a result of the analysis, experimental group 4, that is, the group using the Mutant-specific Forward Primer and Wild-specific Forward Primer (non-complementary nucleotide: 2 nt) set, can detect the SNV of KRAS gene G12D with high sensitivity compared to other experimental groups. It was found that, specifically, it was shown to have a PCR sensitivity of ^{MT template 10 2 copy (Ct value. 59) in less than 60 cycles of PCR.}

In addition, PCR sensitivity was also confirmed through gel electrophoresis using the primers of Experimental Group 3 and Experimental Group 4 of Table 3 for various copies of the synthesized mutant type ultramer template. As a result, as shown in FIG. 3, Mutant type It was shown to show PCR sensitivity of ultramer template 10 ² copy (Ct value. 59).

<Example 2>

SNV-PCR performance and specificity confirmation using the primer set according to the present invention

Furthermore, the present inventors analyzed specificity for SNV detection using the primer set designed in the present invention. For this purpose, real-time PCR was performed using Mutant-specific Forward Primer and Wild-specific Forward Primer (non-complementary nucleotide: 0, 2 nt) targeting the wild type ultramer template. The composition for the experimental group is as described in Tables 5 and 6 below, and each experimental group consists of a sample according to the template concentration. A schematic diagram of SNV-PCR performance for sensitivity analysis according to the present invention described above is shown in FIG. 4 .

As a result of SNV detection specificity analysis of the primer set designed in the present invention, as shown in Table 7 below, Experimental Group 4, that is, Mutant-specific Forward Primer + Wild-specific Forward Primer (non-complementary nucleotide: 2 nt) set The group used was found to be able to detect SNV of KRAS gene G12D with higher specificity compared to other experimental groups, and this result showed that ^{the PCR sensitivity of MT template 10 2} copy (Ct value. 59) in less than 60 cycles of PCR considering, was found that at least 60 PCR cycles indicating a specificity of 10 ^three times, it was found that in less than 60 PCR cycles shows the specificity of the 10 ^four times.

Therefore, through these results, it was found that, when the primer set designed in the present invention is used, rare SNVs, especially SNVs present in very small amounts, can be quickly and accurately detected with very good sensitivity and specificity through a simple method of PCR. .

<Example 3>

Confirmation of sensitivity and specificity according to SNV-PCR using the primer set according to the present invention

In addition, the present inventors analyzed the sensitivity and specificity for SNV detection using the primers designed in the present invention for the mixed template DNA in which the mutant type T30 Ultramer template and wild type Ultramer template were mixed. For this purpose, real-time using various ratios of template mixture (mutant type T30 Ultramer template + wild type Ultramer template mixture) and Mutant-specific Forward Primer and Wild-specific Forward Primer (non-complementary nucleotides: 0, 2 nt) PCR was performed, and the composition and real-time PCR conditions for each experimental group are as shown in Tables 8 and 9 below, and RT-PCR for the sensitivity and specificity analysis according to the present invention described above. A schematic diagram is shown in FIG. 5 .

In addition, in this example, a mutant type T30 Ultramer template was used, which is a PCR product by MT ultramer and a PCR by WT ultramer when two types of ultramer templates (MT / WT) are used together in a real-time PCR reaction. Because TaqMan probes can be annealed in common to all products to generate a signal, for more accurate amplification, it is determined whether amplified by MT ultramer template or WT ultramer template by different lengths. It was made to be easily confirmed through gel electrophoresis.

Sensitivity and specificity through real-time PCR analysis using MT primers for a mixture of MT T30 Ultramer template and WT ultramer template synthesized with nucleotide (T) 30 mer in various ratios to make a difference in amplicon size It was confirmed through Ct vlaue value analysis, and also analyzed through gel electrophoresis.

As a result, as shown in Table 10 and Figure 6, even in the result of using a mixed template of MT and WT using the primer of the present invention (Mismatch) regardless of WT template copy (Perfect-match) MT template 10 ² copy of PCR sensitivity, and 10 ^3- fold PCR specificity (VAF. 0.1%).

Therefore, from these results, the present inventors found that, when using the primers designed in the present invention, a very small amount of SNV can be detected with high sensitivity and specificity by a simple method through PCR.

So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

<110> Industry Academic Cooperation Foundation of Korea University <120> Primer for detecting the minimum rare single nucleotide variant and method for specific and sensitive detecting of the minimum rare single nucleotide variant using the same <130> NPDC82281.01 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 195 <212> DNA <213> Artificial Sequence <220> <223> Mutant type Ultramer template sequence <400> 1 aaggcctgct gaaaatgact gaatataaac ttgtggtagt tggagctgat ggcgtaggca 60 agagtgcctt gacgatacag ctaattcaga atcattttgt ggacgaatat gatccaacaa 120 tagaggtaaa tcttgtttta atatgcatat tactggtgca ggaccattct ttgatacaga 180 taaaggtttc tctga 195 <210> 2 <211> 195 <212> DNA <213> Artificial Sequence <220> <223> Wild type Ultramer template sequence <400> 2 aaggcctgct gaaaatgact gaatataaac ttgtggtagt tggagctggt ggcgtaggca 60 agagtgcctt gacgatacag ctaattcaga atcattttgt ggacgaatat gatccaacaa 120 tagaggtaaa tcttgtttta atatgcatat tactggtgca ggaccattct ttgatacaga 180 taaaggtttc tctga 195 <210> 3 <211> 195 <212> DNA <213> Artificial Sequence <220> <223> Mutant typeT30 Ultramer template sequence <400> 3 aatataaact tgtggtagtt ggagctgatg gcgtaggcaa gagtgccttg acgatacagc 60 taattcagaa tcattttgtg gacgaatatg atccaacaat agaggtattt tttttttttt 120 tttttttttt tttttttaat cttgttttaa tatgcatatt actggtgcag gaccattctt 180 tgatacagat aaagg 195 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer sequence <400> 4 ttgtggtagt tggagctgat 20 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer sequence <400> 5 ctgtatcaaa gaatggtcct gcac 24 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Mutant-specific Forward Primer (2nt) <400> 6 ttgtggtagt tggagctgat tt 22 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Wild-specific Forward Primer (2nt) <400> 7 ttgtggtagt tggagctggt tt 22

Claims (7)

A primer composition for detecting single base variants, comprising:
For a detection target DNA fragment containing a single nucleotide variant (SNV) to be detected, a nucleotide sequence complementary to the detection target DNA fragment is included, but at the 3' end of the primer, the detection target DNA fragment and A primer composition for detecting a single base variant, comprising a first primer comprising a complementary 2 to 6 base sequence. According to claim 1,
A nucleotide sequence complementary to a DNA fragment to be detected, wherein the DNA fragment to be detected is a wild-type DNA fragment that does not contain a single nucleotide mutation, and 2 to 6 bases non-complementary to the wild-type DNA fragment at the 3' end of the primer A primer composition for detecting single base variants, characterized in that it further comprises a second primer comprising a sequence and phosphorylated at the 3' end. According to claim 1,
The primer composition for detecting single base variants is a primer composition for detecting single base variants, characterized in that it is for polymerase chain reaction (PCR). A kit for detecting single base variants, comprising the composition of claim 1. Including the step of performing a polymerase chain reaction (PCR) using the primer composition of claim 1 using a DNA fragment to be detected containing a single nucleotide mutation (SNV, Single Nucleotide Variant) as a template,
A method for detecting a very small amount of a rare single nucleotide variant (SNV). 6. The method of claim 5,
The polymerase chain reaction (PCR) method, characterized in that it is performed using a DNA polymerase without 3'->5' exonuclease (exonuclease) activity. 6. The method of claim 5,
If a single nucleotide mutation exists in the DNA fragment to be detected, an amplification product by polymerase chain reaction (PCR) is generated, and if there is no single nucleotide mutation in the DNA fragment to be detected, the polymerase chain reaction (PCR) is used. A method characterized in that no amplification products are produced by

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