Single-stranded nucleic acid for real-time detection of genetic mutations in a single target gene and detection method using the same

Abstract

The present invention relates to a single-stranded nucleic acid for real-time detection of genetic mutations in a single target gene and a detection method using the same, and more particularly to a method and kit for real-time detection of genetic mutations in a single target gene using a single-stranded nucleic acid having an XYZ structure and a base sequence capable of complementary binding to part or all of the base sequence of a single target gene containing a genetic mutation such as a single nucleotide polymorphism (SNP), a point mutation, or an miRNA isoform. The method for detecting genetic mutations in a single target gene in real time using a single-stranded nucleic acid that is cleaved only by the cleavage reagent according to the present invention has the advantage of being more accurate than conventional methods for detecting genetic mutations using probes. Furthermore, it can accurately distinguish various point mutations that occur in cancer-related genes such as KRAS and EGFR, and is useful for the diagnosis and prognosis of various diseases including cancer.

Description

[Technical field]

[0001] The present invention relates to a single-stranded nucleic acid for real-time detection of genetic mutations in a single target gene and a detection method using the same, and more particularly to a method and kit for detecting genetic mutations in a single target gene in real-time using a single-stranded nucleic acid having a base sequence that has an XYZ structure and is capable of complementary binding to a part or all of the base sequence of a single target gene containing a genetic mutation such as a single nucleotide polymorphism, a point mutation, or an miRNA isoform. [Background technology]

[0002] In genetic engineering, single nucleotide polymorphism (SNP) mutations are the most common form and act as a cause of various diseases (Barreiro LB, et al., Methods Mol. Biol., 578:255-276, 2009; Beaudet L. et al., Genome Res., 11(4):600-608, 2001). Therefore, in order to diagnose various diseases caused by genetic recombination at an early stage, a diagnostic method based on detection of SNPs is extremely efficient and allows for rapid diagnosis. Therefore, many methods for accurately detecting SNPs have been presented, and many related studies are still ongoing (Ermini ML. et al., Biosen. & Bioele., 61:28-37, 2014; K. Chang et al., Biosen. & Bioele., 66:297-307, 2015).

[0003] Specifically, the most commonly used method for multiple gene analysis includes a method using polymerase chain reaction (PCR), a multiplex polymerase chain reaction (Multiplex PCR) method.

[0004] The polymerase chain reaction has an advantage that it can precisely amplify only the desired region of a gene to be detected by arbitrarily designing primers or probes that can bind to template DNA and have fluorescent and quenching substances bound thereto. However, since only one gene of interest can be amplified in one reaction, if there are multiple genes of interest to be amplified, the same process must be repeated.

[0005] The multiplex polymerase chain reaction has the advantage that multiple gene regions can be analyzed simultaneously by performing several polymerase chain reactions in one tube. However, the use of several primers or probes simultaneously in one tube can cause cross-reactions between primers or primers, limiting the number of gene regions that can be amplified at one time. In addition, it requires a lot of effort and time to find the reaction conditions, and it is difficult to obtain good results in terms of sensitivity and specificity (Hardenbol P. et al., Nat. Biotechnol., 21(6):673-678, 2003).

[0006] In recent years, there has been active research into the use of common primers to simultaneously amplify multiple gene regions, enabling large-scale analysis, without using multiplex polymerase chain reaction. Representative techniques include SNPlex, which can simultaneously analyze single nucleotide polymorphisms (SNPs) in several gene regions, Golden Gate assay, and molecular inversion primers. lobes (molecular inversion probes (MIPs)).

[0007] The SNPlex method involves a purification process using exonuclease after oligonucleotide ligation assay (OLA), polymerase chain reaction amplification using common primer sequences on both ends of the probe, and finally analysis on a DNA chip using the ZipCode sequence contained in the probe (Tobler et al., J. Biomol. Tech., 16(4):398-406, 2005).

[0008] The Golden Gate assay involves performing allele-specific primer extension reaction with an upstream probe on genomic DNA immobilized on a solid surface, followed by DNA ligation reaction with a downstream probe, and then removing the probe that was not ligated through a washing process. Similar to SNPlex, the assay amplifies the DNA using a common primer sequence contained in the probe, and analyzes the amplified polymerase chain reaction product using an Illumina BeadChip (Shen R. et al., Mutat. Res., 573(1-2):70-82, 2005).

[0009] Molecular inversion probes (MIPs) are a method for analyzing single nucleotide polymorphisms by performing gap ligation using a padlock probe, removing the probe and genomic DNA that are not ligated using exonuclease, linearizing the padlock probe using uracil-N-glycosylase, and then performing polymerase chain reaction using a common primer sequence contained in the probe and hybridizing it to GenFlex Tag Array (Affymetrix) (Hardenbol P. et al., Nat. Biotechnol., 21(6):673-678, 2003).

[0010] However, these methods have problems in that a part of the product of the reaction in the first tube must be transferred to the second tube for reaction or several kinds of enzymes must be used, which may cause contamination between different samples and complicate the experimental method. Moreover, since only the number of single nucleotide polymorphisms that can be detected is the same as the number of fluorescently labeled probes, the cost of analysis increases as the number of single nucleotide polymorphisms to be analyzed increases.

[0011] Point mutations are generally known to occur during DNA replication. DNA replication occurs when one double-stranded DNA molecule produces two single-stranded DNA strands, each of which serves as a template for the production of a complementary strand. A single point mutation can change the entire DNA sequence, and by changing one purine or pyrimidine, the amino acid that the nucleotide codes for can be changed.

[0012] Point mutations arise from spontaneous mutations during DNA replication, and the mutation rate can be elevated due to such causes.

[0013] In 1959, Ernst Freese coined the term "transitions" or "transversions" to classify different types of point mutations. Transitions are mutations that result in the substitution of one purine base for another, or the replacement of a purine base with another purine. A pyrimidine is a substitution of another pyrimidine for a purine. A transversion is a substitution of a purine for a pyrimidine or vice versa. The mutation rates for transitions (alpha) and transversions (beta) are different, with transition mutations usually occurring about 10 times more frequently than transversions.

[0014] The functional classification of point mutations is as follows: nonsense mutations, which shorten or add abnormally the production of a protein, such as stop-gain and start-loss, and missense mutations, which code for other amino acids (such as valine replacing glutamic acid in the BRAF gene). When the nucleotide sequence is changed to a nucleotide sequence (nucleotide 1), this leads to the activation of the RAF protein, which causes unlimited proliferation signals in cancer cells. nucleotide sequences (nucleotide 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 5

[0015] Such point mutations are also known to be the cause of certain diseases. Point mutations in multiple tumor suppressor proteins cause cancer, and neurofibromatosis occurs due to point mutations in the neurofibromin 1 or neurofibromin 2 genes. Sickle cell anemia Anemia is caused by a point mutation in the β-globin chain of hemoglobin, which replaces the hydrophilic amino acid glutamic acid at position 6 with the hydrophobic amino acid valine. It is also found in Tay-Sachs disease and color blindness.

[0016] Such point mutation analysis is particularly important for diagnosing cancer and for selecting therapeutic agents. Accompanying diagnosis is utilized as an important basis for selecting an optimal anticancer drug or eliminating a specific anticancer drug according to cancer mutation, and in recent years, the development of anticancer drugs targeting specific mutations has been increasing.

[0017] Such mutation analysis methods are performed by PCR, next-generation sequencing (NGS), droplet digital PCR (ddPCR), etc., and the demand and interest in liquid biopsy continues to increase, so more precise measurement methods are required. However, currently known methods do not have sufficient analytical functions, or if they do, they require expensive separate equipment and complicated analytical processes, so there is a demand for the development of methods that allow simpler analysis.

[0018] Therefore, the present inventors have been studying for a long time how to increase the low sensitivity and specificity in detecting genetic modifications such as SNP, point mutation, miRNA isoforms, etc. in real time, and can diagnose cancer simply and accurately. As a result, the present inventors have found that when the present inventors' single-stranded nucleic acid is used, high sensitivity and high specificity are exhibited, and it is extremely useful for diagnosing various diseases caused by genetic modifications, such as cancer, and have completed the present invention. Summary of the Invention [Problem to be solved by the invention]

[0019] One object of the present invention is to provide a single-stranded nucleic acid for detecting a genetic mutation in a single target gene, characterized in that i) it has a structure of XYZ, ii) it binds complementary to a part or all of the base sequence of a single target gene containing a genetic mutation, and iii) it has the same or at least two different detectable markers attached to both ends or inside, and the Y is an RNA composed of one or two base sequences located in the single target gene.

[0020] Another object of the present invention is to provide a method for determining whether a genetic variation in a single target gene is a single nucleotide polymorphism (SNP). The present invention provides a single-stranded nucleic acid for detecting a genetic mutation in a single target gene, wherein, when X is a nucleotide polymorphism (SNP), the single-stranded nucleic acid is characterized in that (a) X is a DNA composed of a 4 to 20 nucleotide sequence, and (b) Z is a DNA composed of a 4 to 20 nucleotide sequence.

[0021] Another object of the present invention is to provide a single-stranded nucleic acid for detecting a genetic mutation in a single target gene, characterized in that, when the genetic mutation in the single target gene is a single nucleotide polymorphism (SNP), a point mutation, or an miRNA isoform, (c) the X is a DNA consisting of a 10-30 base sequence, and (d) the Z is a DNA consisting of a 1-5 base sequence.

[0022] It is still another object of the present invention to provide a kit for real-time detection of a genetic mutation in a single target gene, comprising the single-stranded nucleic acid.

[0023] Another object of the present invention is to provide a method for detecting a genetic mutation in a single target gene, the method comprising: a) obtaining a target nucleic acid containing a genetic mutation to be detected from a biological sample; b) preparing a single-stranded nucleic acid for detecting a genetic mutation in the single target gene; c) mixing the target nucleic acid obtained in step a), the single-stranded nucleic acid prepared in step b), a primer set having a base sequence complementary to the target nucleic acid obtained in step a), and a cleavage reagent, and then amplifying a target nucleic acid-single-stranded nucleic acid complex containing the genetic mutation by an extension reaction; and d) measuring the amount of single-stranded nucleic acid fragment separated from the target nucleic acid-single-stranded nucleic acid complex containing the genetic mutation amplified in step c). [Means for solving the problem]

[0024] To achieve the above object, the present invention provides a single-stranded nucleic acid for detecting a genetic mutation in a single target gene.

[0025] In detail, the single-stranded nucleic acid i) has a structure of XYZ, ii) binds complementarily to a part or all of the base sequence of a single target gene containing a genetic mutation, and iii) has the same or at least two different detectable markers attached to both ends or inside, and Y is an RNA composed of one or two base sequences located in the single target gene, and is cleaved by a cleavage reagent when hybridized with the single target gene.

[0026] In this case, the single-stranded nucleic acid is characterized in that, when the genetic mutation of the single target gene is a single nucleotide polymorphism (SNP), (a) the X is a DNA composed of a 4 to 20 base sequence, and (b) the Z is a DNA composed of a 4 to 20 base sequence; or, when the genetic mutation of the single target gene is a single nucleotide polymorphism (SNP), a point mutation, or an miRNA isoform, (c) the X is a DNA composed of a 10 to 30 base sequence, and (d) the Z is a DNA composed of a 1 to 5 base sequence.

[0027] In addition, when the genetic mutation of the single target gene is a single nucleotide polymorphism (SNP), the single-stranded nucleic acid hybridizes with the single target gene, and when the Y is cleaved by a cleavage reagent, the X and Z are also separated from the single target gene and act as a probe. Alternatively, when the genetic mutation of the single target gene is a single nucleotide polymorphism (SNP), a point mutation, or miRNA, In the case of an isoform, when Y is cleaved by a cleavage reagent after hybridizing with a single target gene, Z is separated from the single target gene, but X is not separated and functions as a primer and a probe simultaneously.

[0028] The present invention also provides a kit for real-time detection of a genetic mutation in a single target gene, comprising the single-stranded nucleic acid.

[0029] The present invention further provides a method for detecting a genetic mutation in a single target gene, the method comprising the steps of: a) obtaining a target nucleic acid containing a genetic mutation to be detected from a biological sample; b) producing a single-stranded nucleic acid for detecting a genetic mutation in the single target gene; c) mixing the target nucleic acid obtained in step a), the single-stranded nucleic acid produced in step b), a primer set having a base sequence complementary to the target nucleic acid obtained in step a), and a cleavage reagent, and then amplifying a target nucleic acid-single-stranded nucleic acid complex containing the genetic mutation by an extension reaction; and d) measuring the amount of single-stranded nucleic acid fragment separated from the target nucleic acid-single-stranded nucleic acid complex containing the genetic mutation amplified in step c). Effect of the Invention

[0030] The method for detecting genetic mutations in a single target gene in real time using the single-stranded nucleic acid according to the present invention has the advantage that it does not require a probe at a separate position for real-time confirmation and can more accurately measure genetic mutations such as SNPs and point mutations, compared to the conventional SNP and point mutation analysis method using qPCR. That is, the single-stranded nucleic acid according to the present invention is cleaved only by the cleavage reagent, and can be more accurately measured than the conventional genetic mutation detection method using a probe.

[0031] In addition, when analyzing genetic mutations such as SNP and point mutations using the single-stranded nucleic acid according to the present invention, mutations can be immediately identified without the need for a separate mutation confirmation process such as melting temperature analysis.

[0032] Therefore, the single-stranded nucleic acid of the present invention and the method for detecting genetic mutations in a single target gene in real time using the same can rapidly and accurately distinguish various point mutations occurring in KRAS, EGFR, etc., and can be usefully used in the diagnosis, selection of therapeutic agents, and prognosis of various diseases including cancer. [Brief description of the drawings]

[0033] [Figure 1] FIG. 1 shows the results of PCR confirming the presence or absence of ApoE gene expression using a single-stranded nucleic acid according to one embodiment of the present invention. In particular, the results show that ApoE single-stranded nucleic acid type 1 (sequence numbers 3 to 6) can be used to distinguish between six phenotypes of the ApoE gene, namely E2 / E2, E3 / E3, E4 / E4, E2 / E3, E2 / E4, and E3 / E4. [Diagram 2] FIG. 1 shows a graph confirming the presence or absence of expression of the G13D mutant gene of the KRAS gene using G13D single-stranded nucleic acid type 1 according to one embodiment of the present invention. In detail, the graph shows the PCR results in which the genomic DNA (gDNA) of the HCT-15 cell line, which is a G13D mutant cell line, was diluted according to concentration, and the presence or absence of expression of the G13D mutant gene depending on the concentration of gDNA was confirmed using G13D single-stranded nucleic acid type 1 (sequence number 7). [Diagram 3] FIG. 1 is a diagram showing whether or not the wild-type KRAS gene is expressed using a KRAS wild-type single-stranded nucleic acid according to one embodiment of the present invention. In detail, the figure shows the PCR results in which gDNA from the NCI-H1975 cell line, which is a KRAS wild-type cell line, was diluted according to concentration, and then the presence or absence of wild-type KRAS gene expression depending on the gDNA concentration was confirmed using KRAS wild-type single-stranded nucleic acid type 1 (sequence number 8). [Figure 4]This is a diagram confirming the presence or absence of expression of wild-type KRAS gene using KRAS wild-type single-stranded nucleic acid type 1 according to one embodiment of the present invention. In detail, the figure shows the PCR results in which the presence or absence of expression of wild-type KRAS gene depending on the concentration of gDNA was confirmed using KRAS wild-type single-stranded nucleic acid type 1 (SEQ ID NO: 8) after diluting genomic DNA (gDNA) of HCT-15 cell line according to the concentration, and it was confirmed that HCT-15 cell line is a cell line that simultaneously has G13D mutation and KRAS wild-type gene as heterozygous type genes. [Diagram 5] FIG. 1 is a diagram showing whether or not the expression of the G13D mutant gene of the KRAS gene mixed with the wild-type KRAS gene was confirmed using a KRAS wild-type single-stranded nucleic acid type 1 according to one embodiment of the present invention. In detail, the genomic DNA (gDNA) of the HCT-15 cell line was diluted to a concentration of 10 to 0.01% relative to the concentration of the genomic DNA (gDNA) of the NCI-H1975 cell line, and the genomic DNAs (gDNA) of these cell lines were mixed, and the PCR results were confirmed using G13D single-stranded nucleic acid type 1 (SEQ ID NO: 7). [Figure 6] FIG. 1 shows the results of confirming the presence or absence of ApoE gene expression using single-stranded nucleic acid type 2 according to one embodiment of the present invention. In particular, the results show the PCR results confirming that ApoE single-stranded nucleic acid type 2 (SEQ ID NOs: 11 to 14) can be used to distinguish between six phenotypes of the ApoE gene, namely E2 / E2, E3 / E3, E4 / E4, E2 / E3, E2 / E4, and E3 / E4. [Figure 7] FIG. 1 is a diagram showing the results of confirming the presence or absence of expression of the G12V mutant gene of the KRAS gene using single-stranded nucleic acid type 2 according to one embodiment of the present invention. In detail, the results are PCR results in which the genomic DNA (gDNA) of the SW620 cell line, a G12V mutant cell line, was diluted according to concentration, and the presence or absence of expression of the G12V mutant gene depending on the concentration of gDNA was confirmed using G12V single-stranded nucleic acid type 2 (sequence number 15). [Figure 8]FIG. 1 is a diagram showing the results of confirming the presence or absence of expression of the G12C mutant gene of the KRAS gene using single-stranded nucleic acid type 2 according to one embodiment of the present invention. In detail, the results are PCR results in which gDNA from the MIA-Paca2 cell line, which is a G12C mutant cell line, was diluted according to concentration, and then the presence or absence of expression of the G12C mutant gene depending on the gDNA concentration was confirmed using G12C single-stranded nucleic acid type 2 (sequence number 16). [Figure 9] FIG. 1 shows the results of confirming the presence or absence of expression of the G12S mutant gene of the KRAS gene using single-stranded nucleic acid type 2 according to one embodiment of the present invention. In detail, the results are PCR results in which gDNA of the A549 cell line, which is a G12S mutant cell line, was diluted according to concentration, and the presence or absence of expression of the G12S mutant gene depending on the gDNA concentration was confirmed using G12S single-stranded nucleic acid type 2 (sequence number 17). [Figure 10] FIG. 2 is a diagram showing whether or not the expression of the T790M mutant gene in axon 20 of the EGFR gene was confirmed using single-stranded nucleic acid type 2 according to one embodiment of the present invention. In detail, the gDNA of the H1975 cell line, which is a T790M mutant cell line, and the A549 cell line, which is a wild-type cell line, were diluted according to concentration, and then the expression of the T790M mutant gene depending on the gDNA concentration was confirmed using T790M single-stranded nucleic acid type 2 (sequence number 24) as a PCR result. [Figure 11] FIG. 1 shows the results of confirming the presence or absence of expression of the let-7a miRNA gene using single-stranded nucleic acid type 2 according to one embodiment of the present invention. In detail, the let-7a cDNA synthesized from let-7a miRNA by RT-PCR was diluted according to concentration, and the presence or absence of expression of the let-7a miRNA gene depending on the cDNA concentration was confirmed using let-7a single-stranded nucleic acid type 2 (sequence number 21) as a PCR result. [Figure 12] FIG. 1 shows the results of confirming the expression or non-expression of the let-7d miRNA gene using single-stranded nucleic acid type 2 according to one embodiment of the present invention. In detail, the let-7d cDNA synthesized from let-7d miRNA by RT-PCR was diluted according to concentration, and the expression or non-expression of the let-7d miRNA gene depending on the cDNA concentration was confirmed using let-7d single-stranded nucleic acid type 2 (sequence number 24) as a result of PCR. [Figure 13] FIG. 1 shows the results of confirming the specific detection ability of the let-7 miRNA gene using single-stranded nucleic acid type 2 according to one embodiment of the present invention. In detail, the PCR results show that let-7a cDNA was diluted according to concentration in the presence of let-7d cDNA (1 pM concentration), and then the presence or absence of expression of the let-7a miRNA gene depending on the cDNA concentration, i.e., the presence or absence of specific detection of the let-7a miRNA gene in the presence of let-7d cDNA, were confirmed using let-7a single-stranded nucleic acid type 2 (sequence number 21). [Figure 14] FIG. 1 is a diagram showing whether or not the expression of miRNA 34a gene is confirmed using single-stranded nucleic acid type 2 according to one embodiment of the present invention, specifically, the PCR results in which miRNA 34a cDNA synthesized from miRNA 34a by RT-PCR was diluted according to concentration, and then the expression of miRNA 34a gene depending on the cDNA concentration was confirmed using miRNA 34a single-stranded nucleic acid type 2 (SEQ ID NO: 27). [Figure 15] FIG. 1 is a diagram showing whether or not the expression of miRNA 34b gene is confirmed using single-stranded nucleic acid type 2 according to one embodiment of the present invention, specifically, the PCR results in which miRNA 34b cDNA synthesized from miRNA 34b by RT-PCR was diluted according to concentration, and then the presence or absence of expression of miRNA 34b gene depending on the cDNA concentration was confirmed using miRNA 34b single-stranded nucleic acid type 2 (SEQ ID NO: 30). [Figure 16] FIG. 1 is a diagram showing whether or not the expression of miRNA 34c gene is confirmed using single-stranded nucleic acid type 2 according to one embodiment of the present invention, specifically, the PCR results in which miRNA 34c cDNA synthesized from miRNA 34c by RT-PCR was diluted according to concentration, and the presence or absence of expression of miRNA 34c gene depending on the cDNA concentration was confirmed using miRNA 34c single-stranded nucleic acid type 2 (SEQ ID NO: 33). [Figure 17]FIG. 1 shows the results of confirming the specific detection ability of miRNA 34 gene using single-stranded nucleic acid type 2 according to one embodiment of the present invention. More specifically, the figure shows the PCR results obtained by diluting miRNA 34c cDNA at different concentrations in the presence of miRNA 34a cDNA (100 pM concentration) and then confirming the presence or absence of expression of miRNA 34c gene depending on the cDNA concentration, i.e., the presence or absence of specific detection of miRNA 34c gene in the presence of miRNA 34a cDNA, using miRNA 34c single-stranded nucleic acid type 2 (SEQ ID NO: 33). [Figure 18] FIG. 1 shows the results of confirming the specific detection ability of miRNA 34 gene using single-stranded nucleic acid type 2 according to one embodiment of the present invention. More specifically, the figure shows the PCR results obtained by diluting miRNA 34c cDNA at different concentrations in the presence of miRNA 34b cDNA (100 pM concentration) and then confirming the presence or absence of expression of miRNA 34c gene depending on the cDNA concentration, i.e., the presence or absence of specific detection of miRNA 34c gene in the presence of miRNA 34b cDNA, using miRNA 34c single-stranded nucleic acid type 2 (SEQ ID NO: 33). [Figure 19] FIG. 1 shows a result of confirming the expression of the G12D mutant gene of the KRAS gene using single-stranded nucleic acid type 2 (G12D-R1DrMR2) of the KRAS gene G12D of a DNA oligo (DNA oligo)-DNA-RNA-mutation-DNA oligo type according to one embodiment of the present invention. In detail, the gDNA of the Aspc-1 cell line, which is a G12D mutant cell line, and the HT-29 cell line, which is a KRAS wild-type cell line, were diluted according to concentration, and the expression of the G12D mutant gene depending on the gDNA concentration was confirmed using G12D single-stranded nucleic acid type 2 (sequence number 36). [Figure 20]FIG. 1 shows a diagram confirming the presence or absence of expression of the G12D mutant gene of the KRAS gene using single-stranded nucleic acid type 2 (G12D-R1rDMR2) of the KRAS gene G12D of a DNA oligo-RNA-DNA-mutation-DNA oligo type according to one embodiment of the present invention. In detail, the gDNA of the Aspc-1 cell line, which is a G12D mutant cell line, and the HT-29 cell line, which is a KRAS wild-type cell line, were diluted at different concentrations, and the presence or absence of expression of the G12D mutant gene depending on the gDNA concentration was confirmed using G12D single-stranded nucleic acid type 2 (sequence number 37) as a PCR result. [Figure 21] FIG. 1 shows the results of confirming the expression of the G12D mutant gene of the KRAS gene using single-stranded nucleic acid type 2 (G12D-R1DMrDR2) of the KRAS gene G12D of a DNA oligo-DNA-mutation-RNA-DNA-DNA oligo type according to one embodiment of the present invention. In more detail, the gDNA of the Aspc-1 cell line, which is a G12D mutant cell line, and the HT-29 cell line, which is a KRAS wild-type cell line, were diluted at different concentrations, and the expression of the G12D mutant gene depending on the gDNA concentration was confirmed using G12D single-stranded nucleic acid type 2 (sequence number 38). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The present invention relates to a single-stranded nucleic acid for real-time detection of genetic mutations in a single target gene and a detection method using the same, and more particularly to a method and kit for detecting genetic mutations in a single target gene in real-time using a single-stranded nucleic acid having a base sequence that has an XYZ structure and is capable of complementary binding to a part or all of the base sequence of a single target gene containing a genetic mutation such as a single nucleotide polymorphism (SNP), a point mutation, or an miRNA isoform.

[0035] According to one embodiment, the present invention provides a single stranded nucleic acid for detecting genetic variations in a single target gene.

[0036] In the present invention, the single-stranded nucleic acid is characterized in that i) it has an XYZ structure, ii) it binds complementarily to a part or all of the base sequence of a single target gene containing a genetic mutation, and iii) it has the same or at least two different detectable markers attached to both ends or inside.

[0037] In this case, the position of the detectable marker is not limited to a specific site, and may be any position where the detectable marker is separated upon cleavage of the Y site by a cleavage reagent.

[0038] The single-stranded nucleic acid is characterized in that it complementarily binds to a part or the whole of the base sequence of a single target gene containing a genetic mutation to be detected in real time to form a complex, which is then amplified.

[0039] In the present invention, the single-stranded nucleic acid is used for detecting genetic mutations, such as single nucleotide polymorphisms (SNPs), point mutations, etc. The term "single-stranded nucleic acid" refers to a nucleic acid that detects the presence of a single nucleotide polymorphism (SNP) or a point mutation or a miRNA isoform. Here, a single-stranded nucleic acid that detects the presence of a single nucleotide polymorphism (SNP) is referred to as "type 1 single-stranded nucleic acid" or "single-stranded nucleic acid type 1", and a single-stranded nucleic acid that detects the presence of a single nucleotide polymorphism (SNP) or a point mutation or a miRNA isoform is referred to as "type 2 single-stranded nucleic acid" or "single-stranded nucleic acid type 2". The type 1 single-stranded nucleic acid can act as a probe, unlike the type 2 single-stranded nucleic acid, and the type 2 single-stranded nucleic acid can be used simultaneously as a primer and a probe, unlike the type 1 single-stranded nucleic acid.

[0040] Specifically, after the type 1 single-stranded nucleic acid hybridizes with a single target gene, when the Y is cleaved by a cleavage reagent, the X and Z are also separated from the single target gene and can function as a probe; and after the type 2 single-stranded nucleic acid hybridizes with a single target gene, when the Y is cleaved by a cleavage reagent, the Z is separated from the single target gene, but the X is not separated and functions as a primer and a probe at the same time.

[0041] The single-stranded nucleic acid of the present invention has the structure XYZ, where each of X, Y and Z may have a variable number of nucleotides.

[0042] The Y is an RNA consisting of one or two base sequences located in a single target gene, and is a site to be cleaved by a cleavage reagent.

[0043] Here, the cleavage is performed by an enzyme such as DNase, RNase, helicase, exonuclease, or endonuclease as a cleavage reagent. is preferred, but other known cleavage reagents may also be used.

[0044] In the case of detecting the presence of a single nucleotide polymorphism (SNP), the X is a DNA consisting of a base sequence of 4 to 20, preferably 4 to 19, more preferably 4 to 18, even more preferably 5 to 18, even more preferably 6 to 18, even more preferably 6 to 17, even more preferably 6 to 16, and most preferably 6 to 15. X in this case belongs to type 1 single-stranded nucleic acid.

[0045] In addition, when detecting the presence of a single nucleotide polymorphism (SNP) or a point mutation or a miRNA isoform, the X is a DNA consisting of a base sequence of 10 to 30, preferably 11 to 30, more preferably 12 to 30, even more preferably 13 to 30, even more preferably 14 to 30, even more preferably 15 to 30, even more preferably 15 to 30, even more preferably 15 to 29, even more preferably 15 to 28, even more preferably 15 to 27, even more preferably 15 to 26, even more preferably 15 to 25, even more preferably 15 to 24, even more preferably 15 to 23, even more preferably 15 to 22, even more preferably 15 to 21, and most preferably 15 to 20. X thus belongs to type 2 single-stranded nucleic acid.

[0046] When detecting the presence of a single nucleotide polymorphism (SNP), the Z is a DNA consisting of a base sequence of 4 to 20, preferably 4 to 19, more preferably 4 to 18, even more preferably 5 to 18, even more preferably 6 to 18, even more preferably 6 to 17, even more preferably 6 to 16, and most preferably 6 to 15. Thus, Z belongs to type 1 single-stranded nucleic acid.

[0047] In addition, when detecting the presence of a point mutation or miRNA isoform, the Z is a DNA composed of 1 to 5, preferably 2 to 5, more preferably 2 to 4, and even more preferably 2 to 3. Thus, Z belongs to type 2 single-stranded nucleic acid.

[0048] In the present invention, the detection of the genetic mutation is to detect the presence of a single nucleotide polymorphism (SNP), a point mutation, or an miRNA isoform, and the detection of each of these genetic mutations can be specifically and sensitively detected depending on the number of bases constituting X and Z in the above-mentioned single-stranded nucleic acid.

[0049] In one embodiment of the present invention, real-time PCR was used to confirm that the ApoE single-stranded nucleic acid (sequence numbers 3 to 6) can accurately distinguish six phenotypes of the ApoE gene, E2 / E2, E3 / E3, E4 / E4, E2 / E3, E2 / E4, and E3 / E4 (Figure 1).

[0050] In one embodiment of the present invention, genomic DNA (gDNA) of SW620 cell line, a cell line with G12V mutation in the KRAS gene, was diluted at various concentrations, and the presence or absence of expression of the G12V mutant gene depending on the concentration of gDNA was confirmed by real-time PCR using G12V single-stranded nucleic acid (sequence number 15) (Figure 7).

[0051] In one embodiment of the present invention, gDNA of MIA-Paca2 cell line, which is a cell line with G12C mutation in KRAS gene, was diluted according to concentration, and the G12C mutation was evaluated according to the concentration of gDNA. The presence or absence of expression of the heterogeneous gene was confirmed by real-time PCR using a G12C single-stranded nucleic acid (SEQ ID NO: 16) (FIG. 8).

[0052] In one embodiment of the present invention, gDNA from the A549 cell line, which is a cell line with a G12S mutant KRAS gene, was diluted at various concentrations, and the presence or absence of expression of the G12S mutant gene depending on the gDNA concentration was confirmed by real-time PCR using G12S single-stranded nucleic acid (sequence number 17) (Figure 9).

[0053] In one embodiment of the present invention, gDNA from the H1975 cell line, which is a T790M mutant cell line in EGFR gene axon 20, and the A549 cell line, which is a wild-type cell line, were diluted at different concentrations, and the presence or absence of expression of the T790M mutant gene depending on the gDNA concentration was confirmed by real-time PCR using the T790M single-stranded nucleic acid (sequence number 19) (FIG. 10).

[0054] In one embodiment of the present invention, let-7a cDNA was diluted according to concentration, and the presence or absence of expression of let-7a miRNA gene depending on the cDNA concentration was confirmed by real-time PCR using let-7a single-stranded nucleic acid (sequence number 21) (Figure 11).

[0055] In one embodiment of the present invention, let-7d cDNA was diluted according to concentration, and the expression or non-expression of let-7d miRNA gene depending on the cDNA concentration was confirmed by real-time PCR using let-7d single-stranded nucleic acid (sequence number 24) (Figure 12).

[0056] In one embodiment of the present invention, an experimental group was conducted in which 2 μl of let-7a cDNA diluted 1 / 10 times to concentrations from 100 fM to 1 aM was added to 2 μl of let-7d cDNA at a concentration of 1 pM, and it was confirmed that let-7a cDNA could be detected at concentrations from 100 fM to 1 fM (Figure 13).

[0057] In one embodiment of the present invention, miRNA 34a cDNA was diluted according to the concentration, and the presence or absence of expression of miRNA 34a gene depending on the cDNA concentration was confirmed by real-time PCR using miRNA 34a single-stranded nucleic acid (SEQ ID NO: 27) (FIG. 14).

[0058] In one embodiment of the present invention, miRNA 34b cDNA was diluted according to the concentration, and the expression of miRNA 34b gene according to the concentration of cDNA was confirmed by real-time PCR using miRNA 34b single-stranded nucleic acid (SEQ ID NO: 30) (FIG. 15).

[0059] In one embodiment of the present invention, miRNA 34c cDNA was diluted according to the concentration, and the presence or absence of expression of miRNA 34c gene depending on the cDNA concentration was confirmed by real-time PCR using miRNA 34c single-stranded nucleic acid (SEQ ID NO: 33) (FIG. 16).

[0060] In one embodiment of the present invention, miRNA 34c cDNA was diluted in various concentrations into miRNA 34a cDNA or miRNA 34b cDNA, and then the expression of miRNA 34c gene was confirmed even at minute concentrations by real-time PCR using miRNA 34c single-stranded nucleic acid (SEQ ID NO: 33) (FIGS. 17 and 18).

[0061] In the present invention, the detectable marker may be a fluorescent substance that binds to single-stranded nucleic acid by a covalent or non-covalent bond, or a fluorescent pair consisting of a fluorescent substance and a quencher.

[0062] The fluorescent substance is, for example, Cy3, Cy5, Cy5.5, Bodipy, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 5 The antibody may be any one selected from the group consisting of, but is not limited to, 94, Alexa 660, rhodamine, TAMRA, FAM, FITC, Fluor X, ROX, Texas Red, ORNAge green 488X, ORNAge green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, Bodipy 630 / 650, Bodipy 650 / 665, Calfluor ORNAge 546, Calfluor red 610, Quasar 670 and biotin.

[0063] Furthermore, the quencher may be, for example, any one selected from the group consisting of DDQ-1, Dabcyl, Eclipse, 6-TAMRA, BHQ-1, BHQ-2, BHQ-3, lowa Black RQ-Sp, QSY-7, QSY-2, and MGBNFQ, but is not necessarily limited thereto.

[0064] When a fluorescent pair is used as a detectable marker in the present invention, the location of the fluorophore and quencher may be at the X or Z or Y site, but is not limited to any one of these locations. For example, the fluorophore may be at X and the quencher at Y or Z.

[0065] The single-stranded nucleic acid of the present invention can be used as: i) an RT primer for synthesizing cDNA from RNA among nucleic acids; ii) a forward primer for amplifying cDNA synthesized from nucleic acid (DNA or RNA); iii) a reverse primer for amplifying cDNA synthesized from nucleic acid (DNA or RNA); iv) a forward primer and a reverse primer for amplifying cDNA synthesized from nucleic acid (DNA or RNA); or v) a probe for confirming the nucleic acid (DNA or RNA) to be detected in real time, when detecting the presence of a point mutation or miRNA isoform as a genetic mutation of a single target gene.

[0066] In particular, when the single-stranded nucleic acid of the present invention is used as an RT primer, or a forward primer and probe, or a reverse primer and probe, for synthesizing and amplifying cDNA from RNA (including miRNA, etc.), the process of forming a loop of the RT primer or the process of forming polyA during cDNA synthesis is unnecessary, and the single-stranded nucleic acid can synthesize cDNA by hybridizing with the RNA to be detected, and the RNA to be detected (including miRNA, etc.) can be amplified and detected in real time.

[0067] In another aspect, the present invention provides a kit for real-time detection of a genetic mutation in a single target gene, comprising the single-stranded nucleic acid.

[0068] When the single-stranded nucleic acid of the present invention is used as a kit for detecting a genetic mutation in a single target gene, it is preferable that the kit further contains, in addition to the single-stranded nucleic acid of the present invention, an enzyme capable of cleaving the Y site of the single-stranded nucleic acid.

[0069] In the present invention, the enzyme capable of cleaving the Y site of the single-stranded nucleic acid may be any enzyme capable of specifically cleaving the Y site of the single-stranded nucleic acid. For example, when the Y site is DNA, it is preferable to use a DNA nuclease (DNase), specifically, DNase I, DNase II, S1 nuclease, nuclease P1, AP endonuclease, UvrABSC nuclease, or the like, and when the Y site is RNA, it is preferable to use an RNA hydrolase (ribonuclease, RNase), specifically, RNase II, RNase III, RNase IV, RNase H, RNase T2, or the like.

[0070] When the single-stranded nucleic acid of the present invention is used as a kit for detecting a genetic mutation in a single target gene, the kit may further contain reagents necessary for a DNA amplification reaction in addition to the single-stranded nucleic acid of the present invention and an enzyme capable of cleaving the Y site of the single-stranded nucleic acid.

[0071] Reagents necessary for the amplification reaction include, for example, an appropriate amount of DNA polymerase (e.g., a thermostable DNA polymerase obtained from Thermus aquatiucus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus litoralis, or Pyrococcus furiosis (Pfu)), a DNA polymerase cofactor (Mg2+), a buffer solution, dNTPs (dATP, dCTP, dGTP, and dTTP), and water (H2O). The buffer solution may include an appropriate amount of Triton X-100 (Triton Examples of suitable solvents include, but are not limited to, X-100, dimethylsufoxide (DMSO), Tween 20, nonidet P40, PEG 6000, formamide, and bovine serum albumin (BSA).

[0072] In another aspect, the present invention provides a method for detecting a genetic mutation in a single target gene, the method comprising: a) obtaining a target nucleic acid containing a genetic mutation to be detected from a biological sample; b) producing the above-mentioned single-stranded nucleic acid; c) mixing the target nucleic acid obtained in step a), the single-stranded nucleic acid produced in step b), a primer set having a base sequence complementary to the target nucleic acid obtained in step a), and a cleavage reagent, and then amplifying the target nucleic acid-single-stranded nucleic acid complex containing the genetic mutation by an extension reaction; and d) measuring the amount of single-stranded nucleic acid fragment separated from the target nucleic acid-single-stranded nucleic acid complex containing the genetic mutation amplified in step c).

[0073] The method for detecting genetic mutations in a single target gene in real time according to the present invention will be described in detail according to each step as follows.

[0074] a) obtaining a target nucleic acid containing a genetic mutation to be detected from a biological sample;

[0075] In the present invention, the target nucleic acid containing the genetic mutation can be RNA or DNA to be detected from a sample, or cDNA obtained by amplifying the RNA with a reverse transcription polymerase.

[0076] The sample may be a biological sample, or may be RNA, DNA, or a fragment thereof isolated from a biological sample. Specifically, the sample may be any one or more selected from the group consisting of blood, saliva, urine, feces, tissue, cells, and biopsy specimens, or may be RNA, DNA, or a fragment thereof isolated from a preserved biological sample, but is not necessarily limited thereto.

[0077] The preserved biological sample may be derived from tissue that has been preserved for one week or more, one year or more, for example, one to ten years, frozen, or stored at room temperature after being fixed in formalin, according to conventional preservation methods known in the art.

[0078] In the present invention, various methods known in the art can be used to extract RNA or DNA from a sample.

[0079] b) Producing the single-stranded nucleic acid.

[0080] In the present invention, the single-stranded nucleic acid is as described above, specifically, characterized in that i) it has an XYZ structure, ii) it binds complementarily to a part or all of the base sequence of a single target gene containing a genetic mutation, and iii) it has the same or at least two different detectable markers attached to both ends or inside.

[0081] In the present invention, the detectable marker may be a fluorescent substance that binds to single-stranded nucleic acid by a covalent or non-covalent bond, or a fluorescent pair consisting of a fluorescent substance and a quencher.

[0082] The fluorescent substance may be any one selected from the group consisting of, for example, Cy3, Cy5, Cy5.5, Bodipy, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, rhodamine, TAMRA, FAM, FITC, Fluor X, ROX, Texas Red, ORNAge green 488X, ORNAge green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, Bodipy 630 / 650, Bodipy 650 / 665, Calfluor ORNAge 546, Calfluor red 610, Quasar 670, and biotin, but is not necessarily limited thereto. Furthermore, the quencher may be, for example, any one selected from the group consisting of DDQ-1, Dabcyl, Eclipse, 6-TAMRA, BHQ-1, BHQ-2, BHQ-3, lowa Black RQ-Sp, QSY-7, QSY-2, and MGBNFQ, but is not necessarily limited thereto.

[0083] c) amplifying the target nucleic acid-single-stranded nucleic acid complex containing the genetic mutation.

[0084] In the present invention, the amplification of the target nucleic acid-single-stranded nucleic acid complex containing the genetic mutation is carried out by mixing the obtained target nucleic acid, the produced single-stranded nucleic acid, a primer set having a base sequence complementary to the obtained target nucleic acid, and a cleavage reagent, and then subjecting the mixture to an extension reaction.

[0085] In the present invention, the cleavage reagent is preferably an enzyme-mediated cleavage reagent, but other known cleavage reagents may be used. In this case, the term "enzyme-mediated cleavage" is used to indicate cleavage of RNA or DNA catalyzed by enzymes such as DNase, RNase, helicase, exonuclease, and endonuclease. In a preferred embodiment of the present invention, it is more preferable that the nicking and cleavage of the hybridized probe is performed by a ribonuclease, which is an endonuclease or exonuclease. It is more preferable that the ribonuclease is a double-stranded ribonuclease that nicks and cleaves ribonucleic acid from the hybridization strand of double-stranded DNA-RNA.

[0086] In the present invention, the cleavage reagent may be, but is not limited to, an RNA hydrolase (ribonuclease, RNase) such as RNase H, RNase II, RNase III, RNase IV, or RNase T2.

[0087] d) measuring the amount of single-stranded nucleic acid fragments separated from the target nucleic acid-single-stranded nucleic acid complex containing the amplified genetic mutation.

[0088] In the present invention, the amount of single-stranded nucleic acid fragments can be measured using various detection methods. Specifically, the amount of single-stranded nucleic acid fragments separated by the present invention can be measured in real time or by a method using a liquid chromatography. is preferably carried out after the reaction has been completed, and can be carried out by measuring the change in fluorescence intensity or chemiluminescence.

[0089] The device for measuring the change in fluorescence intensity or chemiluminescence may be any device known in the art that is capable of detecting a fluorescent marker, such as a TRIAD Multimode Detector, a Wallac / Victor fluorescence, or a Perkin-Elmer LB50B luminescence spectrometer. Examples of the PCR thermocycler that can be used include, but are not limited to, a LightCycler 96 (LightCycler 96), Applied Biosystems 7500, or Biorad CFX96 real-time PCR thermocycler.

[0090] The method for measuring and detecting the amount of single-stranded nucleic acid fragments cleaved according to the present invention may vary depending on the type of single-stranded nucleic acid or label or detectable marker added to the reaction solution.

[0091] In the single-stranded nucleic acid of the present invention, the Y site is cleaved and then amplified, making it easy to distinguish the genetic mutation of the Y site, and thus enabling the confirmation of the mutation by the subsequent nucleic acid amplification reaction. That is, in the single-stranded nucleic acid of the present invention, when hybridization is formed between the Y site and the genetic mutation site of the target nucleic acid, the Y site is cleaved only when it exactly binds complementary to the genetic mutation site, and then an amplification reaction is performed, so that the presence or absence of a genetic mutation can be clearly confirmed. Specifically, in the single-stranded nucleic acid of the present invention, even if the Y site and the genetic mutation site of the target nucleic acid are made to form hybridization, if the Y site does not bind complementary, the Y site is not cleaved, and therefore no amplification reaction is caused, which means that the target nucleic acid does not have the genetic mutation to be measured. On the other hand, even if the Y site in the single-stranded nucleic acid of the present invention and the site where the genetic mutation of the target nucleic acid occurs that is not mutated are made to form hybridization, if the Y site does not bind complementary, the Y site is not cleaved, and therefore no amplification reaction is caused, which means that the target nucleic acid has a genetic mutation.

[0092] In the present invention, the detection of the genetic mutation means detecting the presence of a single nucleotide polymorphism (SNP), a point mutation, or an miRNA isoform.

[0093] In one embodiment of the present invention, gDNA of the Aspc-1 cell line, which is a G12D mutant cell line, and the HT-29 cell line, which is a KRAS wild-type cell line, were diluted at different concentrations, and the presence or absence of expression of the G12D mutant gene depending on the concentration of gDNA was confirmed by real-time PCR using a single-stranded nucleic acid of the KRAS gene G12D of DNA oligo-DNA-RNA-mutation-DNA oligo type (G12D-R1DrMR2; sequence number 36) (FIG. 19).

[0094] In one embodiment of the present invention, gDNA from the Aspc-1 cell line, which is a G12D mutant cell line, and the HT-29 cell line, which is a KRAS wild-type cell line, were diluted at different concentrations, and the presence or absence of expression of the G12D mutant gene depending on the gDNA concentration was confirmed by real-time PCR using a single-stranded nucleic acid of the KRAS gene G12D of DNA oligo-RNA-DNA-mutation-DNA oligo type (G12D-R1rDMR2; sequence number 37) (Figure 20).

[0095] In one embodiment of the present invention, the Aspc-1 cell line, which is a G12D mutant cell line, and the KR gDNA from the HT-29 cell line, an AS wild-type cell line, was diluted at various concentrations, and the presence or absence of expression of the G12D mutant gene depending on the concentration of gDNA was confirmed by real-time PCR using a single-stranded nucleic acid of the KRAS gene G12D of the DNA oligo-DNA-mutation-RNA-DNA-DNA oligo type (G12D-R1DMrDR2; sequence number 38) (Figure 21).

[0096] The extension reaction, i.e., nucleic acid amplification reaction, which can be used in the present invention, is known to those skilled in the art to which the present invention pertains. That is, the amplification of the target nucleic acid includes, but is not limited to, polymerase chain reaction (PCR), rolling circle amplification (RCA), strand displacement amplification (SDA) or nucleic acid sequence based amplification (NASBA). The product of the nucleic acid amplification is DNA or RNA.

[0097] In general, the reaction mixture contains a target nucleic acid, a single-stranded nucleic acid, components of a nucleic acid amplification reaction, and a cleavage enzyme, so that the amplification of the target nucleic acid and the detection by cleavage of the single-stranded nucleic acid described above can be performed simultaneously. Each amplification reaction requires individual optimization of buffer conditions, primers, reaction temperature, and cleavage conditions of the single-stranded nucleic acid. By using the detection method of the present invention in combination with a nucleic acid amplification reaction, the sensitivity and speed of detecting the target nucleic acid can be significantly improved.

[0098] Meanwhile, it has been confirmed that the single-stranded nucleic acid for real-time detection of genetic mutations in a single target gene of the present invention can rapidly and accurately detect and detect point mutations in genes such as KRAS and EGFR associated with cancer. The single-stranded nucleic acid can be applied to a cancer diagnostic kit or composition, and can be usefully used to diagnose cancer in real time and provide information on the occurrence or non-occurrence of cancer. EXAMPLES

[0099] The configuration and effects of the present invention will be described in more detail below with reference to examples. These examples are intended solely to illustrate the present invention, and the scope of the present invention is not limited to these examples.

[0100] Example 1: Analysis of ApoE using type 1 single-stranded nucleic acid Type 1 single-stranded nucleic acid was used to analyze six phenotypes of the apolipoprotein E (ApoE) gene: E2 / E2, E3 / E3, E4 / E4, E2 / E3, E2 / E4, and E3 / E4.

[0101] The ApoE gene located on human chromosome 19 is a gene associated with cardiovascular disease and Alzheimer's disease. The ApoE gene has three allele isoforms, ApoEε2, ApoEε3, and ApoEε4, due to DNA single nucleotide polymorphisms (SNPs) at codons 112 (cys / arg) and 158 (cys / arg) [genomic DNA positions 586 (T / C), 724 (T / C)], and six phenotypes (E2 / E2, E3 / E3, E4 / E4, E2 / E3, E2 / E4, E3 / E4) are obtained depending on the combination of alleles. In order to distinguish each of the six phenotypes of the ApoE gene, four types of improved type 1 single-stranded nucleic acids with fluorescent dyes attached to the 5'-end were analyzed in 4-plex. Conventional analytical methods generally have difficulty in satisfying the sensitivity and specificity of 4-plex analytical methods, but the present invention has demonstrated results that meet these requirements.

[0102] In detail, the SN for the allele types of codon 112 and codon 158 of the ApoE gene To measure the presence or absence of P, type 1 single-stranded nucleic acid and primers according to the present invention were manufactured by IDT (Integrated DNA Technologies, USA) as shown in Table 1 below. Here, type 1 single-stranded nucleic acid is a probe having a structure of XYZ, and 6-FAM, HEX, TexasRed, and Cy5 were attached to the 5'-end, and IABkFQ was attached to each 3'-end. Ribonucleotides (RNA) are indicated with the subscript "r" before the sequence to distinguish them from deoxyribonucleotides (DNA).

[0103] [Table 1]

[0104] For the analysis, human cell lines PC3 (E2 / E2), A549 (E3 / E3), and U937 (E4 / E4) were provided by the Korean Cell Line Bank, and genomic DNA was obtained from them. For the analysis of the six phenotypes, PC3 (E2 / E2), A549 (E3 / E3), and U937 (E4 / E4) were used for the homozygous phenotypes, and PC3 + ​​A549 (E2 / E3), PC3 + ​​U937 (E2 / E4), and A549 + U937 (E3 / E4), which are mixtures of each genomic DNA, were used for the heterozygous phenotypes, at a high concentration of 32 ng (approximately 104 copies) per reaction.

[0105] The quantified genomic DNA, 0.5 U in the presence of final concentrations of 0.2 μM, 0.15 μM, 0.15 μM, and 0.075 μM of ApoE single-stranded nucleic acids 1, 2, 3, and 4 in Table 1, respectively, and 0.35 μM of ApoE forward and reverse primers, respectively. After adjusting the total volume to 20 μl with RNase-H, 4 μl of AptaTaq DNA Master (Roche), 3 μl of GC rich solution (Roche), and nuclease-free water, a polymerase chain reaction (PCR) was performed. The PCR reaction conditions were 95°C for 5 minutes, 95°C for 15 seconds, and 65°C for 70 seconds, with 45 cycles. The results are shown in Figure 1.

[0106] As a result, it was confirmed that six combinations of ApoE alleles can be analyzed in one reaction well. This indicates that when there is a full or half mutation, such as a congenital mutation, the improved probe format provides excellent discrimination ability.

[0107] Example 2: Analysis of KRAS mutations using type 1 single-stranded nucleic acid

[0108] Example 2-1: Real-time analysis of G13D mutation in KRAS gene using single-stranded nucleic acid according to the present invention In order to determine the presence or absence of the G13D mutation (mutant) of the KRAS gene using the type 1 single-stranded nucleic acid according to the present invention, IDT was asked to prepare the single-stranded nucleic acid (type 1) of the G13D mutation of the KRAS gene, the KRAS wild type single-stranded nucleic acid (type 1), and the forward and reverse primers of the G13D mutation of the KRAS gene as shown in Table 2 below. In this case, the single-stranded nucleic acid thus prepared had HEX and FAM attached to the 5'-end and IABkFQ attached to the 3'-end. In addition, ribonucleotides (RNA) are indicated by the subscript "r" before the sequence to distinguish them from deoxyribonucleotides (DNA).

[0109] [Table 2]

[0110] The KRAS gene to be measured was detected from total genomic DNA extracted from HCT-15 cell line and NCI-H1975 cell line cultured for a certain period of time. Total genomic DNA was extracted from 5×106 cells of each cell line using PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific, Cat No. K1820-00) and quantified using NanoDrop One (Thermo Fisher Scientific). The quantified total genomic DNA was diluted to 15ng / μl and serially diluted 1 / 10 to use 2μl of genomic DNA with concentrations of 15ng / μl to 1.5pg / μl. The HCT-15 cell line is a G13D mutant cell line, and NCI-H1975 is known to be a KRAS wild-type cell line.

[0111] Thereafter, 0.3 μl of the G13D single-stranded nucleic acid of SEQ ID NO: 7 at a concentration of 10 μM prepared above was prepared, and 0.5 μl of primers of SEQ ID NO: 9 and 10 at a concentration of 10 μM were prepared. 0.5 U RNase-H, 4 μl of AptaTaq DNA Master (Roche), 30 ng of total genomic DNA extracted from NCI-H1975 cell line, 30 ng, 3 ng, 300 pg, and 30 pg of total genomic DNA extracted from HCT-15 cell line were added thereto, and the total volume was adjusted to 20 μl with triple distilled water, and then polymerase chain reaction was performed to measure G13D mutations. At this time, the reaction conditions were 95° C. for 10 minutes, initial denaturation was followed by 40 cycles of 95° C. for 10 seconds and 64° C. for 60 seconds, and the signal (HEX) was measured in real time for each cycle to derive the results. The results are shown in FIG.

[0112] Example 2-2: Real-time analysis of KRAS wild-type gene using single-stranded nucleic acid according to the present invention Under the same conditions as in Example 2-1, in the presence of wild-type single-stranded nucleic acid of KRAS gene of SEQ ID NO: 8 and primers of SEQ ID NO: 9 and 10, 30ng, 300pg, and 30pg of total genomic DNA extracted from NCI-H1975 cell line and 30ng, 300pg, and 30pg of total genomic DNA extracted from HCT-15 cell line were added, respectively, and then the KRAS wild-type gene was measured. The results are shown in Figure 3. The HCT-15 cell line is a heterozygous type gene, and is a cell line that simultaneously has the G13D mutation (see the results in Figure 2) and the KRAS wild-type gene, and even when it has only half of the wild type, the results shown in Figure 4 are obtained.

[0113] Example 2-3: Analysis of the G13D mutation mixed with the KRAS wild-type gene in real time using single-stranded nucleic acid according to the present invention The KRAS gene to be measured was detected from total genomic DNA extracted from HCT-15 cell line and NCI-H1975 cell line cultured for a certain period of time. Total genomic DNA was extracted from 5 × 106 cells of each cell line using PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific, Cat No. K1820-00) and quantified using NanoDrop One (Thermo Fisher Scientific). The quantified NCI-H1975 DNA was diluted to 30ng / μl, and the HCT-15 DNA was diluted to 30ng / μl, and then serial dilutions were carried out in 1 / 10 increments to obtain NCI-H1975 genomic DNA and HCT-15 genomic DNA with concentrations of 3ng / μl to 3pg / μl. Then, 2 μl of each genomic DNA was mixed and used in the experiment so that the concentration of HCT-15 was 10 to 0.01% of the concentration of NCI-H1975. The HCT-15 cell line is a G13D mutant cell line, and NCI-H1975 is a KRAS wild-type cell line.

[0114] Thereafter, 0.3 μl of the G13D single-stranded nucleic acid of 10 μM concentration of SEQ ID NO: 7 prepared above was used, and 0.5 μl of primers of 10 μM concentration of SEQ ID NO: 9 and 10 were prepared. 0.5 U RNase-H, 4 μl of AptaTaq DNA Master (Roche), and DNA diluted to 10 to 0.01% were added thereto, and then the total volume was adjusted to 20 μl with triple distilled water, and a polymerase chain reaction was performed to measure G13D mutations. At this time, the reaction conditions were 95°C for 10 minutes, and after initial denaturation, 40 cycles of 95°C for 10 seconds and 64°C for 60 seconds were performed, and the signal (HEX) was measured in real time for each cycle to derive the results. The results are shown in FIG. 5.

[0115] As shown in Figures 2 to 5, when G13D single-stranded nucleic acid (type 1) was used to detect the G13D mutation, cross-reaction with NCI-H1975 wild-type genomic DNA was not induced, and the specificity was excellent. However, when HCT-15 genomic DNA was less than 600 pg, the detection was inaccurate. In the case of an analysis including wild-type and point mutation genes, it was confirmed that an analysis including point mutations at less than 10% was difficult.

[0116] Example 3: Analysis of ApoE using type 2 single-stranded nucleic acid The type 2 single-stranded nucleic acid according to the present invention was used to determine the presence or absence of SNPs for the allele types of codons 112 and 158 of the ApoE gene. As shown in Table 3 below, type 2 single-stranded nucleic acid was produced by IDT (Integrated DNA Technologies, USA).

[0117] Here, in the case of type 2 single-stranded nucleic acid, the primer and probe have the structure XYZ, and FAM, HEX, and Texas Red are attached to the 5'-end, respectively, and IABkFQ is attached to the 3'-end. In addition, ribonucleotides (RNA) are indicated with the subscript "r" before the sequence to distinguish them from deoxyribonucleotides (DNA).

[0118] [Table 3]

[0119] Genomic DNA was obtained from human cell lines PC3(E2 / E2), A549(E3 / E3), and U937(E4 / E4) provided by the Korean Cell Line Bank for analysis. For the analysis of the six phenotypes, PC3(E2 / E2), A549(E3 / E3), and U937(E4 / E4) were used for homozygous phenotypes, and PC3+A549(E2 / E3), PC3+U937(E2 / E4), and A549+U937(E3 / E4) were used for heterozygous phenotypes, with a high concentration of 32ng (approximately 104 copies) per reaction.

[0120] In the presence of final concentrations of 0.375 μM, 0.1 μM, 0.25 μM, and 0.25 μM of ApoE single-stranded nucleic acids 1, 2, 3, and 4 in Table 3, respectively, the genomic DNA, 0.1 ng of heat-stable RNase H, 4 μl of AptaTaq DNA master w / o MgCl2 (Roche), 4 μl of GC-rich solution (Roche), 2.75 mM MgCl2, and 62.5 nM Low ROX were adjusted with nuclease-free water to a total volume of 20 μl, and then polymerase chain reaction (PCR) was performed. At this time, the PCR reaction conditions were 95° C. for 10 minutes, 95° C. for 15 seconds, and 64° C. for 55 seconds, and 40 cycles were performed. The results are shown in FIG. 6.

[0121] As a result, in the analysis of six combinations of each allele of ApoE using type 2 single-stranded nucleic acid according to the present invention, the 586T mutation at codon 112 and the 724T mutation at codon 158 were limited in that they could be distinguished by the difference in the fluorescence value at the end point using the same fluorescent dye, not by the difference in the fluorescent dye. Due to such limitations, there may be problems in interpreting the analysis results based on the concentration of each DNA sample. This shows that in the case of ApoE, which is a congenital mutation, it is easier to analyze using type 1 single-stranded nucleic acid than using type 2 single-stranded nucleic acid, as shown in Example 1.

[0122] Example 4: Analysis of KRAS mutations using type 2 single-stranded nucleic acid

[0123] Example 4-1: Real-time analysis of G12V, G12C, and G12S mutations using type 2 single-stranded nucleic acid according to the present invention Using type 2 single-stranded nucleic acid, the presence or absence of three types of mutations (G12V, G12C, G12S) in the 12 codon mutation of the KRAS gene was measured. The single-stranded nucleic acid and universal reverse primer according to the present invention were manufactured by IDT as shown in Table 4 below. The single-stranded nucleic acid had HEX, FAM, and Cy5 attached to the 5'-end and IABkFQ attached to the 3'-end. Ribonucleotides (RNA) are indicated with the subscript "r" before the sequence to distinguish them from deoxyribonucleotides (DNA).

[0124] [Table 4]

[0125] In order to confirm the detectability of KRAS point mutation using the synthesized single-stranded nucleic acid, wild-type cell lines and mutant cell lines were cultured, and total genomic DNA was extracted from 5 x 106 cells of each cell line using PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific, Cat No. K1820-00). Meanwhile, after quantification using NanoDrop One (Thermo Fisher Scientific), it was used as a template. The cell lines used are as shown in Table 5 below.

[0126] [Table 5]

[0127] Thereafter, 0.5 μl of the prepared G12V single-stranded nucleic acid of SEQ ID NO: 15 and primer of SEQ ID NO: 18 were used at a concentration of 10 μM. 3.6 μl of 5x AptaTaq DNA Master (Roche) and 0.2 μl of heat-stable RNase H were added with 30 ng of Colo201 (KRAS wild-type cell line) and 3 ng, 300 pg, 30 pg, and 3 pg of total genomic DNA extracted from SW620 (G12V mutant) cell line, respectively, and then diluted with triple distilled water. After adjusting the total volume to 20 μl, polymerase chain reaction (PCR) was performed to measure G12V mutation. At this time, the reaction conditions were 95°C for 10 minutes, initial denaturation, 95°C for 15 seconds, 66°C for 30 seconds for 40 cycles to perform a primary PCR reaction, and then 85°C for 15 seconds, 64°C for 40 seconds for 40 cycles to perform a secondary PCR reaction, and the signal (HEX) was measured in real time for each cycle to derive the results. The results are shown in FIG. 7.

[0128] In addition, 0.5 μl of the prepared G12C single-stranded nucleic acid of SEQ ID NO: 16 and primer of SEQ ID NO: 18 were used at a concentration of 10 μM, and 7 ng, 700 pg, 70 pg, and 7 pg of total genomic DNA extracted from Colo201 (KRAS wild-type cell line) and MIA-Paca2 (G12C mutation) cell line were added to 2.8 μl of 5X AptaTaq DNA Master (Roche), 1.2 μl of 5X Apta Fast buffer, 0.4 μl of 1 U / μl heat-stable RNase H, and 0.5 μl of 25 mM MgCl2, respectively, and the total volume was adjusted to 20 μl with triple distilled water, followed by polymerase chain reaction to measure the G12C mutation. The reaction conditions were 95°C for 10 minutes, followed by initial denaturation, followed by 40 cycles of 95°C for 10 seconds and 64°C for 60 seconds, and the signal (Cy5) was measured in real time for each cycle to derive the results. The results are shown in Figure 8.

[0129] In addition, the G12S single-stranded nucleic acid of SEQ ID NO: 17 and the primer of SEQ ID NO: 18 were used at 0.5 μl of 10 μM concentration, and 30 ng of Colo201 (KRAS wild-type cell line) and 3 ng, 300 pg, 30 pg, and 7 pg of total genomic DNA extracted from A549 (G12S mutation) cell line were added to 3.6 μl of 5x AptaTaq DNA Master (Roche) and 0.2 μl of 10 ng / μl heat-resistant RNase H, respectively, and the total volume was adjusted to 20 μl with triple distilled water, and then a polymerase chain reaction was performed to measure the G12S mutation. At this time, the reaction conditions were 95°C for 10 minutes, and after initial denaturation, 40 cycles of 95°C for 10 seconds and 64°C for 60 seconds were performed, and the signal (FAM) was measured in real time for each cycle to derive the results. The results are shown in FIG. 9.

[0130] As can be seen from FIG. 7 to FIG. 9, the single-stranded nucleic acid according to the present invention was able to analyze mutated genes with excellent specificity and sensitivity even when the point mutation gene was contained at less than 0.01%.

[0131] Example 5: Method for analyzing EGFR mutations using type 2 single-stranded nucleic acid EGFR (Epidermal growth factor receptor) is overexpressed in non-small cell lung cancer and is a target of EGFR tyrosine kinase (TKI). By analyzing mutations occurring in the tyrosine kinase domain corresponding to axons 18, 19, 20, and 21 of the EGFR gene, drug responsiveness of non-small cell lung cancer patients to therapeutic agents can be predicted, so mutation analysis is useful for drug prescription and treatment of patients. Among these, the presence or absence of mutations was analyzed for the most frequently used T790M (C2369T) mutation.

[0132] The type 2 single-stranded nucleic acid and primer according to the present invention were manufactured by IDT (Integrated DNA Technologies, USA) as shown in Table 6 below. Here, the T790M single-stranded nucleic acid had FAM attached to the 5'-end and IABkFQ attached to the 3'-end. Meanwhile, ribonucleotides (RNA) are indicated with the subscript "r" before the sequence to distinguish them from deoxyribonucleotides (DNA).

[0133] [Table 6]

[0134] For the analysis, genomic DNA was obtained from the T790M mutant cell line H1975 and the wild-type cell line A549. H1975 and A549 genomic DNA were quantified at 30 ng (approximately 1 × 104 copies) and then diluted 10-fold for use.

[0135] In the presence of the final concentrations of 0.25 μM T790M single-stranded nucleic acid and 0.25 μM T790M primer in Table 6, the genomic DNA, 0.5 units of heat-resistant RNase H, and 3.6 μl of 5x AptaTaq DNA Master (Roche) were adjusted with nuclease-free water to a total volume of 20 μl, and then polymerase chain reaction (PCR) was performed. At this time, the PCR reaction conditions were 95° C. for 10 minutes, 95° C. for 15 seconds, and 64° C. for 60 seconds, with 45 cycles. The results are shown in FIG. 10.

[0136] As a result, it was confirmed that in the analysis of EGFR mutations using the single-stranded nucleic acid according to the present invention, it was possible to distinguish between each mutant type and the wild type to a degree of 0.1% or less. As a result, it was confirmed that, as described above, in the case of acquired mutations such as cancer point mutations, detection using type 2 single-stranded nucleic acid is advantageous.

[0137] Example 6: Analysis of let-7 miRNA and miRNA 34 isoforms using type 2 single-stranded nucleic acids

[0138] Example 6-1: Real-time analysis of let-7 miRNA using type 2 single-stranded nucleic acid according to the present invention It is known that let-7 miRNA has the most isoforms among miRNAs. It is a very difficult analysis to distinguish between such let-7 isoforms, and it is known that it is particularly difficult to distinguish between isoforms with a specificity of less than 1%. In this experiment, in particular, in order to measure the presence or absence of gene expression of let-7a (5'-UGAGGUAGUAGGUUGUAUAGUU) and let-7d (5'-AGAGGUAGUAGGUUGCAUAGUU), the type 2 single-stranded nucleic acid, primer, and RT-primer according to the present invention were manufactured by IDT (Integrated DNA Technologies, USA) as shown in Table 7 below. In addition, for accurate quantification, the let-7 miRNA was manufactured by IDT. Here, the single-stranded nucleic acid had FAM (fluorescein succinimidyl ester) attached to the 5' end and 3IABkFG attached to the 3' end. On the other hand, ribonucleotides (RNA) are indicated with the subscript "r" before the sequence to distinguish them from deoxyribonucleotides (DNA).

[0139] [Table 7]

[0140] cDNA was synthesized by reacting 1 μl of each synthesized miRNA at a concentration of 20 pM, Ambion's poly-(A) tailing kit, and Roche's Nxtscript RT kit (total 20 μl) at 45° C. for 30 minutes in the presence of 1 μl of each RT primer at a concentration of 10 μM in Table 7.

[0141] The synthesized cDNA was diluted to concentrations ranging from 100 fM to 1 aM.

[0142] In the presence of 0.5 μl of each of the single-stranded nucleic acids and primers in Table 7 at a concentration of 10 μM, 2 μl of each of the cDNAs diluted after synthesis, 1U heat-resistant RNase H, and 4 μl of AptaTaq DNA Master (Roche) were added, and the total volume was adjusted to 20 μl with triple distilled water, and then polymerase chain reaction (PCR) was performed. At this time, the PCR reaction conditions were 95° C. for 5 minutes, 63 to 64° C. for 60 seconds, and 95° C. for 10 seconds, and 45 cycles were performed. The results are shown in FIG. 11 and FIG. 12.

[0143] As a result, it was confirmed that analysis of each miRNA was possible using only 2 μl (approximately 1 copy) of diluted cDNA at a miRNA concentration of 1 aM.

[0144] Example 6-2: Real-time detection of let-7 miRNA specificity using type 2 single-stranded nucleic acid according to the present invention In the presence of 0.5 μl of 10 μM concentration of each of the single-stranded nucleic acid and primer in Table 7, 2 μl of let-7a cDNA diluted 1 / 10 times from 100 fM to 1 aM was added to 2 μl of let-7d cDNA at 1 pM concentration, 1U heat-resistant RNase H, 4 μl of AptaTaq DNA Master (Roche), and adjusted to a total volume of 20 μl with triple distilled water, and then polymerase chain reaction (PCR) was performed. At this time, the PCR reaction conditions were 95 ° C for 5 minutes, 63 to 64 ° C for 60 seconds, and 95 ° C for 10 seconds, and 45 cycles were performed. The results are shown in FIG. 13.

[0145] As a result, in an experimental group in which 2 μl of let-7a cDNA diluted 1 / 10 times from 100 fM to 1 aM was added to 2 μl of 1 pM let-7d cDNA, it was confirmed that concentrations from 100 fM to 1 fM could be stably analyzed. This indicates that isoform miRNAs can be analyzed while maintaining specificity down to 0.1%.

[0146] Example 6-3: Analysis of miRNA 34a, miRNA 34b, and miRNA 34c in real time using type 2 single-stranded nucleic acid according to the present invention miRNA 34 has three isoforms, but it is known that it is quite difficult to distinguish between these isoforms. In this example, in order to measure the presence or absence of gene expression of miRNA 34a, miRNA 34b, and miRNA 34c, we requested IDT to synthesize and manufacture single-stranded nucleic acids, primers, and RT-primers as shown in Table 8. The single-stranded nucleic acids were attached with FAM (fluorescein succinimidyl ester) and HEX (hexachloro-fluorescein) at the 5' end and 3IABkFQ at the 3' end. In addition, ribonucleotides (RNA) are indicated with the subscript "r" before the sequence to distinguish them from deoxyribonucleotides (DNA).

[0147] [Table 8]

[0148] For each miRNA, cDNA was synthesized using Invitrogen's poly-(A) tailing kit and Roche's Nxtscript RT kit (total 20 μl) at 40°C in the presence of 1 μl of RT primer at a concentration of 10 μM for 60 minutes. The synthesized cDNA was then diluted to 7 concentrations ranging from 1 pM to 1 aM (approximately 106 to 100 copies). The PCR was carried out by diluting each of the cDNAs in 1 / 10 increments, adding heat-stable RNase H, AptaTaq DNA Master (Roche), single-stranded nucleic acid, and primers, adjusting the total volume to 20 μl with triple distilled water, and carrying out 45 cycles of reaction conditions of 95°C for 5 minutes, 95°C for 10 seconds, and 65°C for 1 minute.

[0149] As a result, it was confirmed that miRNA 34a, miRNA 34b, and miRNA 34c could all be measured with normal PCR efficiency at concentrations from 1 pM to 10 aM (approximately 106 to 101 copies). The results are shown in Figures 14 to 16.

[0150] Example 6-4: Specific detection of miRNA 34a, miRNA 34b, and miRNA 34c in real time using type 2 single-stranded nucleic acid according to the present invention In the above-mentioned Example 6-3, it was confirmed that the single-stranded nucleic acids of miRNA 34a, miRNA 34b, and miRNA 34c can distinguish each isoform, and in this Example, it was confirmed at what ratio the desired isoform can be distinguished when the miRNA to be amplified is mixed with miRNA of other isoforms. 108 (100 pM) miRNA 34a or miRNA 34b isoform was mixed with miRNA 34c diluted 1 / 10 from 107 to 104 or 103 (10 pM to 10 fM or 10 pM to 1 fM), and specificity detection was carried out by polymerase chain reaction (PCR) using the single-stranded nucleic acid and primers of miR-34c.

[0151] PCR was performed by adding each synthetic cDNA, heat-stable RNase H, AptaTaq DNA Master (Roche), single-stranded nucleic acid, and primer, adjusting the total volume to 20 μl with triple distilled water, and performing 45 cycles under the following reaction conditions: 95°C for 5 minutes, 95°C for 10 seconds, and 65°C for 1 minute.

[0152] The results are shown in FIGS.

[0153] As a result, it was confirmed that miRNA 34c can distinguish isoforms with a specificity of up to 0.001%.

[0154] Example 7: Analysis method based on the structure of single-stranded nucleic acid In order to increase the specificity of the detection of mutations in single-stranded nucleic acids (type 2) according to the present invention, three types of G12D, specifically, R1DrMR2, R1rDMR2, and R1DMrDR2 types were manufactured by IDT (Integrated DNA Technologies, USA) to examine the ability to detect mutations according to the position of ribonucleotides (RNA) in single-stranded nucleic acids, as shown in Table 9 below. FAM was attached to the 5'-end of each single-stranded nucleic acid, and 3IABkFQ was attached to the 3'-end. Ribonucleotides (RNA) are indicated with the subscript "r" before the sequence to distinguish them from deoxyribonucleotides (DNA).

[0155] [Table 9]

[0156] Example 7-1: Analysis of G12D mutant gene and KRAS wild-type gene by G12D single-stranded nucleic acid type (G12D-R1DrMR2, G12D-R1rDMR2, G12D-R1DMrDR2) Single-stranded nucleic acid SEQ ID NO:36 to 38 and primer SEQ ID NO:18 were prepared in 0.5 μl each at a concentration of 10 μM, and 0.5 U RNase H, 3.6 μl AptaTaq Master (Roche), and 30 ng total genomic DNA extracted from Aspc-1 (G12D mutant type) and HT-29 (KRAS wild type) were added, and the total volume was adjusted to 20 μl with triple distilled water, and then polymerase chain reaction (PCR) was performed. At this time, the reaction conditions were 95°C for 10 minutes, and after initial denaturation, 95°C for 10 seconds and 64°C for 60 seconds were reacted for 45 cycles, and the signal (FAM) was measured in real time for each cycle to derive the results. The results are shown in Figures 19 to 21.

[0157] Although the specificity for the KRAS wild-type gene varied depending on the three structures of the single-stranded nucleic acid, it was confirmed that all three structures were capable of distinguishing point mutations.

Claims

1. A kit for real-time detection of genetic variations in a single target gene, comprising: the target gene is ApoE, The kit comprises: (i) a primer set of SEQ ID NOs: 1 and 2, single-stranded nucleic acids of SEQ ID NOs: 3 to 6, and an enzyme; or (ii) a single-stranded nucleic acid of SEQ ID NO: 11 to 14, and an enzyme; Including, The enzyme is an RNase (ribonuclease, RNase) selected from the group consisting of RNase H, RNase II, RNase III, RNase IV, and RNase T2.

2. A method for detecting a genetic mutation in a single target gene, comprising: the target gene is ApoE, a) obtaining a target nucleic acid containing a genetic mutation to be detected from a biological sample, the genetic mutation being a single nucleotide polymorphism; b) producing (i) a primer set of SEQ ID NOs: 1 and 2 and a single-stranded nucleic acid of SEQ ID NOs: 3 to 6, or (ii) a single-stranded nucleic acid of SEQ ID NOs: 11 to 14; c) in the case of (i) above, mixing the target nucleic acid obtained in step a) with the single-stranded nucleic acids of SEQ ID NOs: 3 to 6 prepared in step b), a primer set of SEQ ID NOs: 1 and 2 having a base sequence complementary to the target nucleic acid obtained in step a), and a cleavage reagent, and amplifying the target nucleic acid-single-stranded nucleic acid complex containing the genetic mutation by an extension reaction; or In the case of (ii) above, the target nucleic acid obtained in step a) is mixed with the single-stranded nucleic acid of SEQ ID NO: 11 to 14 prepared in step b) and a cleavage reagent, and then the target nucleic acid-single-stranded nucleic acid complex containing the genetic mutation is amplified by an extension reaction; d) measuring the amount of single-stranded nucleic acid fragments separated from the target nucleic acid-single-stranded nucleic acid complex containing the genetic mutation amplified in step c), The cleavage reagent in step c) is RNase H, RNase II, RNase III ... A method for detecting genetic mutations in a single target gene, the target gene being an RNA hydrolase (ribonuclease, RNase) of RNase IV or RNase T2.

3. The method for detecting a genetic mutation in a single target gene described in claim 2, wherein the target nucleic acid containing a genetic mutation in step a) is RNA or DNA to be detected from a sample, or cDNA obtained by amplifying the RNA with a reverse transcription polymerase.

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