A method for amplifying target nucleic acids using a guide probe and a clamping probe, and a composition for amplifying target nucleic acids containing the same.

Abstract

The present invention relates to a method for amplifying a target nucleic acid, which can amplify a very low concentration of a target nucleic acid with high specificity, and a composition for amplifying a target nucleic acid using the same. More specifically, the present invention relates to a method for generating an amplicon of a target nucleic acid with high specificity using a guide probe that binds to the target nucleic acid when the target nucleic acid is present, a partial primer that can amplify the target nucleic acid by binding to the guide probe, and a clamping probe that suppresses the amplification of other nucleic acids except the target nucleic acid, and a composition for polymerase chain reaction (PCR) therefor. The method for amplifying a target nucleic acid according to the present invention uses a guide probe to bind to all nucleic acids present in a sample that can be hybridized, while helping the partial primer to bind to the detection site of the target nucleic acid, thereby enabling amplification of a target nucleic acid present at a very low concentration, and has the advantages that the amplification rate of nucleic acids other than the target nucleic acid differs due to the sequence specificity of the partial primer, and the amplification of nucleic acids other than the target nucleic acid is suppressed by the clamping probe, allowing the target nucleic acid to be detected with high specificity, making it useful for molecular diagnosis, prenatal diagnosis, early diagnosis, cancer diagnosis, genetic-related diagnosis, genotype diagnosis, diagnosis of infectious bacteria, discrimination of drug-resistant bacteria, forensic medicine, species discrimination of organisms, etc.

Description

Technical Field

[0001] The present invention relates to a method for amplifying a target nucleic acid that can amplify a very low concentration of a target nucleic acid with high specificity, and a composition for target nucleic acid amplification using the same. More specifically, when a target nucleic acid is present, a guide probe bound to the target nucleic acid, a partial primer that can bind to the guide probe and amplify the target nucleic acid, and a clamping probe that suppresses the amplification of other nucleic acids excluding the target nucleic acid are used to generate an amplification product (amplicon) of the target nucleic acid with high specificity, and a composition for polymerase chain reaction (PCR) for implementing the above method.

Background Art

[0002] The genetic information possessed by all living organisms on Earth is the source of the unique attributes of each individual, and this is recorded in a substance called nucleic acid (DNA or RNA) in the order of the bases adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). Therefore, clarifying or confirming the order (base sequence) of these bases can be a process for confirming the attributes of living organisms and understanding the inherent metabolic mechanisms.

[0003] Recently, molecular diagnostics methods for confirming the presence or absence or attributes of living organisms by detecting or confirming specific base sequences of living organisms have been widely used. Representative examples of molecular diagnostics used medically include detecting gene mutations related to human cancer development and detecting causative pathogens of infectious diseases that may occur in humans. In addition, tests for detecting harmful microorganisms present in food also belong to molecular diagnostics.

[0004] Among the various molecular diagnostic methods for specifically identifying a particular base sequence, most utilize polymerase chain reaction (PCR) using nucleic acid polymerases (DNA polymerases). This polymerase chain reaction is carried out via a composition containing a pair of primers that can be specifically hybridized with a target nucleic acid containing a specific base sequence site, a thermo-stable nucleic acid polymerase that can initiate a polymerase chain reaction using the primers as a starting material and the target nucleic acid as a template, and a thermal cycler that can apply a predetermined temperature to the composition in a stepwise and repeated manner. Furthermore, molecular diagnostics via polymerase chain reaction utilize nucleic acid-binding dyes or probes to detect specific base sequences within a large amount of formed (amplified) target nucleic acids in real time; these are called real-time polymerase chain reactions (real-time PCR) (Higuchi, R. et al., Biotechnology 1992. 10:413-417, Higuchi, R. et al., Biotechnology 1993. 11:1026-1030).

[0005] In the aforementioned molecular diagnostics, detecting gene mutations associated with human cancer development requires high sensitivity and specificity. This is because the mutations to be detected belong to the category of somatic mutations, which exist in very small amounts mixed with wild-type normal DNA. Furthermore, if the target mutation is not accurately recognized, it can lead to false positives, as it may be a point mutation with little difference in base sequence from the normal gene, or a complex insertion / deletion mutation or gene fusion mutation.

[0006] In other words, the essential requirements for tumor-specific mutation detection tests are (1) sensitivity to detect mutant DNA present in a low proportion within normal DNA, and (2) specificity to minimize the rate of false-positives, where normal DNA is mistakenly identified as mutant DNA.

[0007] Various diagnostic methods have been developed and are used to detect tumor-specific mutations. Representative methods include direct sequencing, allele-specific PCR (AS-PCR), restriction fragment length polymorphism (RFLP), TaqMan probe method, and ARMS (amplification refractory mutation system) PCR. However, these methods have not been able to produce satisfactory results in terms of sensitivity and specificity. Direct sequencing has the highest specificity and the lowest false-positive rate, but it has the drawback of only being able to detect mutations when 20-30% or more of the DNA is mutated. On the other hand, AS-PCR, RFLP, and TaqMan probe methods have high sensitivity but low specificity, and therefore always suffer from the problem of false positives.

[0008] Recently, methods that significantly improve sensitivity and specificity, such as PNA (peptide nucleic acid)-mediated PCR clamping (Sun, X., et al., 2002. Nat Biotechnol, 20: 186-189), LNA (locked nucleic acid)-mediated PCR clamping (Dominguez, PL, et al., 2005. Oncogene, 24: 6830-6834), and COLD-PCR (co-amplification at lower denaturation temperature PCR) (Li, J., et al., 2008. Nat. Med, 14: 579-584), have been developed, and the ability to analyze base sequences with high specificity is gradually being emphasized.

[0009] ARMS-PCR is a method developed to improve the specificity, which is a drawback of AS-PCR. Like AS-PCR, it is based on the principle that PCR is performed only after the primers are composed of sequences that are perfectly complementary to the template sequence ((Newton, CR, et al., 1989. Nucl Acids Res, 17; 2503-2516), but it has the drawback that experimental trial and error is required to determine the optimal primer with superior specificity (Drenkard, E., et al., 2000. Plant Physiol. 124: 1483-1492).

[0010] Korean Patent Application No. 10-2020-0066712 describes a method for selectively enriching a DNA population from a mixed sample containing multiple populations by contacting it with multiple blocking oligonucleotides to enrich the nucleic acid ratio of a specific population. However, it has the drawback of requiring additional aliphatic molecules to suppress amplification reactions in addition to the blocking oligonucleotides. Furthermore, Korean Patent No. 10-2019800 describes a method for detecting the methylation state of a target gene using a reduction probe and the difference in Tm (melting temperature) values. However, it has the drawback of being unable to detect gene mutations and only being able to detect the methylation state.

[0011] Therefore, the present inventors have made diligent efforts to develop a method that can dramatically increase the specificity of target nucleic acid amplification at very low concentrations. As a result, they have confirmed that when PCR is performed using a method that generates amplification products using a guide probe containing a sequence that can hybridize with a site other than the detection site of the target nucleic acid and a sequence that does not hybridize with the target nucleic acid, a partial primer containing a sequence complementary to the non-hybridizable sequence of the guide probe and a sequence complementary to the detection site of the target nucleic acid, a clamping probe that suppresses the amplification of other nucleic acids excluding the target nucleic acid, and a specific primer that can amplify the target nucleic acid in conjunction with the partial primer, it is possible to detect target nucleic acids at very low concentrations with high specificity, thus completing the present invention.

[0012] The information described in this background section is intended solely to deepen understanding of the background of the present invention and may not include information that constitutes prior art already known to a person with ordinary skill in the art to which the present invention belongs. [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] Korean Patent Application No. 10-2020-0066712 [Patent Document 2] Korean Patent No. 10-2019800 [Overview of the project]

[0014] The object of the present invention is to provide a method for amplifying target nucleic acids at very low concentrations with high specificity. Another object of the present invention is to provide a polymerase chain reaction (PCR) composition for target nucleic acid amplification that can amplify target nucleic acids at very low concentrations with high specificity.

[0015] To achieve the above objective, the present invention provides a method for amplifying a target nucleic acid, comprising the steps of: (a) a guide probe comprising a sequence that can hybridize with a site other than the detection site of the target nucleic acid and a sequence that does not hybridize with the target nucleic acid; ii) a partial primer comprising a sequence complementary to the sequence of the guide probe that does not hybridize with the target nucleic acid and a sequence complementary to the detection site of the target nucleic acid; iii) a clamping probe that suppresses the amplification of other nucleic acids excluding the target nucleic acid; and iv) a specific primer that can amplify the target nucleic acid in conjunction with the partial primer; and (b) a step of determining whether or not an amplification product is present.

[0016] The present invention also provides a PCR composition for target nucleic acid amplification comprising: i) a guide probe including a sequence that can hybridize with a site other than the detection site of the target nucleic acid and a sequence that does not hybridize with the target nucleic acid; ii) a partial primer including a sequence complementary to the sequence of the guide probe that does not hybridize with the target nucleic acid and a sequence complementary to the detection site of the target nucleic acid; iii) a clamping probe that suppresses the amplification of other nucleic acids excluding the target nucleic acid; and iv) a specific primer that can amplify the target nucleic acid in conjunction with the partial primer. [Brief explanation of the drawing]

[0017] [Figure 1] This figure shows a guide probe, clamping probe, partial primer, specific primer, and the hybridization relationships between components necessary for one aspect of the present invention. [Figure 2]This figure illustrates the process by which the present invention elicits a difference in amplification efficiency due to the difference in base sequences between a target nucleic acid and a non-target nucleic acid. (a) shows that, in the presence of the target nucleic acid, with the help of a guide probe that hybridizes with a specific site of the target nucleic acid, when a partial primer that hybridizes with a part of the guide probe approaches the target amplification site of the target nucleic acid, hybridization and extension amplification occur because a part of the 3' end of the partial primer is complementary to the base sequence of the target amplification site of the target nucleic acid. At this time, the binding of the guide probe to the target nucleic acid and the guide probe to the partial primer occurs first because the Tm is high, while the binding of the partial primer to the target nucleic acid occurs first because the complementary sequence length is short and the Tm is low. (b) indicates that, because the complementarity is low, hybridization occurs later, the complementarity between the base sequence of the clamping probe and the target nucleic acid is low, and the binding of the partial primer to the target nucleic acid cannot be prevented. (b) indicates that, in the presence of a non-target nucleic acid in which some of the base sequences differ from the target nucleic acid, the guide probe hybridizes with a specific site of the non-target nucleic acid, and even if the partial primer that hybridizes with a part of the guide probe is close to the non-target nucleic acid, the complementarity between a part of the 3' end of the partial primer and the base sequence of the non-target nucleic acid is low, and the clamping probe hybridizes with the non-target nucleic acid, preventing the binding of the partial primer to the non-target nucleic acid, thus suppressing extension amplification. [Figure 3] This is a conceptual diagram illustrating a method for detecting specific mutations by distinguishing them from the wild type, according to one aspect of the present invention. [Figure 4]This figure shows the results of detecting the G13D mutation in the KRAS gene within the human genome with high sensitivity and specificity according to one aspect of the present invention. When a partial primer is prepared so that a portion of the 3' end is hybridized with the genotype of the G13D mutation and a clamping probe is prepared so as to hybridize with the wild-type without the mutation, high amplification efficiency is observed and a relatively low Ct value is calculated when the G13D mutation is present. On the other hand, when only the wild-type is present, low amplification efficiency is observed and a relatively high Ct value is calculated, or no amplification occurs. As a result, even G13D mutations present in very small amounts at the 0.1% level can be detected by comparing the Ct values. [Figure 5] This figure compares the results of detecting the G13D mutation in the KRAS gene within the human genome under one embodiment of the present invention (a) and under conditions without a clamping probe (b). It shows that when a partial primer, constructed so that a portion of its 3' end hybridizes with the genotype of the G13D mutation, is used together with a clamping probe that hybridizes with the wild type, a very large difference in Ct values ​​is observed between the conditions in which the G13D mutation is present and the conditions in which only the wild type is present. In contrast, under conditions without a clamping probe, the difference in Ct values ​​between the presence of the G13D mutation and the presence of the wild type is relatively small. [Figure 6] This document compares the results of detecting G13D mutations in the KRAS gene within the human genome under one embodiment of the present invention (a) and under conditions without a clamping probe (b). It shows that when a partial primer, designed so that a portion of the 3' end hybridizes with the genotype of the G13D mutation, is used together with a clamping probe that hybridizes with the wild type, even G13D mutations present at trace amounts of 0.1% can be detected through the ΔCt value, which indicates the difference between the amplified Ct value of the control site and the amplified Ct value of the target site. On the other hand, under conditions without a clamping probe, G13D mutations present at 0.5% can be detected through the ΔCt value, but it is difficult to distinguish G13D mutations present at even smaller amounts (0.1-0.25%) from the wild type. [Figure 7] An example of a linker that may be included in the guide probe of the present invention.

Mode for Carrying Out the Invention

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

[0019] In the present invention, it was attempted to confirm whether a very low concentration of target nucleic acid can be detected using a guide probe, a clamping probe, and a partial primer.

[0020] That is, in one embodiment of the present invention, in order to specifically amplify and detect the G13D mutation, which is a GGC>GAC base sequence mutation located at exon 2, codon 13 of the human KRAS gene, a guide probe that can specifically hybridize to a site near codon 13 of the KRAS gene, a clamping probe that can specifically hybridize to codon 13 and its vicinity of the wild-type KRAS gene, a partial primer whose partial 3'-end can specifically hybridize to codon 13 and its vicinity of the KRAS gene where the G13D mutation has occurred, and a specific primer that can hybridize to a part of the base sequence of the KRAS gene so as to be able to amplify the target nucleic acid in pair with the partial primer were designed. At this time, a part of the base sequence at the C-terminus of the guide probe and a part of the base sequence at the 5'-terminus of the partial primer were made capable of hybridizing.

[0021] PCR was performed using the guide probe, the clamping probe, the partial primer, and the specific primer to determine whether the G13D mutation present in trace amounts in the presence of the wild-type nucleic acid can be detected.

[0022] As a result, we confirmed that under conditions where the target nucleic acid of the G13D mutation was present, the increase in the fluorescence signal on the amplification curve appeared quickly and a low Ct value was calculated, while under conditions where only wild-type nucleic acid was present, there was no increase in the fluorescence signal on the amplification curve, or it appeared very slowly and a large Ct value was calculated (Figure 4).

[0023] Therefore, in one view, the present invention, (a) i) A guide probe containing a sequence that can be hybridized with a site other than the detection site of the target nucleic acid and a sequence that does not hybridize with the target nucleic acid; ii) A partial primer comprising a sequence complementary to the sequence of the guide probe that does not hybridize with the target nucleic acid, and a sequence complementary to the detection site of the target nucleic acid; iii) Clamping probes that suppress the amplification of other nucleic acids while excluding the target nucleic acid; and iv) A specific primer that, in conjunction with the aforementioned partial primer, can amplify the target nucleic acid. A step of amplifying the isolated nucleic acid by performing a polymerization enzyme chain reaction (PCR) in the presence of; and (b) The step of determining whether or not an amplification product is present.

[0024] In this invention, the term "target nucleic acid" means any type of nucleic acid containing the base sequence of interest to be amplified or detected. The target nucleic acid includes gene base sequences from multiple species, subspecies, or variants, or gene mutations within the same species, and may, but is not limited to, any type of DNA, including genomic DNA, mitochondrial DNA, and viral DNA, or any type of RNA, including mRNA, ribosomal RNA, non-coding RNA, tRNA, and viral RNA. The target nucleic acid may be hybridized with a guide probe, a partial primer (part of the 3' end), and a specific primer under conditions for a polymerization enzyme chain reaction.

[0025] In this invention, the term "hybridization" refers to the formation of a double-stranded nucleic acid through hydrogen bonding between single-stranded nucleic acids having complementary base sequences, and is used in a similar sense to annealing. However, in a broader sense, hybridization includes not only cases where the base sequences of the two single strands are perfectly complementary (perfect match), but also exceptional cases where some base sequences are not complementary (mismatch).

[0026] In this invention, the term "guide probe" is characterized by being able to be hybridized simultaneously with the target nucleic acid and a partial primer, and by the 3' end of the partial primer assisting in the hybridization of the target base sequence of the target nucleic acid. In other words, sequences of the guide probe that do not hybridize with the target nucleic acid can be hybridized with the partial primer. The guide probe may be manufactured from one or more materials that can hybridize with the target nucleic acid (e.g., DNA, RNA, LNA (locked nucleic acid), PNA (peptide nucleic acid), etc.).

[0027] The term "clamping probe" in this invention is characterized by being able to be hybridized with non-target nucleic acids that are not to be amplified, and preventing the 3' end of a partial primer from hybridizing with the non-target nucleic acid. The clamping probe may be prepared by mixing one or more of any materials that can be hybridized with non-target nucleic acids (e.g., DNA, RNA, LNA (locked nucleic acid), PNA (peptide nucleic acid), etc.).

[0028] In this invention, the term "partial primer" is characterized in that a portion of the 5' end is hybridized with a guide probe, and the 3' end is hybridized with the target base sequence of the target nucleic acid, allowing for synthetic extension by nucleic acid polymerase.

[0029] In a preferred embodiment of the present invention, the target nucleic acid, guide probe, and partial primer are hybridized with each other (target nucleic acid-guide probe, guide probe-partial primer, partial primer-target nucleic acid). The guide probe binds to the vicinity of the target base sequence of the target nucleic acid, while the hybridized partial primer is simultaneously positioned near the target base sequence. This allows the 3' end of the partial primer to further easily hybridize with the target base sequence, initiating synthetic extension by nucleic acid polymerase and amplification of the target site of the target nucleic acid.

[0030] Hybridization between target nucleic acid-guide probe and guide probe-partial primer occurs because complementary parts hybridize first due to their higher Tm compared to the partial primer-target nucleic acid, while the partial primer-target nucleic acid hybridizes later due to its relatively lower Tm.

[0031] In this invention, the term "specific primer" is characterized by being hybridized with a target nucleic acid without the assistance of the guide probe, and by which synthesis and extension by nucleic acid polymerases occur.

[0032] In the present invention, the site that hybridizes with the target nucleic acid in the guide probe may be located at the 5' end or N-terminus, and the site that hybridizes with the partial primer may be located at the 3' end or C-terminus, but the invention is not limited thereto. In the present invention, the guide probe may further include a linker between the hybridized sequence of the target nucleic acid and the sequence that does not hybridize with the target nucleic acid.

[0033] In the present invention, the linker may be any compound that is located between the target nucleic acid hybridization sequence and the partial primer hybridization sequence and does not contain nucleic acid bases that link the two sites. Preferably, the linker may be one or more compounds selected from the group consisting of β-alanine (β-Ala-OH, C3), aminobutyric acid (C4), aminohexanoic acid (C6), aminolauric acid (C12), acetoacetoxyethyl acrylate (AAEA (O-linker)), aminoethoxyethoxyethoxyacetic acid (2-[2-[2-[2-(amino)ethoxy]ethoxy]ethoxy]acetic acid, AEEEA), AEEEEA, DL15, and L35, but is not limited to these.

[0034] In the present invention, each of the guide probe, clamping probe, partial primer, and specific primer may be characterized by being composed of one or more links of oligonucleotides, locked nucleic acid (LNA), and peptide nucleic acid (PNA).

[0035] More specifically, each of the guide probe and the clamping probe may be prepared by combining one or more oligonucleotides, LNAs, and PNAs. For example, if the guide probe is 20 bp, the 5' end 10 bp may be composed of PNA and the 3' end 10 bp may be composed of oligonucleotide.

[0036] Each of the aforementioned partial primers and specific primers may be prepared by combining one or more oligonucleotides, LNAs, or PNAs, but the amplification reaction may be induced by constructing at least five base sequences at the 3' end from oligonucleotides.

[0037] In the present invention, the guide probe may be any nucleic acid that can be hybridized with the target nucleic acid and partial primer, but it may preferably be a PNA prepared with a base sequence length of 10 to 500 bases, and more preferably a PNA prepared with a base sequence length of 20 to 150 bases.

[0038] In the present invention, the clamping probe may be any nucleic acid that can be hybridized with a non-target nucleic acid, but it may preferably be a PNA prepared with a base sequence length of 5 to 500, and more preferably a PNA prepared with a base sequence length of 10 to 100. In the present invention, the clamping probe may be characterized by preventing the partial primer from binding to other nucleic acids, excluding the target nucleic acid.

[0039] In the present invention, the clamping probe may be characterized by containing a sequence complementary to the detection site of the target nucleic acid of the partial primer, and having 1 to 10 bases different.

[0040] In the present invention, the clamping probe is characterized in that, because the binding sequences of the partial primer and the detection site of the target nucleic acid are different, the partial primer does not bind to other nucleic acids present in the sample other than the target nucleic acid, and therefore does not bind to the target nucleic acid. In the present invention, the guide probe and clamping probe may be characterized by hybridizing to the opposite strand of the target nucleic acid to which the specific primer is hybridized.

[0041] In the present invention, the partial primer may further include a spacer, which is a single-strand oligonucleotide with a length of 1 to 100 bases, between a sequence complementary to a sequence that does not hybridize with the target nucleic acid of the guide probe and a sequence complementary to the detection site of the target nucleic acid.

[0042] In the present invention, the spacer refers to a sequence that excludes the sequence complementary to the guide probe contained in the partial primer and the sequence complementary to the target nucleic acid. If necessary, a probe that can bind to the spacer can be used to enable detection with the same probe even if the base sequence of the target nucleic acid is changed.

[0043] In the present invention, the spacer and the linker of the guide probe can play a role in regulating the efficiency by which a partial primer located near the target site of the target nucleic acid binds to the target site by the guide probe. This means that if the guide probe hybridizes with the target nucleic acid at a location slightly further away from the target site, increasing the length of the spacer and linker can help the partial primer bind to the target site.

[0044] In the present invention, the sequence complementary to the detection site of the target nucleic acid of the partial primer may be characterized by being a sequence of 3 to 15 bases. In the present invention, the length of the amplification product may be characterized as being 50 bp to 1 kbp.

[0045] In the present invention, the step of determining the presence or absence of an amplification product may be characterized by using a nucleic acid-binding dye or probe that can bind to the amplification product.

[0046] In the present invention, the nucleic acid-binding dye may be any intercalating substance or DNA minor groove-binding substance without limitation, but may preferably be selected from the group consisting of ethidium bromide, SYBR Green I, SYBR Gold, EvaGreen, YO-PRO-1, SYTO, BEBO, and BEXTO.

[0047] In the present invention, the probe capable of binding to the amplification product may be selected from the group consisting of one or more combinations of oligonucleotides, LNAs, and PNAs.

[0048] PNA (Peptide nucleic acid) is a DNA-like substance in which nucleic acid bases are linked to a peptide backbone rather than a sugar-phosphate backbone. It was first synthesized by Nielsen et al. in 1991. Like LNA (Locked nucleic acid) or MNA (Mopholino nucleic acid), PNA is one of the artificially synthesized gene recognition substances, and its basic backbone is composed of polyamide.

[0049] PNA exhibits excellent affinity and selectivity, high stability against nucleolytic enzymes, and is not degraded by existing restriction enzymes. Furthermore, it has the advantage of high thermal and chemical stability, making long-term storage easy.

[0050] PNA forms double helix through hybridization reactions with native nucleic acids of complementary base sequences. For the same length, PNA / DNA double helix is ​​more stable than DNA / DNA double helix, and PNA / RNA double helix is ​​more stable than DNA / RNA double helix. Furthermore, because PNA is more susceptible to single base mismatches, it has a superior ability to detect single nucleotide polymorphisms (SNPs) compared to native nucleic acids.

[0051] In other words, PNA-DNA binding strength is significantly superior to DNA-DNA binding strength, and even a single nucleotide mismatch typically results in a difference of 15-20°C in melting temperature (Tm). This difference in binding strength can be used to detect changes in base sequences such as SNPs (single-nucleotide polymorphisms) and In / Del (insertion / deletion).

[0052] In the present invention, the probe capable of binding to the amplification product may be characterized by having a base sequence that is partially or completely complementary to any base sequence in the amplification product and the base sequence of the target nucleic acid, and preferably by having a reporter and a quencher linked to both ends.

[0053] In the present invention, the probe suppresses signal generation when the distance between the reporter and the extinguisher is short, but the signal intensity increases as the distance between the reporter and the extinguisher increases. Generally, when the probe is hybridized with a complementary base sequence, the distance between the reporter and the extinguisher is greatest, allowing for the detection of a specific base sequence through signal generation or an increase in signal intensity.

[0054] In the present invention, the reporter may be characterized by being one or more fluorescent substances selected from the group consisting of fluorescein, fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, FITC, Oregon green, Alexa Fluor, FAM, JOE, ROX, HEX, Texas Red, TET, TRITC, TAMRA, cyanine dyes, and thiadicarbocyanine dyes.

[0055] In the present invention, the extinction agent may be one or more selected from the group consisting of Dabcyl, TAMRA, Eclipse, DDQ, QSY, Blackberry Quencher, Black Hole Quencher, Qxl, Iowa Black FQ, Iowa Black RQ, and IRDye QC-1.

[0056] In a preferred embodiment of the present invention, the detection of the amplification product by the nucleic acid polymerase is performed via real-time polymerase chain reaction (real-time PCR), and at this time, an amplification curve is obtained as the amplification product increases, and the Ct (threshold cycle) value is measured, but the invention is not limited to this method. In the method using the Ct value, the principle is utilized that the more the target nucleic acid (target base sequence) is present in the sample and the faster the amplification product is generated and increases, the faster the signal generated by the detection probe increases, the fewer cycles are needed to reach the threshold, and the lower the measured Ct value.

[0057] In the present invention, the isolated nucleic acid may be any nucleic acid that is free from other substances that can inhibit the polymerization enzyme chain reaction, and may preferably be characterized by being isolated from a specimen sample, but is not limited thereto.

[0058] From another perspective, the present invention i) A guide probe containing sequences that can be hybridized with sites other than the detection site of the target nucleic acid, and sequences that do not hybridize with the target nucleic acid; ii) A partial primer comprising a sequence complementary to the sequence of the guide probe that does not hybridize with the target nucleic acid, and a sequence complementary to the detection site of the target nucleic acid; iii) Clamping probes that suppress the amplification of other nucleic acids while excluding the target nucleic acid; and iv) A specific primer that, in conjunction with the aforementioned partial primer, can amplify the target nucleic acid. This relates to PCR compositions for target nucleic acid amplification, including those containing the specified components.

[0059] In the present invention, "sample" includes a variety of samples, preferably a biological sample analyzed using the method of the present invention. More preferably, it may be a sample mixed with a virus species, or a sample of an individual infected with the virus (e.g., humans, mammals, and fish), and biological samples of plant, animal, human, fungal, bacterial, and viral origin may be analyzed. When analyzing a mammalian or human-derived sample, the sample may originate from a specific tissue or organ. Typical examples of tissues include connective tissue, skin, muscle, or nerve tissue. Typical examples of organs include the eye, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, small intestine, testes, ovaries, uterus, rectum, nervous system, glands, and internal blood vessels. The biological sample to be analyzed also includes any cells, tissues, fluids, or any other medium from a biological source that can be well analyzed by the present invention, including samples obtained from humans, animals, or food produced for human or animal consumption. Furthermore, the biological samples to be analyzed include, but are not limited to, bodily fluid samples, which include blood, serum, plasma, lymph, breast milk, urine, feces, ocular lactate, saliva, semen, brain extracts (e.g., cerebral fragments), cerebrospinal fluid, appendix, spleen, and tonsil tissue extracts.

[0060] In other words, the present invention relates to a kit for detecting a target nucleic acid comprising the composition. In the present invention, the kit may selectively contain reagents necessary for carrying out a target nucleic acid amplification reaction (e.g., a polymerase chain reaction), such as a buffer, DNA polymerase, DNA polymerase cofactor, and deoxyribonucleotide-5-triphosphate (dNTP). Selectively, the kit of the present invention may also contain various oligonucleotide molecules, reverse transcriptase, various buffers and reagents, and antibodies that inhibit DNA polymerase activity. Furthermore, the optimal amount of reagents used in a particular reaction of the kit may be readily determined by a person skilled in the art who has mastered the disclosures herein. Typically, the equipment of the present invention may be prepared in a separate package or compartment containing the aforementioned components.

[0061] In one embodiment, the kit may include a partitioned carrier means for holding a sample, a container for reagents, a container for a guide probe, a clamping probe and primers, and a container for a probe for detecting the amplified product.

[0062] The carrier means is suitable for comprising one or more containers, such as bottles or tubes, each container comprising an independent component used in the method of the present invention. In the specification of the present invention, a person with ordinary skill in the art can easily dispense the required formulation from the container.

[0063] The present invention will be described in more detail below with reference to examples. It will be obvious to those with ordinary skill in the art that these examples are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention.

[0064] Example 1. High-sensitivity amplification and detection of the KRAS gene with a G13D mutation in the human genome. 1.1 Preparation of primers and probes The human KRAS gene encodes a GTP-based switch protein that functions in the body's signaling pathway, and certain mutations in the KRAS gene are known to be associated with cancer development (Waters, AM and Der, CJ 2018. Cold Spring Harb. Perspect. Med.8:a031435.). The mutation in the GGC>GAC base sequence located at exon 2, codon 13 of the human KRAS gene is called the G13D mutation.

[0065] To specifically amplify and detect the G13D mutation in the KRAS gene through the novel target nucleic acid amplification method of the present invention, a guide probe 1 that can be specifically hybridized to the vicinity of codon 13 of the KRAS gene, a clamping probe 1 that can be hybridized to codon 13 and the vicinity of a wild-type KRAS gene without the G13D mutation, a partial primer 1 that can be specifically hybridized to a portion of the 3' end of codon 13 and the vicinity of a KRAS gene with the G13D mutation, and a specific primer 1 that can be hybridized to a portion of the base sequence of the KRAS gene in pair with the partial primer 1 to amplify the target nucleic acid, with the hybridization being made possible by hybridizing a portion of the base sequence of the C-terminus of the guide probe 1 and a portion of the base sequence of the 5' end of the partial primer 1. Furthermore, a detection probe 1 was designed and manufactured to detect the product amplified through the designed guide probe and primer (PANAGENE Inc., South Korea). The base sequences of each primer and probe are disclosed in Tables 1 and 2.

[0066] [Table 1] [Table 2]

[0067] The partial primer 1 is prepared using a single-chain oligonucleotide with a length of 19 base pairs. The underlined base pairs are complementary to a portion of the base pair of guide probe 1, and the italicized base pairs have complementary properties to the base pair of codon 13 and neighboring regions of the KRAS gene where the G13D mutation occurred.

[0068] The guide probe 1 is made of PNA with a length of 23 base pairs. The base pairs shown in italics are complementary to the base pair of the KRAS gene near codon 13, and the base pairs shown underline have complementary characteristics to a portion of the base pair of the partial primer 1. In addition, a [K (lysine)] tag is attached to the N-terminus of the guide probe 1, so that the manufacturer's (PANAGENE Inc., South Korea) L35 linker is linked between the region complementary to the KRAS gene and the region complementary to a portion of the partial primer 1's base pair.

[0069] The clamping probe 1 was fabricated from PNA with a length of 15 base pairs, and had [K (lysine)] attached to its N-terminus.

[0070] The detection probe 1 is made from PNA with a length of 14 base pairs, with a quencher [Dabcyl] attached to the N-terminus and a reporter [FAM] attached to the C-terminus. The probe is constructed so that a [O linker] and [K (lysine)] from the manufacturer (PANAGENE Inc., South Korea) are linked between the PNA and [FAM]. When the detection probe 1 is hybridized with an amplification product having complementary base pairs, the quencher and reporter move to the furthest distance from each other, at which point the signal (fluorescence) value from the reporter is maximized, and the amplification product can be detected by measuring this signal.

[0071] The partial primer 1 has a base sequence length of only 8 that is complementary to the base sequence of the target site in the KRAS gene, making it difficult to hybridize to the target site by itself under polymerase chain reaction (PCR) conditions. However, if a guide probe 1 that hybridizes is present near the target site, a portion of the base sequence of the guide probe 1 and a portion of the base sequence of the partial primer 1 can hybridize and easily hybridize to the target site.

[0072] The partial primer 1 has a hybridizable base sequence length of only 8, which is shorter than that of a typical primer with 18 to 30 hybridizable base sequences. Therefore, if there is one or more mismatched base sequences between the partial primer 1 and the target nucleic acid hybridization site, these mismatched base sequences will have a greater impact on hybridization efficiency than a typical primer, and as a result, subtle changes in the base sequence can be detected better than with a typical primer.

[0073] Furthermore, if one or more non-complementary nucleotide sequences exist between the partial primer 1 and the codon 13 and neighboring regions of the wild-type KRAS gene without the G13D mutation, resulting in low hybridization efficiency, the clamping probe 1 will hybridize with the codon 13 and neighboring regions of the wild-type KRAS gene, further inhibiting hybridization of the partial primer 1. This allows for the detection of subtle changes in the nucleotide sequence with even higher sensitivity than when the clamping probe is not used.

[0074] 1.2 Generation and Confirmation of Amplification Products Approximately 10,000 copies of the wild type. To form the intended amplification product under conditions where KRAS gene target nucleic acids are present, or under conditions where approximately 10,000 wild-type KRAS gene target nucleic acids are mixed with approximately 500 KRAS gene target nucleic acids with G13D mutations (5%), or under conditions where approximately 10,000 wild-type KRAS gene target nucleic acids are mixed with approximately 100 KRAS gene target nucleic acids with G13D mutations (1%), or under conditions where approximately 10,000 wild-type KRAS gene target nucleic acids are mixed with approximately 50 KRAS gene target nucleic acids with G13D mutations (0.5%), or under conditions where approximately 10,000 wild-type KRAS gene target nucleic acids are mixed with approximately 25 KRAS gene target nucleic acids with G13D mutations (0.25%), or under conditions where approximately 10,000 wild-type KRAS gene target nucleic acids are mixed with approximately 10 KRAS gene target nucleic acids with G13D mutations (0.1%), 10 A polymerization enzyme chain reaction composition was prepared containing a pmole guide probe 1, a 5 pmole clamping probe 1, a 10 pmole partial primer 1, a 4 pmole specific primer 1, and a 3 pmole detection probe 1.

[0075] The aforementioned polymerase chain reaction composition typically contains a nucleic acid polymerase (DNA polymerase) used in polymerase chain reactions, along with elements such as a buffer, deoxyribonucleotide-5-triphosphate (dNTP), potassium chloride (KCl), magnesium chloride (MgCl2), and a surfactant (detergent).

[0076] Temperature control for the polymerization enzyme chain reaction was performed using a standard thermal cycler. After initial denaturation [95°C, 5 min], the first step of 15 cycles consisted of denaturation [95°C, 20 sec] - annealing [63°C, 20 sec] - extension [72°C, 20 sec], followed by the second step of 35 cycles: denaturation [95°C, 10 sec] - measure [50°C, 10 sec] - denaturation [95°C, 20 sec] - annealing [58°C, 20 sec] - extension [72°C, 20 sec]. The fluorescence value of the FAM channel was measured at each cycle during the measure step of the second step to derive the amplification curve.

[0077] As a result, a clear increase in the fluorescence signal on the amplification curve was observed under all conditions in which KRAS target nucleic acids containing the G13D mutation were mixed. On the other hand, under conditions in which only wild-type KRAS target nucleic acids were present, no increase in the fluorescence signal on the amplification curve was observed, or it was observed very weakly. Specifically, Ct values ​​of 13.6, 14.9, 16.0, 17.9, and 18.9 were observed on average under conditions in which approximately 500, 100, 50, 25, and 10 G13D mutated target nucleic acids were mixed, respectively. In contrast, under conditions in which only wild-type was present, the Ct value was either not calculated (ND; not determined) or a relatively large Ct value of 29.1 was observed. Consequently, the presence of very small amounts of the G13D mutation could be specifically identified through the difference in Ct values ​​(Figure 4).

[0078] Example 2. Confirmation of the detection limit of the KRAS gene in which the G13D mutation occurs in the human genome. 2.1 Preparation of primers and probes To specifically amplify and detect the G13D mutation in the KRAS gene through the novel target nucleic acid amplification method of the present invention, a guide probe 1 that can be specifically hybridized to the region near codon 13 of the KRAS gene, a clamping probe 1 that can be hybridized to codon 13 and the surrounding region of a wild-type KRAS gene without the G13D mutation, a partial primer 1 that can be specifically hybridized to a portion of the 3' end of codon 13 and the surrounding region of a KRAS gene with the G13D mutation, and a specific primer 1 that can be hybridized with a portion of the base sequence of the KRAS gene in pair with the partial primer 1 to amplify the target nucleic acid, with the hybridization being made possible by hybridizing a portion of the base sequence of the C-terminus of the guide probe 1 and a portion of the base sequence of the 5' end of the partial primer 1. Furthermore, a detection probe 1 was designed and manufactured to detect the product amplified through the designed guide probe and primer (PANAGENE Inc., South Korea).

[0079] On the other hand, in order to specifically amplify and detect the KRAS gene in a sample regardless of the presence or absence of the G13D mutation in the KRAS gene, a guide probe 2 that can specifically hybridize with exon 6 of the KRAS gene, a partial primer 2 that can specifically hybridize a portion of the 3' end with exon 6 of the KRAS gene, and a specific primer 2 that can hybridize with a portion of the exon 6 base sequence of the KRAS gene so that it can amplify the target nucleic acid in conjunction with the partial primer 2 were designed, and at this time, a portion of the base sequence of the C-terminus of the guide probe 2 and a portion of the base sequence of the 5' end of the partial primer 2 were made hybridizable. Furthermore, a detection probe 2 was designed and manufactured to detect the product amplified through the designed guide probe and primer (PANAGENE Inc., South Korea). The base sequences of each primer and probe are disclosed in Tables 3 and 4.

[0080] [Table 3] [Table 4]

[0081] The partial primer 1 is prepared using a single-chain oligonucleotide with a length of 19 base pairs. The underlined base pairs are complementary to a portion of the base pair of guide probe 1, and the italicized base pairs have complementary properties to the base pair of codon 13 and neighboring regions of the KRAS gene where the G13D mutation occurred.

[0082] The partial primer 2 is prepared using a single-chain oligonucleotide with a length of 19 base pairs. The underlined base pairs are complementary to a portion of the base pair of guide probe 2, and the italicized base pairs are complementary to a portion of the exon 6 base pair of the KRAS gene.

[0083] The guide probe 1 is made of PNA with a length of 23 base pairs. The base pairs shown in italics are complementary to the base pair of the region adjacent to codon 13 of the KRAS gene, and the base pairs shown underline have complementary characteristics to a portion of the base pair of the partial primer 1. In addition, [K (lysine)] is attached to the N-terminus of the guide probe 1, so that the manufacturer's (PANAGENE Inc., South Korea) L35 linker is linked between the region complementary to the base pair of the KRAS gene and the region complementary to a portion of the base pair of the partial primer 1.

[0084] The guide probe 2 is made of PNA with a length of 25 base pairs. The base pairs shown in italics are complementary to a portion of the exon 6 base pair sequence of the KRAS gene, and the base pairs shown underline have complementary characteristics to a portion of the base pair sequence of the partial primer 2. In addition, [K (lysine)] is attached to the N-terminus of the guide probe 2, so that the manufacturer's (PANAGENE Inc., South Korea) L35 linker is linked between the region complementary to the base pair sequence of the KRAS gene and the region complementary to a portion of the base pair sequence of the partial primer 2.

[0085] The clamping probe 1 was fabricated from PNA with a length of 15 base pairs, and had [K (lysine)] attached to its N-terminus.

[0086] The detection probe 1 was fabricated using PNA with a length of 14 base pairs, with a quencher [Dabcyl] attached to the N-terminus and a reporter [FAM] attached to the C-terminus. The manufacturer's [O linker] and [K (lysine)] were linked between the PNA and [FAM].

[0087] The detection probe 2 was fabricated using PNA with a length of 13 base pairs, with a quencher [Dabcyl] attached to the N-terminus and a reporter [HEX] attached to the C-terminus. The probe was fabricated so that the manufacturer's (PANAGENE Inc., South Korea) [O linker] and [K (lysine)] were linked between the PNA and [HEX].

[0088] When detection probe 1 and detection probe 2 are mixed with an amplification product having complementary base sequences, the quencher and reporter move to the furthest distance from each other, and at this time the signal (fluorescence) value from the reporter is maximized. The amplification product can be detected by measuring this signal.

[0089] The partial primer 1 has a base sequence length of only 8 that is complementary to the base sequence of the target site in the KRAS gene, making it difficult to hybridize to the target site by itself under polymerase chain reaction (PCR) conditions. However, if a guide probe 1 that hybridizes is present near the target site, a portion of the base sequence of the guide probe 1 and a portion of the base sequence of the partial primer 1 hybridize, allowing for easy hybridization to the target site.

[0090] The partial primer 1 has a hybridizable base sequence length of only 8, which is shorter than that of a typical primer with 18 to 30 base sequences that can be hybridized with the target nucleic acid. Therefore, if there is one or more mismatched base sequences between the partial primer 1 and the target nucleic acid hybridization site, these mismatched base sequences will have a greater impact on hybridization efficiency than a typical primer, and as a result, subtle changes in the base sequence can be detected better than with a typical primer.

[0091] Furthermore, if one or more non-complementary sequences exist between the partial primer 1 and the codon 13 and neighboring regions of the wild-type KRAS gene without the G13D mutation, resulting in low hybridization efficiency, the clamping probe 1 will hybridize with the codon 13 and neighboring regions of the wild-type KRAS gene, further inhibiting hybridization of the partial primer 1. This allows for the detection of subtle changes in the base sequence with even higher sensitivity than when the clamping probe is not used.

[0092] The partial primer 2 has a base sequence length of only 9 that is complementary to the base sequence of the target site in the KRAS gene, making it difficult to hybridize to the target site by itself under polymerase chain reaction (PCR) conditions. However, if a guide probe 2 that hybridizes is present near the target site, a portion of the base sequence of the guide probe 2 and a portion of the base sequence of the partial primer 2 hybridize, allowing for easy hybridization to the target site.

[0093] The 3' end of the partial primer 2 hybridizes with a portion of the exon 6 base sequence of the KRAS gene, and this can occur regardless of the presence or absence of the G13D mutation in the KRAS gene. Therefore, considering this characteristic, amplification using partial primer 2 may be used as a control.

[0094] In other words, if the G13D mutation in the KRAS gene is not present in the sample, amplification by partial primer 1 (Target amplification) is suppressed, while amplification by partial primer 2 (Control amplification) is performed smoothly, resulting in a large difference (ΔCt) between the calculated Ct values ​​of the two amplification curves. On the other hand, if the G13D mutation in the KRAS gene is present in the sample, amplification is performed smoothly by both partial primer 1 and partial primer 2, resulting in a small difference between the Ct values ​​calculated through the two amplification curves. This allows for easy determination of the presence or absence of the G13D mutation.

[0095] 2.2 Generation and Confirmation of Amplification Products First, to confirm the detection limit of G13D mutations under conditions where clamping probes are not injected, conditions were set up with approximately 10,000 wild-type KRAS gene target nucleic acids present, or a mixture of approximately 10,000 wild-type KRAS gene target nucleic acids and approximately 1,000 KRAS gene target nucleic acids with G13D mutations (10%), or a mixture of approximately 10,000 wild-type KRAS gene target nucleic acids and approximately 500 KRAS gene target nucleic acids with G13D mutations (5%), or approximately 10,000 wild-type KRAS gene target nucleic acids and approximately 100 G A polymerase chain reaction composition was prepared containing a 10 pmole guide probe 1 and a 15 pmole guide probe 2, a 10 pmole partial primer 1, a 4 pmole partial primer 2, a 4 pmole specific primer 1, a 2 pmole specific primer 2, a 3 pmole detection probe 1, and a 3 pmole detection probe 2, which can form the intended amplification product under conditions of a mixture of KRAS gene target nucleic acids with 13D mutations (1%), a mixture of approximately 10,000 wild-type KRAS gene target nucleic acids and approximately 50 KRAS gene target nucleic acids with G13D mutations (0.5%), a mixture of approximately 10,000 wild-type KRAS gene target nucleic acids and approximately 25 KRAS gene target nucleic acids with G13D mutations (0.25%), or a mixture of approximately 10,000 wild-type KRAS gene target nucleic acids and approximately 10 KRAS gene target nucleic acids with G13D mutations (0.1%).

[0096] The aforementioned polymerase chain reaction composition typically contained, along with the nucleic acid polymerase (DNA polymerase) used in polymerase chain reactions, elements such as a buffer, deoxyribonucleotide-5-triphosphate (dNTP), potassium chloride (KCl), magnesium chloride (MgCl2), and a surfactant (detergent).

[0097] Temperature control for the polymerization enzyme chain reaction was performed using a standard thermal cycler. After initial denaturation [95°C, 5 min], the first step of the 15-cycle reaction consisted of denaturation [95°C, 20 sec] - annealing [63°C, 20 sec] - extension [72°C, 20 sec], followed by the second step of the 35-cycle reaction: denaturation [95°C, 10 sec] - measure [50°C, 10 sec] - denaturation [95°C, 20 sec] - annealing [58°C, 20 sec] - extension [72°C, 20 sec]. The fluorescence values ​​of the FAM and HEX channels were measured at each cycle during the measure step of the second step to derive the amplification curve.

[0098] Subsequently, the Ct value was calculated from the amplification curve derived from the FAM channel and referred to as "Target site amplification Ct," and the Ct value was calculated from the amplification curve derived from the HEX channel and referred to as "Control site amplification Ct." When the ΔCt value was obtained by subtracting the Target site amplification Ct from the Control site amplification Ct, a clear difference in ΔCt value was observed between the mutation injection condition and the condition in which only wild-type was injected under conditions in which 0.5-10% of G13D mutations were injected. Under conditions in which 0.1-0.25% of G13D mutations were injected, a difference in ΔCt value similar to that of the wild-type-only injection condition appeared in some reactions, making distinction difficult. Therefore, the detection limit under conditions in which the clamping probe was not injected was confirmed to be at the 0.5% level.

[0099] On the other hand, in order to allow for further experiments under conditions in which clamping probes were injected and to compare with the detection results of G13D, conditions were set up such as: conditions in which approximately 10,000 wild-type KRAS gene target nucleic acids were present, or conditions in which approximately 10,000 wild-type KRAS gene target nucleic acids and approximately 1,000 KRAS gene target nucleic acids with G13D mutations were mixed (10%), or conditions in which approximately 10,000 wild-type KRAS gene target nucleic acids and approximately 500 KRAS gene target nucleic acids with G13D mutations were mixed (5%), or conditions in which approximately 10,000 wild-type KRAS gene target nucleic acids and approximately 100 The intended amplification product can be formed under conditions where the KRAS gene target nucleic acid containing the G13D mutation is mixed (1%), or under conditions where approximately 10,000 wild-type KRAS gene target nucleic acids are mixed with approximately 50 KRAS gene target nucleic acids containing the G13D mutation (0.5%), or under conditions where approximately 10,000 wild-type KRAS gene target nucleic acids are mixed with approximately 25 KRAS gene target nucleic acids containing the G13D mutation (0.25%), or under conditions where approximately 10,000 wild-type KRAS gene target nucleic acids are mixed with approximately 10 KRAS gene target nucleic acids containing the G13D mutation (0.1%). A polymerization enzyme chain reaction composition was prepared containing pmole guide probe 1, 15 pmole guide probe 2, 5 pmole clamping probe 1, 10 pmole partial primer 1, 4 pmole partial primer 2, 4 pmole specific primer 1, 2 pmole specific primer 2, 3 pmole detection probe 1, and 3 pmole detection probe 2.

[0100] The aforementioned polymerase chain reaction composition typically contains a nucleic acid polymerase (DNA polymerase) used in polymerase chain reactions, along with elements such as a buffer, deoxyribonucleotide-5-triphosphate (dNTP), potassium chloride (KCl), magnesium chloride (MgCl2), and a surfactant (detergent).

[0101] Temperature control for the polymerization enzyme chain reaction was performed using a standard thermal cycler. After initial denaturation [95°C, 5 min], the first step of 15 cycles consisted of denaturation [95°C, 20 sec] - annealing [63°C, 20 sec] - extension [72°C, 20 sec], followed by the second step of 35 cycles: denaturation [95°C, 10 sec] - measure [50°C, 10 sec] - denaturation [95°C, 20 sec] - annealing [58°C, 20 sec] - extension [72°C, 20 sec]. The fluorescence values ​​of the FAM and HEX channels were measured at each cycle during the measure step of the second step to derive the amplification curve.

[0102] Subsequently, the Ct value was calculated from the amplification curve derived from the FAM channel and referred to as the "Target site amplification Ct," and the Ct value was calculated from the amplification curve derived from the HEX channel and referred to as the "Control site amplification Ct." If no increase in the amplification curve occurred and the Ct value could not be calculated, the Ct value was arbitrarily set to 35, referring to the number of cycles in the second step of temperature control for the polymerization enzyme chain reaction.

[0103] By subtracting the Target site amplification Ct from the Control site amplification Ct to determine the ΔCt value, a clear difference in ΔCt values ​​was confirmed between the condition in which the G13D mutation was injected and the condition in which only the wild type was injected. Specifically, while the ΔCt value was -10 or higher in all conditions in which the G13D mutation was injected (0.1-10%), the ΔCt value was less than -20 when only the wild type was injected, creating a clear distinction. Therefore, the detection limit in the condition in which the clamping probe was injected was confirmed to be at a level of 0.1% or less.

[0104] Based on these results, a high level of sensitivity was confirmed in the targeted mutant nucleic acid detection method using a polymerase chain reaction composition containing a guide probe, clamping probe, partial primer, and specific primer, enabling detection even at a low concentration of 0.1% or an extremely small amount corresponding to approximately 10 copies per reaction of the target nucleic acid. In particular, it was confirmed that the injection of the clamping probe more effectively suppressed the amplification of non-target wild-type nucleic acids, thereby improving sensitivity (Figures 5 and 6).

[0105] Although specific parts of the present invention have been described in detail above, it is clear to those with ordinary skill in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents. [Industrial applicability]

[0106] The method for amplifying target nucleic acids according to the present invention has the advantage of enabling amplification of target nucleic acids even at very low concentrations, as it uses a guide probe to bind to all hybridizable nucleic acids present in the sample while assisting the binding of the partial primer to the detection site of the target nucleic acid. Furthermore, due to the sequence specificity of the partial primer, nucleic acids other than the target nucleic acid will have different amplification rates, and the clamping probe prevents the partial primer from binding to nucleic acids other than the target nucleic acid, allowing for detection of the target nucleic acid with high specificity. Therefore, it is useful for molecular diagnosis, prenatal diagnosis, early diagnosis, cancer diagnosis, genetic diagnosis, genotypic diagnosis, diagnosis of infectious bacteria, identification of drug-resistant bacteria, forensic medicine, and species identification of organisms.

Claims

1. Methods for amplifying target nucleic acids, including mutations, which include the following steps: (a) i) A guide probe comprising a sequence that can be hybridized with a site other than the target nucleic acid detection site, which includes a mutation site of the target nucleic acid; a sequence that does not hybridize with the target nucleic acid; and a linker between the sequence that can hybridize with the site other than the target nucleic acid detection site and the sequence that does not hybridize with the target nucleic acid; ii) A partial primer comprising a sequence complementary to the target nucleic acid sequence of the guide probe that does not hybridize with the target nucleic acid, and a sequence complementary to the detection site of the target nucleic acid including the mutation site; iii) A clamping probe containing a sequence complementary to the wild-type sequence corresponding to the mutant sequence of the target nucleic acid, which binds to a non-target nucleic acid and suppresses the amplification of other nucleic acids excluding the target nucleic acid; and iv) A specific primer that, in conjunction with the aforementioned partial primer, can amplify the target nucleic acid; A step of amplifying the isolated nucleic acid by performing a polymerization enzyme chain reaction (PCR) in the presence of; and (b) A step to determine whether or not an amplification product is present; Here, the partial primer further includes a spacer, which is a single-stranded oligonucleotide with a base sequence length of 1 to 100, between a sequence complementary to the sequence that does not hybridize with the target nucleic acid of the guide probe and a sequence complementary to the target nucleic acid detection site. The area other than the target nucleic acid detection site in i) above is not adjacent to the target nucleic acid detection site.

2. The method for amplifying a target nucleic acid according to claim 1, characterized in that the sequence that hybridizes with the target nucleic acid using the guide probe is located at the 5' end or N-terminus, and the sequence that does not hybridize with the target nucleic acid is located at the 3' end or C-terminus.

3. The method for amplifying a target nucleic acid according to claim 1, characterized in that the linker is one or more selected from the group consisting of β-alanine (β-Ala-OH), aminobutyric acid, aminohexanoic acid, aminolauric acid, acetoacetoxyethyl acrylate, and aminoethoxyethoxyethoxyacetic acid (2-[2-[2-[2-(amino)ethoxy]ethoxy]ethoxy]acetic acid).

4. The method for amplifying a target nucleic acid according to claim 1, characterized in that the guide probe, partial primer, clamping probe, and specific primer consist of one or more combinations of oligonucleotides, locked nucleic acid (LNA), and peptide nucleic acid (PNA).

5. The method for amplifying a target nucleic acid according to item 4, characterized in that the guide probe is a PNA prepared with a length of 10 to 500 base sequences.

6. The method for amplifying a target nucleic acid according to claim 5, characterized in that the guide probe is a PNA prepared with a length of 20 to 150 base sequences.

7. The method for amplifying a target nucleic acid according to claim 1, characterized in that the guide probe and clamping probe are hybridized to a strand complementary to the target nucleic acid to which the specific primer binds.

8. The method for amplifying a target nucleic acid according to claim 1, characterized in that the sequence complementary to the detection site of the target nucleic acid of the partial primer is a sequence of 3 to 15 bases.

9. The method for amplifying a target nucleic acid according to claim 1, characterized in that the clamping probe is a PNA prepared with a length of 5 to 500 base sequences.

10. The method for amplifying a target nucleic acid according to claim 1, characterized in that the clamping probe prevents the partial primer from binding to other nucleic acids excluding the target nucleic acid, thereby suppressing the amplification of other nucleic acids excluding the target nucleic acid.

11. The method for amplifying a target nucleic acid according to claim 1, characterized in that the length of the amplified product is 50 bp to 1 kbp.

12. The method for amplifying a target nucleic acid according to claim 1, characterized in that the step of determining the presence or absence of the amplification product in step (b) above is performed by using a nucleic acid-binding dye or probe that can bind to the amplification product.

13. The method for amplifying a target nucleic acid according to claim 12, characterized in that the probe that can bind to the amplification product is selected from the group consisting of one or more combinations of oligonucleotides, LNAs, and PNAs.

14. The method for amplifying a target nucleic acid according to claim 13, characterized in that the probe, which can bind to the amplification product, has a reporter and a quencher attached to both ends.

15. The method for amplifying a target nucleic acid according to claim 14, characterized in that the reporter is one or more fluorescent substances selected from the group consisting of fluorescein, fluorescein chlorotriazinyl, tetramethylrhodamine, FITC, cyanine dyes, and thiadicarbocyanine dyes.

16. The method for amplifying a target nucleic acid according to claim 1, characterized in that the isolated nucleic acid was separated from a specimen sample.

17. i) A guide probe comprising a sequence that can hybridize with a site other than the detection site of the target nucleic acid, which includes a mutation site of the target nucleic acid; a sequence that does not hybridize with the target nucleic acid; and a linker between the sequence that can hybridize with a site other than the detection site of the target nucleic acid and the sequence that does not hybridize with the target nucleic acid; ii) A partial primer comprising a sequence complementary to the target nucleic acid sequence of the guide probe that does not hybridize with the target nucleic acid, and a sequence complementary to the detection site of the target nucleic acid including the mutation site; iii) A clamping probe containing a sequence complementary to the wild-type sequence corresponding to the mutant sequence of the target nucleic acid, which binds to a non-target nucleic acid and suppresses the amplification of other nucleic acids excluding the target nucleic acid; and iv) A specific primer that, in conjunction with the aforementioned partial primer, can amplify the target nucleic acid; PCR composition for target nucleic acid amplification containing; Here, the partial primer further includes a spacer, which is a single-stranded oligonucleotide with a base sequence length of 1 to 100, between a sequence complementary to the sequence that does not hybridize with the target nucleic acid of the guide probe and a sequence complementary to the target nucleic acid detection site. The area other than the target nucleic acid detection site in i) above is not adjacent to the target nucleic acid detection site.

Images