Highly specific target nucleic acid amplification method and composition for amplifying target nucleic acid using the same

Abstract

To provide a target nucleic acid amplification method capable of amplifying very low concentrations of a target nucleic acid with high specificity, and to provide a composition for amplifying a target nucleic acid using the same.SOLUTION: Provided are a method for generating a target nucleic acid amplification product with high specificity through a guide probe that binds to the target nucleic acid and a partial primer that can bind to the guide probe and amplify the target nucleic acid, and a polymerase chain reaction composition for carrying out the method. This method is capable of amplifying target nucleic acids present at very low concentrations, and the sequence specificity of the partial primers results in differences in an amplification rate of nucleic acids other than the target nucleic acid, allowing for high specificity detection of the target nucleic acid. Therefore, the method is useful in molecular diagnostics, prenatal diagnostics, early diagnostics, cancer diagnostics, genetic-related diagnostics, genotypic diagnostics, infectious disease diagnostics, drug-resistant bacterial discrimination, forensic medicine, and biological species identification.SELECTED DRAWING: Figure 1

Description

Field of invention

[0001] The present invention relates to a method for amplifying a target nucleic acid, which can amplify a target nucleic acid at a very low concentration 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 when the target nucleic acid is present, using a guide probe that binds to the target nucleic acid and a partial primer that can amplify the target nucleic acid by binding to the guide probe, and a composition for polymerase chain reaction (PCR) for realizing the method. [Background technology]

[0002] The genetic information possessed by all living organisms on Earth is the source of each individual's unique attributes, and this information is recorded in the order in which the bases adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U) are arranged in a substance called nucleic acid (DNA or RNA). Therefore, clarifying and confirming the order of these bases (base sequence) is a process for identifying the attributes of living organisms and understanding their underlying metabolic mechanisms.

[0003] Recently, molecular diagnostics, which detect or confirm specific base sequences of living organisms to determine their existence or their attributes, have been widely used. Representative examples of molecular diagnostics used in medicine include detecting gene mutations related to the development of human cancer and detecting pathogens that cause infectious diseases in humans. Tests to detect harmful microorganisms in food also fall under the category of molecular diagnostics.

[0004] Among the various molecular diagnostic techniques for specifically identifying specific base sequences, most utilize the polymerase chain reaction (PCR) using a DNA polymerase. The PCR is carried out using a composition containing a pair of primers capable of specifically hybridizing to a target nucleic acid containing a specific base sequence and a thermostable nucleic acid polymerase capable of initiating a PCR using the primers as a starting material and the target nucleic acid as a template, and a thermal cycler capable of repeatedly and stepwise heating the composition. Furthermore, molecular diagnostics using the PCR utilizes nucleic acid-binding dyes or probes to detect specific base sequences in the amplified target nucleic acid in real time. This is called real-time PCR.

[0005] In molecular diagnostics, detecting genetic mutations related to human cancer development requires high sensitivity and specificity. The mutations being detected are somatic mutations, which exist in very small amounts mixed with wild-type normal DNA. Furthermore, if the target mutation is not accurately identified, it can result in false positives, as it can be a point mutation with only a small difference in base sequence from the normal gene, or an insertion / deletion or gene fusion mutation with complex base sequence mutations.

[0006] In other words, the important requirements for a tumor-specific mutation detection test are (1) sensitivity to detect mutant DNA that exists in low proportions in normal DNA, and (2) specificity to minimize the rate of false positives, in which normal DNA is mistakenly identified as mutant DNA.

[0007] Various testing methods for detecting tumor-specific mutations have been developed and are in use, including direct sequencing, allele-specific PCR (AS-PCR), multiple restriction fragment length polymorphism (RFLP), TaqMan probe, and amplification refractory mutation system (ARMS) PCR. However, these methods have not demonstrated satisfactory results in terms of sensitivity and specificity. Direct sequencing has the highest specificity and lowest false-positive rate, but it has the disadvantage of only being able to detect mutations when 20–30% or more of the DNA is present. On the other hand, AS-PCR, RFLP, and TaqMan probe methods have high sensitivity but low specificity, and are constantly plagued by false-positives.

[0008] Recently, methods with significantly improved sensitivity and specificity have been developed, such as the peptide nucleic acid (PNA)-mediated PCR clamp method (Sun, X., et al., 2002. Nat. Biotechnol., 20: 186-189), the locked nucleic acid (LNA)-mediated PCR clamp method (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). The ability to perform highly specific base sequence analysis is gradually being emphasized.

[0009] ARMS-PCR was developed to address the shortcoming of AS-PCR, which is its low specificity. Like AS-PCR, ARMS-PCR is based on the principle that PCR will not occur unless the primers are perfectly complementary to the template sequence (Newton, C.R., et al., 1989. Nucl Acids Res., 17; 2503-2516). However, it has the disadvantage that experimental trial and error is required to determine the optimal primers with excellent specificity (Drenkard, E., et al., 2000. Plant Physiol., 124: 1483-1492).

[0010] Therefore, the present inventors have made extensive efforts to develop a method for dramatically increasing the specificity of amplification of target nucleic acids present at very low concentrations. As a result, they have confirmed that target nucleic acids present at very low concentrations can be detected with high specificity when PCR is performed using a guide probe capable of hybridizing with a site other than the detection site of the target nucleic acid, a partial primer including a sequence that binds to a site other than the hybridization site of the target nucleic acid of the guide probe and a sequence that binds to the detection site of the target nucleic acid, and a specific primer that can amplify the target nucleic acid by pairing with the partial primer, thereby generating an amplification product, thereby completing the present invention.

[0011] The information provided in this Background section is intended solely to provide a better understanding of the background of the present invention and may not include information that constitutes prior art already known to a person of ordinary skill in the art to which the present invention pertains. Summary of the Invention

[0012] An object of the present invention is to provide a method for amplifying target nucleic acids at very low concentrations with high specificity.

[0013] Another object of the present invention is to provide a polymerase chain reaction (PCR) composition for amplifying target nucleic acids that is capable of amplifying very low concentrations of target nucleic acids with high specificity.

[0014] In order to achieve the above object, the present invention provides: (a) a guide probe capable of hybridizing a nucleic acid separated from a specimen sample with i) a site other than the detection site of a target nucleic acid; ii) a partial primer including a sequence that binds to a site other than the target nucleic acid hybridization site of the guide probe and a sequence that binds to the target nucleic acid detection site; and iii) mixing the partial primer with a specific primer capable of amplifying the target nucleic acid by pairing with the partial primer, and performing a polymerase chain reaction (PCR); (b) determining the presence or absence of an amplification product.

[0015] The present invention also provides a PCR composition for amplifying a target nucleic acid, comprising: i) a guide probe capable of hybridizing to a site other than the detection site of the target nucleic acid; ii) a partial primer comprising a sequence that binds to a site other than the target nucleic acid hybridization site of the guide probe and a sequence that binds to the target nucleic acid detection site; and iii) a specific primer that can pair with the partial primer to amplify the target nucleic acid. [Brief explanation of the drawings]

[0016] FIG. 1 shows the components necessary for one embodiment of the present invention, namely, a guide probe, a partial primer, and a specific primer, and the hybridization relationship between the components.

[0017] FIG. 2 shows the principle of maximizing the difference in amplification efficiency due to the difference in base sequence between target nucleic acid and non-target nucleic acid through the present invention. (a) shows that the length of the portion of the partial primer of the present invention that hybridizes with the target nucleic acid is short, so the difference in hybridization efficiency due to mismatch in base sequence is large, thereby maximizing the difference in amplification efficiency between normal DNA and mutant DNA. (b) shows that the length of the portion of the normal primer that hybridizes with the target nucleic acid is long, so the difference in hybridization efficiency due to mismatch in base sequence is small, making it difficult to distinguish between normal DNA and mutant DNA.

[0018] FIG. 3 shows the process of deriving the difference in amplification efficiency due to the difference in base sequence between target nucleic acid and non-target nucleic acid according to the present invention. (a) shows that when a target nucleic acid is present, a guide probe hybridizes to a specific site of the target nucleic acid, and a partial primer hybridized with a part of the guide probe approaches the target amplification site of the target nucleic acid. Since 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, hybridization and extension amplification occur. At this time, the guide probe and target nucleic acid and the guide probe and partial primer (b) indicates that when a non-target nucleic acid having a base sequence different from that of the target nucleic acid is present, the guide probe hybridizes to a specific site of the non-target nucleic acid, and even if the partial primer hybridized to a portion of the guide probe is close to the non-target nucleic acid, the complementarity between a portion of the 3'-end portion of the partial primer and the base sequence of the non-target nucleic acid is low, thereby suppressing hybridization and extension amplification.

[0019] FIG. 4 is a conceptual diagram showing a method for detecting a specific mutant and distinguishing it from the wild type according to one embodiment of the present invention.

[0020] FIG. 5 shows the results of detecting the V600E mutation in the BRAF gene in a human genome with high specificity according to one embodiment of the present invention. When partial primers designed to hybridize to the genotype of the V600E mutation at a portion of the 3' end were used, high amplification efficiency was observed and a relatively low Ct value was calculated when the V600E mutation was present. On the other hand, low amplification efficiency was observed and a relatively high Ct value was calculated when the wild-type and non-targeted mutants (V600A, V600D, V600G, V600K, V600M, V600R, K601E) were present. As a result, the presence or absence of the V600E mutation can be determined by comparing the Ct values.

[0021] Figure 6 shows the results of highly sensitive detection of the G12C mutation in the KRAS gene in a human gene according to one embodiment of the present invention. When using partial primers designed so that a portion of the 3' end hybridizes with the genotype of the G12C mutation, when a very small amount (1% and 0.5%) of the G12C mutation is present, high amplification efficiency is observed and a relatively low Ct value is calculated. However, when only the wild type is present, no amplification curve is formed. As a result, the presence or absence of the G12C mutation can be determined by comparing the Ct values.

[0022] FIG. 7 shows the results of detecting the D1002N, D1002G, Y1003F, Y1003N, Y1003*(1), and Y1003*(2) mutations in the MET gene of a human genome in a single reaction solution according to one embodiment of the present invention. Six partial primers were prepared and mixed so that portions of the 3' end hybridized with the D1002N, D1002G, Y1003F, Y1003N, Y1003*(1), and Y1003*(2) mutation genotypes. When the target mutation was present, high amplification efficiency was observed and a relatively low Ct value was calculated. However, when only the wild type was present, low amplification efficiency was observed and a relatively high Ct value was calculated. As a result, the presence or absence of the target mutation can be determined by comparing the Ct values.

[0023] FIG. 8 shows an example of a linker contained in a guide probe of the present invention.

[0024] 9 shows the results of measuring the detection limit for detecting the G12C mutation of the KRAS gene in the human genome according to one embodiment of the present invention, demonstrating that the method has very high sensitivity and excellent specificity, even when the target mutant nucleic acid is present at only about 0.5% (20 copies) per reaction.

[0025] 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. Generally, the nomenclature used herein is well known and commonly used in the art.

[0026] In the present invention, we attempted to confirm whether very low concentrations of target nucleic acid can be detected using a guide probe and partial primers.

[0027] That is, in one embodiment of the present invention, in order to specifically amplify and detect the V600E mutation, which is a GTG>GAG base sequence mutation located in codon 600 of exon 15 of the human BRAF gene, a guide probe capable of specifically hybridizing to a region surrounding codon 600 of the BRAF gene, a partial primer capable of specifically hybridizing a portion of its 3' end to codon 600 and a surrounding region of the BRAF gene where the V600E mutation occurs, and a specific primer capable of hybridizing to a portion of the base sequence of exon 15 of the BRAF gene so as to pair with the partial primer and amplify the target nucleic acid were designed, wherein a portion of the base sequence at the C-terminus of the guide probe and a portion of the base sequence at the 5' end of the partial primer were hybridized.

[0028] PCR was performed using the guide probe, partial primers and specific primers to determine whether the V600E gene could be detected in the presence of wild-type nucleic acid and various mutations at the V600 position.

[0029] As a result, an increase in the fluorescent signal on the amplification curve was observed under all target nucleic acid injection conditions, but it was confirmed that the increase in the fluorescent signal on the amplification curve occurred more rapidly under the condition in which the V600E mutant target nucleic acid was mixed compared to the other conditions, and a lower Ct value was calculated (Figure 5).

[0030] Therefore, in one aspect, the present invention provides: (a) a guide probe capable of hybridizing a nucleic acid separated from a specimen sample with i) a site other than the detection site of a target nucleic acid; ii) a partial primer including a sequence that binds to a site other than the target nucleic acid hybridization site of the guide probe and a sequence that binds to the target nucleic acid detection site; and iii) mixing the partial primer with a specific primer capable of amplifying the target nucleic acid by pairing with the partial primer, and performing a polymerase chain reaction (PCR); (b) a method for amplifying a target nucleic acid, comprising determining the presence or absence of an amplification product;

[0031] As used herein, the term "target nucleic acid" refers to any type of nucleic acid containing a sequence of interest to be amplified or detected. The target nucleic acid includes gene sequences from multiple species, subspecies, or variants, or genetic variations within the same species. This includes, 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 (a portion of the 3' end), or a specific primer under conditions for polymerase chain reaction.

[0032] In the present invention, the term "hybridization" refers to the formation of double-stranded nucleic acids by hydrogen bonding between single-stranded nucleic acids having complementary base sequences, and is used in the same sense as annealing. However, in a broader sense, hybridization includes not only cases where the base sequences between two single-stranded nucleic acids are completely complementary (perfect match), but also exceptional cases where some base sequences are not complementary (mismatch).

[0033] In the present invention, the term "guide probe" refers to a probe that can be hybridized simultaneously with a target nucleic acid and a partial primer, allowing the 3' end of the partial primer to hybridize with the target base sequence of the target nucleic acid. The guide probe can be prepared from any one or a mixture of two or more materials that can hybridize with the target nucleic acid (e.g., DNA, RNA, LNA (locked nucleic acid), PNA (peptide nucleic acid), etc.).

[0034] In the present invention, the term "partial primer" is characterized in that a part of the 5'-end region is hybridized with a guide probe, and the 3'-end region is hybridized with a target base sequence of a target nucleic acid, and synthetic extension is performed by nucleic acid polymerase.

[0035] 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 and guide probe, guide probe and partial primer, partial primer and target nucleic acid). While the guide probe binds to the periphery of the target base sequence of the target nucleic acid, the hybridized partial primer is positioned at the periphery of the target base sequence, so that the 3' end of the partial primer is easily hybridized with the target base sequence, initiating synthetic extension by nucleic acid polymerase and amplification of the target site of the target nucleic acid.

[0036] The hybridization between the target nucleic acid and the guide probe, and between the guide probe and the partial primer, occurs first because the complementary portions are longer than those between the partial primer and the target nucleic acid, resulting in a higher Tm, while the hybridization between the partial primer and the target nucleic acid occurs later because the Tm is relatively low.

[0037] In the present invention, the term "specific primer" is characterized in that it hybridizes to a target nucleic acid without the aid of the guide probe and undergoes synthetic extension by a nucleic acid polymerase.

[0038] In the present invention, the site of the guide probe that hybridizes with the target nucleic acid is located at the 5'-end or N-end, and the site that hybridizes with the partial primer is located at the 3'-end or C-end.

[0039] In the present invention, the guide probe further comprises a linker between the target nucleic acid hybridization site and the partial primer hybridization site.

[0040] In the present invention, the linker may be any compound that is present between the target nucleic acid hybridization site and the partial primer hybridization site and does not contain a nucleic acid base linking the two sites, and is preferably at least one selected from the group consisting of beta-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 thereto.

[0041] In the present invention, the guide probe, partial primer, and specific primer are characterized by being composed of any one of oligonucleotide, LNA (locked nucleic acid), and PNA (peptide nucleic acid), or a mixture thereof.

[0042] In the present invention, the guide probe can be any nucleic acid that can hybridize with the target nucleic acid and the partial primer, but is preferably a PNA having a base sequence length of 10 to 500, more preferably a PNA having a base sequence length of 20 to 150.

[0043] In the present invention, the guide probe is characterized in that it hybridizes to the opposite strand of the target nucleic acid to which the specific primer hybridizes.

[0044] In the present invention, the partial primer further comprises a spacer, which is a single-stranded oligonucleotide having a length of 1 to 100 bases, between the sequence that binds to a site other than the target nucleic acid hybridization site of the guide probe and the sequence that binds to the target nucleic acid detection site.

[0045] In the present invention, the spacer refers to a sequence excluding the sequence contained in the partial primer that hybridizes to the guide probe and the sequence that hybridizes to the target nucleic acid, and if necessary, a probe that can bind to the spacer can be used to detect the target nucleic acid with the same probe even if the base sequence of the target nucleic acid is changed.

[0046] In the present invention, the spacer and the linker of the guide probe can play a role in adjusting the efficiency of hybridization of the partial primer located around the target site of the target nucleic acid to the target site by the guide probe. This means that when the guide probe hybridizes to the target nucleic acid at a position slightly distant from the target site, increasing the length of the spacer and the linker can help the partial primer to hybridize to the target site.

[0047] In the present invention, the sequence of the partial primer that binds to the target nucleic acid detection site is a 3 to 15 base sequence.

[0048] In the present invention, the length of the amplification product is 50 bp to 1 kbp.

[0049] In the present invention, the step (b) of determining the presence or absence of the amplification product is characterized by using a nucleic acids-binding dye or a probe capable of binding to the amplification product.

[0050] In the present invention, the nucleic acid binding dye may be any intercalating substance or DNA minor groove binding substance without limitation, but is preferably ethidium bromide, and is selected from the group consisting of SYBR® Green I, SYBR® Gold, EvaGreen, YO-PRO-1, SYTO, BEBO, and BEXTO.

[0051] In the present invention, the probe capable of binding to the amplification product is selected from the group consisting of oligonucleotides, LNA, PNA, and mixtures thereof.

[0052] PNA (peptide nucleic acid) is a DNA-like molecule in which nucleic acid bases are linked to a peptide backbone rather than a sugar-phosphate backbone, and was first synthesized by Nielsen et al. in 1991. PNA is one of the artificially synthesized gene recognition materials, such as LNA (locked nucleic acid) or MNA (mopholino nucleic acid), and its basic backbone is composed of polyamide.

[0053] PNA has excellent affinity and selectivity, is highly stable against nucleases, is not degraded by existing restriction enzymes, and has the advantage of being easily stored for a long period of time due to its high thermal and chemical stability.

[0054] PNA forms a double strand through hybridization with natural nucleic acids of complementary base sequence. When the lengths are the same, a PNA / DNA duplex is more stable than a DNA / DNA duplex, and a PNA / RNA duplex is more stable than a DNA / RNA duplex. Furthermore, PNA has a superior ability to detect single nucleotide polymorphisms (SNPs) compared to natural nucleic acids because the duplex is more unstable due to single base mismatches.

[0055] In other words, the PNA-DNA binding strength is much stronger than the DNA-DNA binding strength, and even a single nucleotide mismatch results in a difference in melting temperature (Tm) of approximately 15-20°C. By utilizing this difference in binding strength, it is possible to detect base sequence changes such as SNPs (single-nucleotide polymorphisms) and In / Del (insertion / deletion).

[0056] In the present invention, the probe capable of binding to the amplification product is characterized by having a base sequence that is partially or entirely complementary to any base sequence in the amplification product and the target nucleic acid base sequence, and is preferably characterized by having a reporter and a quencher attached to both ends.

[0057] In the present invention, the probe suppresses signal generation when the distance between the reporter and the quencher is short, and the signal intensity increases as the distance between the reporter and the quencher increases. Generally, when the probe hybridizes with a complementary base sequence, the distance between the reporter and the quencher is the greatest, and a specific base sequence can be detected through signal generation or an increase in signal intensity.

[0058] In the present invention, the reporter is 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.

[0059] In the present invention, the quencher is 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.

[0060] In a preferred embodiment of the present invention, the detection of the amplification product by the nucleic acid polymerase is performed through a real-time polymerase chain reaction (real-time PCR), and in this case, the amplification product can be detected by obtaining an amplification curve based on the increase in the amplification product and measuring the cycle threshold (Ct) value, but is not limited to this. In the method using the Ct value, the faster the amplification product is generated and increased due to the presence of a target base sequence in the target nucleic acid in the sample, the faster the amount of signal generated by the detection probe increases, the fewer cycles are required to reach the threshold, and the lower the Ct value is measured.

[0061] In another aspect, the present invention relates to a PCR composition for amplifying a target nucleic acid, comprising: i) a guide probe capable of hybridizing with a site other than the detection site of a target nucleic acid; ii) a partial primer comprising a sequence that binds to a site other than the target nucleic acid hybridization site of the guide probe and a sequence that binds to the target nucleic acid detection site; and iii) a specific primer that can pair with the partial primer to amplify the target nucleic acid.

[0062] The term "sample" as used herein encompasses a variety of samples, and preferably, biological samples are analyzed using the methods of the present invention. More preferably, the sample may be mixed with a virus species or may be a sample from an individual (e.g., human, mammal, fish, etc.) infected with the virus. Biological samples of plant, animal, human, fungal, bacterial, and viral origin can be analyzed. When analyzing samples from mammals or humans, the sample is derived from a specific tissue or organ. Representative examples of tissues include connective, skin, muscle, or nervous tissue. Representative 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 other media from a biological source that can be analyzed by the present invention, including samples obtained from humans, animals, or foods manufactured for human or animal consumption. Additionally, biological samples that may be analyzed include bodily fluid samples, including, but not limited to, blood, serum, plasma, lymph, breast milk, urine, feces, ocular fluid, saliva, semen, brain extracts (e.g., brain homogenates), spinal fluid, appendix, spleen, and tonsil tissue extracts.

[0063] From another aspect, the present invention relates to a kit for detecting a target nucleic acid, which comprises the composition.

[0064] In the present invention, the kit may optionally contain reagents necessary for carrying out a target nucleic acid amplification reaction (e.g., polymerase chain reaction), such as a buffer, DNA polymerase, a DNA polymerase cofactor, and deoxyribonucleotide-5-tripolyphosphate (dNTP). Optionally, the kit may also contain various oligonucleotide molecules, reverse transcriptase, various buffers and reagents, and an antibody that inhibits DNA polymerase activity. Furthermore, the optimal amounts of reagents to be used in a particular reaction of the kit may be readily determined by one of ordinary skill in the art after learning the disclosure herein. Typically, the device of the present invention may be fabricated with separate packages or compartments containing the aforementioned components.

[0065] In one embodiment, the kit may comprise a compartmentalized carrier means for holding a sample, a container containing reagents, a container containing guide probes and primers, and a container containing probes for detecting the amplification products.

[0066] The carrier means is suitable for containing one or more containers such as bottles, tubes, etc., each containing an individual component used in the method of the present invention. With the present specification, a person skilled in the art can easily dispense the required formulations in the containers. [Example]

[0067] The present invention will be described in more detail below with reference to examples. These examples are provided solely for the purpose of more specifically illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention.

[0068] Example 1 Amplification and detection of the BRAF gene in which the V600E mutation has occurred in the human gene 1.1 Preparation of primers and probes The human BRAF gene encodes a growth signal transduction protein kinase that functions in the body's signal transduction system, and specific mutations in the BRAF gene are known to be associated with cancer development (Zaman, A. et al., 2019. Cancers 11:1197.). The GTG>GAG mutation located in exon 15, codon 600 of the human BRAF gene is called the V600E mutation.

[0069] To specifically amplify and detect the V600E mutation in the BRAF gene using the novel target nucleic acid amplification method of the present invention, we designed a guide probe 1 that specifically hybridizes to a region surrounding codon 600 of the BRAF gene, a partial primer 1 whose 3' end specifically hybridizes to codon 600 and a surrounding region of the BRAF gene where the V600E mutation occurs, and a specific primer 1 that hybridizes to a portion of the base sequence of exon 15 of the BRAF gene so as to pair with the partial primer 1 and amplify the target nucleic acid, where a partial base sequence at the C-terminus of the guide probe 1 hybridizes to a partial base sequence at the 5' end of the partial primer 1. Additionally, we designed and manufactured a detection probe 1 (PANAGENE Inc., South Korea) to detect the product amplified by the designed guide probe and primers. The base sequences of each primer and probe are shown in Tables 1 and 2.

[0070] [Table 1]

[0071] [Table 2]

[0072] The partial primer 1 was prepared as a single-stranded oligonucleotide having a length of 24 bases, and the underlined base sequence is complementary to a part of the base sequence of guide probe 1, and the italicized base sequence has the characteristics of being complementary to the base sequence of codon 600 and the surrounding region of the BRAF gene where the V600E mutation occurred.

[0073] The guide probe 1 was made of a 25-base PNA, and the italicized base sequence is complementary to the base sequence of the region surrounding codon 600 of the BRAF gene, and the underlined base sequence is complementary to a portion of the base sequence of the partial primer 1. In addition, [K (lysine)] was attached to the N-terminus of the guide probe 1, and an L35 linker (PANAGENE Inc., South Korea) was attached between the region complementary to the BRAF gene sequence and the region complementary to a portion of the partial primer 1 sequence.

[0074] The detection probe 1 was made of a 15-base sequence PNA, with a quencher [Dabcyl] attached to the N-terminus and a reporter [FAM] attached to the C-terminus, and was manufactured so that an [O linker] and [K (lysine)] (manufactured by PANAGENE Inc., South Korea) were connected between the PNA and [FAM]. When the detection probe 1 hybridizes to an amplification product with a complementary base sequence, the quencher and reporter are at their furthest distance from each other, and the signal (fluorescence) from the reporter is maximized. The amplification product can be detected by measuring this signal.

[0075] The partial primer 1 has a base sequence complementary to the base sequence of the target site in the BRAF gene that is only 10 bases long, making it difficult for it to hybridize to the target site alone under polymerase chain reaction (PCR) conditions. However, if a hybridizable guide probe 1 is present near the target site, a portion of the base sequence of the guide probe 1 hybridizes with a portion of the base sequence of the partial primer 1, allowing it to easily hybridize to the target site.

[0076] The partial primer 1 has a base sequence length of only 10 that can hybridize with the target nucleic acid base sequence of the BRAF gene, which is shorter than that of a typical primer, which has a base sequence length of 18 to 30 that can hybridize with the target nucleic acid. Therefore, if one or more non-complementary base sequences exist between partial primer 1 and the target nucleic acid hybridization site, the non-complementary base sequences have a greater effect 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.

[0077] 1.2 Generation and confirmation of amplification products The conditions include a condition in which about 10,000 copies of wild-type BRAF gene target nucleic acids are present, a condition in which about 10,000 copies of wild-type BRAF gene target nucleic acids are mixed with about 500 copies of V600E mutated BRAF gene target nucleic acids, a condition in which about 10,000 copies of wild-type BRAF gene target nucleic acids are mixed with about 500 copies of V600A (codon 600 GTG>GCG) mutated BRAF gene target nucleic acids, a condition in which about 10,000 copies of wild-type BRAF gene target nucleic acids are mixed with about 500 copies of V600D (codon 600 GTG>GAT) mutated BRAF gene target nucleic acids, and a condition in which about 10,000 copies of wild-type BRAF gene target nucleic acids are mixed with about 500 copies of V600G (codon 600 a mixture of about 10,000 wild-type BRAF gene target nucleic acids and about 500 V600K (codon 600 GTG>AAG) mutated BRAF gene target nucleic acids; a mixture of about 10,000 wild-type BRAF gene target nucleic acids and about 500 V600M (codon 600 GTG>ATG) mutated BRAF gene target nucleic acids; a mixture of about 10,000 wild-type BRAF gene target nucleic acids and about 500 V600R (codon 600 GTG>AGG) mutated BRAF gene target nucleic acids; or a mixture of about 10,000 wild-type BRAF gene target nucleic acids and about 500 K601E (codon 601 AAA>GAA) mutated BRAF gene target nucleic acids. A polymerase chain reaction composition was prepared containing pmoles of guide probe 1, 10 pmoles of partial primer 1, 4 pmoles of specific primer 1, and 3 pmoles of detection probe 1.

[0078] The polymerase chain reaction composition typically contains a nucleic acid polymerase (DNA polymerase) used in polymerase chain reaction, as well as other components such as a buffer, deoxyribonucleotide-5-tripolyphosphate (dNTP), potassium chloride (KCl), magnesium chloride (MgCl), and a detergent.

[0079] The temperature for the polymerase chain reaction was controlled using a standard thermal cycler. After initial denaturation (95°C, 5 minutes), 15 cycles of the first step (denaturation (95°C, 20 seconds) - annealing (63°C, 20 seconds) - extension (72°C, 20 seconds)) were performed, followed by 50 cycles of the second step (denaturation (95°C, 20 seconds) - measurement (50°C, 30 seconds) - annealing and extension (60°C, 1 minute). Fluorescence intensity of the FAM channel was measured every cycle during the measurement step of the second step to derive an amplification curve.

[0080] As a result, an increase in the fluorescence signal on the amplification curve was observed under all target nucleic acid injection conditions. However, the increase in the fluorescence signal on the amplification curve occurred more rapidly and a lower Ct value was calculated under the condition where the V600E mutant target nucleic acid was mixed compared to the other conditions. That is, an average Ct value of 22.5 was observed under the condition where the V600E mutant target nucleic acid was mixed, while an average Ct value of 29 or more was observed under the condition where only the wild-type nucleic acid or target nucleic acids with non-target mutations (V600A, V600D, V600G, V600K, V600M, V600R, K601E) were mixed. As a result, the difference in Ct values allowed specific identification of the V600E mutation (Figure 5).

[0081] Example 2 Highly sensitive amplification and detection of the KRAS gene in which the G12C mutation has occurred in the human gene 2.1 Preparation of primers and probes The human KRAS gene encodes a GTP-based switch protein that functions in the body's signal transduction system, and specific mutations in the KRAS gene are said to be associated with cancer development (Waters, AM and Der, CJ 2018. Cold Spring Harb. Perspect. Med.8:a031435.). A mutation in the GGT>TGT base sequence located in exon 2, codon 12 of the human KRAS gene is called a G12C mutation.

[0082] To specifically amplify and detect the G12C mutation in the KRAS gene using the novel target nucleic acid amplification method of the present invention, we designed a guide probe 2 that specifically hybridizes to a region surrounding codon 12 of the KRAS gene, a partial primer 2 that specifically hybridizes at a portion of its 3' end to codon 12 and the surrounding region of the KRAS gene where the G12C mutation occurs, and a specific primer 2 that hybridizes to a portion of the base sequence of exon 2 of the KRAS gene so that it can pair with the partial primer 2 to amplify the target nucleic acid, where a partial base sequence at the C-terminus of guide probe 2 can hybridize with a partial base sequence at the 5' end of partial primer 2. Additionally, we designed and manufactured a detection probe 2 (PANAGENE Inc., South Korea) to detect the product amplified by the designed guide probe and primers. The base sequences of each primer and probe are shown in Tables 3 and 4.

[0083] [Table 3]

[0084] [Table 4]

[0085] The partial primer 2 was prepared as a single-stranded oligonucleotide having a length of 18 bases, and the underlined base sequence is complementary to a part of the base sequence of guide probe 2, and the italicized base sequence has the characteristics of being complementary to the base sequence of codon 12 and the surrounding region of the KRAS gene in which the G12C mutation occurred.

[0086] The guide probe 2 was made of a PNA having a length of 24 bases, and the base sequence shown in italics is complementary to the base sequence of the region surrounding codon 12 of the KRAS gene, and the underlined base sequence is complementary to a portion of the base sequence of the partial primer 2. In addition, [K (lysine)] was attached to the N-terminus of the guide probe 2, and an L35 linker (manufactured by PANAGENE Inc., South Korea) was attached between the region complementary to the KRAS gene sequence and the region complementary to a portion of the partial primer 2 sequence.

[0087] Detection probe 2 was made of a 12-base PNA with a quencher [Dabcyl] attached to the N-terminus and a reporter [FAM] attached to the C-terminus, with an [O linker] and [K (lysine)] (manufactured by PANAGENE Inc., South Korea) attached between the PNA and [FAM]. When detection probe 2 hybridizes to an amplification product with a complementary base sequence, the quencher and reporter are at their furthest distance from each other, maximizing the signal (fluorescence) from the reporter. The amplification product can be detected by measuring this signal.

[0088] The partial primer 2 has a base sequence complementary to the base sequence of the target site in the KRAS gene that is only 8 bases long, making it difficult to hybridize to the target site alone under polymerase chain reaction (PCR) conditions. However, if a hybridizable guide probe 2 is present near the target site, a portion of the base sequence of the guide probe 2 hybridizes with a portion of the base sequence of the partial primer 2, allowing it to easily hybridize to the target site.

[0089] The partial primer 2 has a base sequence length of only 8 that can hybridize with the target nucleic acid base sequence of the KRAS gene, which is shorter than that of a typical primer, which has a base sequence length of 18 to 30 that can hybridize with the target nucleic acid. Therefore, if one or more non-complementary base sequences exist between partial primer 2 and the target nucleic acid hybridization site, the non-complementary base sequences have a greater effect 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.

[0090] 2.2 Generation and confirmation of amplification products A polymerase chain reaction composition containing 20 pmoles of guide probe 2, 10 pmoles of partial primer 2, 4 pmoles of specific primer 2, and 3 pmoles of detection probe 2 was prepared to form the intended amplification product under the conditions of the presence of about 10,000 copies of wild-type KRAS gene target nucleic acid, a mixture of about 10,000 copies of wild-type KRAS gene target nucleic acid and about 100 copies of KRAS gene target nucleic acid having G12C mutation, or a mixture of about 10,000 copies of wild-type KRAS gene target nucleic acid and about 50 copies of KRAS gene target nucleic acid having G12C mutation.

[0091] The polymerase chain reaction composition typically contains a nucleic acid polymerase (DNA polymerase) used in polymerase chain reaction, as well as other components such as a buffer, deoxyribonucleotide-5-tripolyphosphate (dNTP), potassium chloride (KCl), magnesium chloride (MgCl), and a detergent.

[0092] The temperature for the polymerase chain reaction was controlled using a standard thermal cycler. After initial denaturation (95°C, 5 minutes), 15 cycles of the first step (denaturation (95°C, 20 seconds) - annealing (63°C, 20 seconds) - extension (72°C, 20 seconds)) were performed, followed by 35 cycles of the second step (denaturation (95°C, 10 seconds) - measurement (50°C, 10 seconds) - denaturation (95°C, 20 seconds) - annealing (58°C, 20 seconds) - extension (72°C, 20 seconds). Fluorescence intensity of the FAM channel was measured every cycle during the measurement step of the second step to derive an amplification curve.

[0093] As a result, a clear increase in the fluorescent signal on the amplification curve was observed under all conditions in which the G12C-mutated KRAS target nucleic acid was mixed, whereas no increase in the fluorescent signal on the amplification curve was observed or it was very weak under conditions in which only wild-type KRAS target nucleic acid was present. That is, under conditions in which approximately 100 or approximately 50 G12C-mutated target nucleic acids were mixed, an average Ct value of 12.4 and an average Ct value of 13.6 were observed, respectively, whereas under conditions in which only wild-type KRAS was present, no Ct value was calculated (ND; not determined). As a result, the difference in Ct values allowed specific identification of the G12C mutation, which was present in very small amounts (Figure 6).

[0094] Example 3 Amplification and detection of MET gene with D1002N, D1002G, Y1003F, Y1003N, and Y1003* mutations in the human gene 3.1 Preparation of primers and probes The human MET gene, also known as the c-Met gene, encodes the hepatocyte growth factor receptor, which functions in the body's signal transduction pathway. Specific mutations and dysregulation of the MET gene are known to be associated with cancer development (Sierra, JR and Tsao, M.-S. 2011. Ther. Adv. Med. Oncol. 3:S21-S35.). The GAC>AAC mutation in exon 14, codon 1002 of the human MET gene is referred to as the D1002N mutation, the GAC>GGC mutation in exon 14, codon 1002 as the D1002G mutation, the TAC>TTC mutation in exon 14, codon 1003 as the TTC mutation, the Y1003F mutation, the TAC>AAC mutation in exon 14, codon 1003 as the Y1003N mutation, and the TAC>TAG or TAC>TAA mutation in exon 14, codon 1003 as the Y1003*(1) and Y1003*(2) mutations, respectively.

[0095] In order to specifically amplify and detect the D1002N, D1002G, Y1003F, Y1003N, Y1003*(1), and Y1003*(2) mutations of the MET gene using a single reaction solution composition according to the novel method for amplifying a target nucleic acid of the present invention, a guide probe 3 capable of specifically hybridizing to codon 1002 and the surrounding region of codon 1003 of the MET gene, partial primer 3 capable of specifically hybridizing a portion of its 3' end to codon 1002 and the surrounding region of the MET gene in which the D1002N mutation has occurred, partial primer 4 capable of specifically hybridizing a portion of its 3' end to codon 1002 and the surrounding region of the MET gene in which the D1002G mutation has occurred, partial primer 5 capable of specifically hybridizing a portion of its 3' end to codon 1003 and the surrounding region of the MET gene in which the Y1003F mutation has occurred, and partial primer 6 capable of specifically hybridizing a portion of its 3' end to codon 1003 and the surrounding region of the MET gene in which the Y1003N mutation has occurred. Partial primer 6, capable of hybridizing specifically to codon 1003 and its surrounding region at a portion of its 3' end; partial primer 7, capable of hybridizing specifically to codon 1003 and its surrounding region at a portion of its 3' end of the MET gene where the Y1003*(1) mutation occurred; partial primer 8, capable of hybridizing specifically to codon 1003 and its surrounding region at a portion of its 3' end of the MET gene where the Y1003*(2) mutation occurred; and specific primer 3, capable of hybridizing to a portion of the base sequence around exon 14 of the MET gene so as to amplify the target nucleic acid by pairing with partial primers 3, 4, 5, 6, 7, and 8, were designed, whereby a partial base sequence at the C-terminus of guide probe 3 was hybridized to a partial base sequence at the 5' end of partial primers 3, 4, 5, 6, 7, and 8. Detection probe 3 was also designed and manufactured (PANAGENE Inc., South Korea) to detect products amplified through the designed guide probes and primers. The base sequences of each primer and probe are disclosed in Tables 5 and 6.

[0096] [Table 5]

[0097] [Table 6]

[0098] The partial primer 3 was constructed as a single-stranded oligonucleotide with a length of 23 bases. The underlined base sequence is complementary to a portion of the base sequence of guide probe 3, and the italicized base sequence is complementary to the base sequence of codon 1002 and the surrounding region of the MET gene where the D1002N mutation occurred.

[0099] The partial primer 4 was constructed as a single-stranded oligonucleotide having a length of 22 bases. The underlined base sequence is complementary to a portion of the base sequence of guide probe 3, and the italicized base sequence is complementary to the base sequence of codon 1002 and the surrounding region of the MET gene where the D1002G mutation occurred.

[0100] The partial primer 5 was constructed as a single-stranded oligonucleotide having a length of 23 bases. The underlined base sequence is complementary to a part of the base sequence of the guide probe 3, and the italicized base sequence is complementary to the base sequence of codon 1003 and the surrounding region of the MET gene where the Y1003F mutation occurred.

[0101] The partial primer 6 was constructed as a single-stranded oligonucleotide with a length of 23 bases. The underlined base sequence is complementary to a portion of the base sequence of guide probe 3, and the italicized base sequence is complementary to the base sequence of codon 1003 and the surrounding region of the MET gene where the Y1003N mutation occurred.

[0102] The partial primer 7 was constructed as a single-stranded oligonucleotide with a length of 23 bases. The underlined base sequence is complementary to a portion of the base sequence of guide probe 3, and the italicized base sequence is complementary to the base sequence of codon 1003 and the surrounding region of the MET gene where the Y1003*(1) mutation occurred.

[0103] The partial primer 8 was constructed as a single-stranded oligonucleotide with a length of 23 bases. The underlined base sequence is complementary to a portion of the base sequence of guide probe 3, and the italicized base sequence is complementary to the base sequence of codon 1003 and the surrounding region of the MET gene where the Y1003*(2) mutation occurred.

[0104] The guide probe 3 was constructed from a 25-base PNA, and the italicized base sequence is complementary to the base sequence of the region surrounding codon 1002 and codon 1003 of the MET gene, while the underlined base sequence is complementary to a portion of the base sequences of the partial primers 3, 4, 5, 6, 7, and 8. In addition, [K (lysine)] was attached to the N-terminus of the guide probe 3, and an L35 linker (PANAGENE Inc., South Korea) was attached between the region complementary to the MET gene sequence and the region complementary to a portion of the base sequences of the partial primers 3, 4, 5, 6, 7, and 8.

[0105] The detection probe 3 was made of a PNA with an 11-base sequence length, with a quencher [Dabcyl] attached to the N-terminus and a reporter [FAM] attached to the C-terminus, and was manufactured so that an [O linker] and [K (lysine)] (manufactured by PANAGENE Inc., South Korea) were connected between the PNA and [FAM]. When the detection probe 3 hybridizes to an amplification product with a complementary base sequence, the quencher and reporter are at their furthest distance from each other, and at this point the signal (fluorescence) from the reporter is at its maximum, allowing the amplification product to be detected by measuring this signal.

[0106] The partial primers 3, 4, 5, 6, 7, and 8 have a base sequence length of only 8 or 9 that is complementary to the base sequence of the target site in the MET gene, making it difficult for them to hybridize to the target site alone under polymerase chain reaction (PCR) conditions. However, when a guide probe 3 that hybridizes to the target site is present around the target site, a portion of the base sequence of the guide probe 3 hybridizes with a portion of the base sequence of the partial primers 3, 4, 5, 6, 7, and 8, allowing them to easily hybridize to the target site.

[0107] The partial primers 3, 4, 5, 6, 7, and 8 have a base sequence length of only 8 to 9 that can hybridize with the target nucleic acid base sequence of the MET gene, which is shorter than that of conventional primers, which have a base sequence length of 18 to 30 that can hybridize with the target nucleic acid. Therefore, if there is one or more non-complementary base sequences between the partial primers 3, 4, 5, 6, 7, and 8 and the target nucleic acid hybridization site, the non-complementary base sequences have a greater effect on hybridization efficiency than conventional primers, and as a result, subtle changes in base sequence can be detected better than conventional primers.

[0108] 3.2 Generation and confirmation of amplification products A condition in which about 10,000 copies of a wild-type MET gene target nucleic acid are present, or a condition in which about 10,000 copies of a wild-type MET gene target nucleic acid and about 500 copies of a MET gene target nucleic acid in which the D1002N mutation has occurred are mixed, or a condition in which about 10,000 copies of a wild-type MET gene target nucleic acid and about 500 copies of a MET gene target nucleic acid in which the D1002G mutation has occurred are mixed, or a condition in which about 10,000 copies of a wild-type MET gene target nucleic acid and about 500 copies of a MET gene target nucleic acid in which the Y1003F mutation has occurred are mixed. A polymerase chain reaction composition was prepared, comprising 30 pmole of guide probe 3, 8 pmole of partial primer 3, 4 pmole of partial primer 4, 4 pmole of partial primer 5, 4 pmole of partial primer 6, 4 pmole of partial primer 7, 4 pmole of partial primer 8, 3 pmole of specific primer 3, and 2 pmole of detection probe 3, so as to form the intended amplification product under the conditions of mixing about 10,000 wild-type MET gene target nucleic acids with about 500 MET gene target nucleic acids having a Y1003N mutation, mixing about 10,000 wild-type MET gene target nucleic acids with about 500 MET gene target nucleic acids having a Y1003*(1) mutation, or mixing about 10,000 wild-type MET gene target nucleic acids with about 500 MET gene target nucleic acids having a Y1003*(2) mutation.

[0109] The polymerase chain reaction composition typically contains a nucleic acid polymerase (DNA polymerase) used in polymerase chain reaction, as well as other components such as a buffer, deoxyribonucleotide-5-tripolyphosphate (dNTP), potassium chloride (KCl), magnesium chloride (MgCl), and a detergent.

[0110] The temperature for the polymerase chain reaction was controlled using a standard thermal cycler. After initial denaturation (95°C, 5 minutes), 15 cycles were performed: denaturation (95°C, 20 seconds), annealing (63°C, 20 seconds), extension (72°C, 20 seconds), and 35 cycles were performed: denaturation (95°C, 10 seconds), measurement (50°C, 10 seconds), denaturation (95°C, 20 seconds), annealing (58°C, 20 seconds), extension (72°C, 20 seconds). Fluorescence intensity of the FAM channel was measured every cycle during the measurement step of the second cycle to derive an amplification curve.

[0111] As a result, an increase in the fluorescent signal on the amplification curve was observed under all target nucleic acid injection conditions. However, compared to the condition in which only the wild-type MET gene target nucleic acid was injected, the increase in the fluorescent signal on the amplification curve appeared more rapidly under each of the conditions in which the D1002N, D1002G, Y1003F, Y1003N, Y1003*(1), and Y1003*(2) mutant target nucleic acids were mixed, and lower Ct values were calculated. That is, an average Ct value of 19.9 was observed when the D1002N mutant target nucleic acid was mixed, an average Ct value of 18.6 when the D1002G mutant target nucleic acid was mixed, an average Ct value of 21.2 when the Y1003F mutant target nucleic acid was mixed, an average Ct value of 17.5 when the Y1003N mutant target nucleic acid was mixed, an average Ct value of 17.5 when the Y1003*(1) mutant target nucleic acid was mixed, and an average Ct value of 20.6 when the Y1003*(2) mutant target nucleic acid was mixed. On the other hand, an average Ct value of 28.0 was observed when only the wild-type target nucleic acid was present. As a result, all of the mutants to be detected could be specifically confirmed using the difference in Ct values with a single reaction solution composition (Figure 7).

[0112] Example 4: Confirmation of the detection limit of the KRAS gene in which the G12C mutation has occurred in the human gene 4.1 Preparation of primers and probes To specifically amplify and detect the G12C mutation (GGT>TGT nucleotide sequence mutation) located in exon 2, codon 12 of the human KRAS gene (TGT nucleotide sequence mutation) according to the novel target nucleic acid amplification method of the present invention, a guide probe 4 capable of specifically hybridizing to a region surrounding codon 12 of the KRAS gene, a partial primer 2 capable of specifically hybridizing at a portion of its 3' end to codon 12 of the KRAS gene where the G12C mutation occurred and a surrounding region, and a specific primer 2 capable of hybridizing to a portion of the exon 2 nucleotide sequence of the KRAS gene so as to pair with the partial primer 2 to amplify the target nucleic acid, were designed, whereby a partial nucleotide sequence at the C-terminus of the guide probe 4 and a partial nucleotide sequence at the 5' end of the partial primer 2 were hybridized. In addition, a detection probe 2 was designed and manufactured (PANAGENE Inc., South Korea) to detect the product amplified by the designed guide probe and primers.

[0113] To specifically amplify and detect the KRAS gene in a sample regardless of the presence or absence of the G12C mutation in the KRAS gene, we designed guide probe 5, which specifically hybridizes to exon 6 of the KRAS gene; partial primer 9, whose 3'-terminal portion specifically hybridizes to exon 6 of the KRAS gene; and specific primer 4, which hybridizes to a portion of the exon 6 sequence of the KRAS gene to pair with partial primer 9 and amplify the target nucleic acid, where a partial base sequence at the C-terminus of guide probe 5 hybridizes to a partial base sequence at the 5'-terminus of partial primer 9. Detection probe 4 was also designed and manufactured (PANAGENE Inc., South Korea) to detect the product amplified by the designed guide probe and primers. The base sequences of each primer and probe are shown in Tables 7 and 8.

[0114] [Table 7]

[0115] [Table 8]

[0116] The partial primer 2 was prepared as a single-stranded oligonucleotide having a length of 18 bases, and the underlined base sequence is complementary to a part of the base sequence of the guide probe 4, and the italicized base sequence has the characteristics of being complementary to the base sequence of codon 12 and the surrounding region of the KRAS gene in which the G12C mutation occurred.

[0117] The partial primer 9 is made of a single-stranded oligonucleotide with a length of 19 bases, and the underlined base sequence is complementary to a portion of the base sequence of the guide probe 5, and the italicized base sequence has the characteristic of being complementary to a portion of the base sequence of exon 6 of the KRAS gene.

[0118] The guide probe 4 was made of a PNA having a length of 24 bases, and the base sequence shown in italics is complementary to the base sequence of the region surrounding codon 12 of the KRAS gene, and the underlined base sequence is complementary to a portion of the base sequence of the partial primer 2. In addition, [K (lysine)] was attached to the N-terminus of the guide probe 4, and an L35 linker (manufactured by PANAGENE Inc., South Korea) was attached between the region complementary to the KRAS gene sequence and the region complementary to a portion of the base sequence of the partial primer 2.

[0119] The guide probe 5 was made of a PNA having a length of 25 bases, and the base sequence shown in italics is complementary to a portion of the base sequence of exon 6 of the KRAS gene, and the underlined base sequence is complementary to a portion of the base sequence of the partial primer 9. In addition, [K (lysine)] was attached to the N-terminus of the guide probe 5, and an L35 linker (manufactured by PANAGENE Inc., South Korea) was attached between the portion complementary to the KRAS gene sequence and the portion complementary to a portion of the base sequence of the partial primer 9.

[0120] The detection probe 2 was made of a PNA having a length of 12 bases, and had a quencher [Dabcyl] attached to the N-terminus and a reporter [FAM] attached to the C-terminus. The PNA and [FAM] were connected with [O linker] and [K (lysine)] from the manufacturer (PANAGENE Inc., South Korea).

[0121] The detection probe 4 was made of a PNA having a length of 13 bases, with the quencher [Dabcyl] attached to the N-terminus and the reporter [HEX] attached to the C-terminus, and was made so that [O linker] and [K(lysine)] from the manufacturer (PANAGENE Inc., South Korea) were connected between the PNA and [HEX].

[0122] When detection probe 2 and detection probe 4 hybridize to their respective amplification products having complementary base sequences, the quencher and reporter are at the furthest distance from each other, at which point the signal (fluorescence) value from the reporter becomes maximum, and by measuring the signal, each amplification product can be detected.

[0123] The partial primer 2 has a base sequence complementary to the base sequence of the target site in the KRAS gene that is only 8 bases long, making it difficult to hybridize to the target site alone under polymerase chain reaction (PCR) conditions. However, if a hybridizable guide probe 4 is present near the target site, a portion of the base sequence of the guide probe 4 hybridizes with a portion of the base sequence of the partial primer 2, allowing it to easily hybridize to the target site.

[0124] The partial primer 2 has a base sequence length of only 8 that can hybridize with the target nucleic acid base sequence of the KRAS gene, which is shorter than that of a typical primer, which has a base sequence length of 18 to 30 that can hybridize with the target nucleic acid. Therefore, if there is one or more non-complementary base sequences between partial primer 2 and the target nucleic acid hybridization site, the non-complementary base sequences have a greater effect 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.

[0125] The partial primer 9 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 alone under polymerase chain reaction (PCR) conditions. However, if a hybridizable guide probe 5 is present near the target site, a portion of the base sequence of the guide probe 5 hybridizes with a portion of the base sequence of the partial primer 9, making it easy to hybridize to the target site.

[0126] The 3' end of partial primer 9 hybridizes with a portion of the exon 6 base sequence of the KRAS gene, which may occur regardless of the presence or absence of the G12C mutation in the KRAS gene. Taking this characteristic into consideration, amplification using partial primer 9 can be used as a control.

[0127] In other words, if the G12C mutation of the KRAS gene is not present in the sample, amplification by partial primer 2 (target amplification) is suppressed, while amplification by partial primer 9 (control amplification) appears smoothly, and the difference (ΔCt) between the calculated Ct values of the two amplification curves appears large. On the other hand, if the G12C mutation of the KRAS gene is present in the sample, amplification by partial primer 2 and partial primer 9 appears smoothly, and the difference between the calculated Ct values of the two amplification curves appears small. Therefore, the presence or absence of the G12C mutation can be easily determined using this.

[0128] 4.2 Generation and confirmation of amplification products The conditions are as follows: there are about 4,000 copies of wild-type KRAS gene target nucleic acid; or the wild-type KRAS gene target nucleic acid and the G12C mutated KRAS gene target nucleic acid are mixed in the ratios of 0:100 (100% G12C), 50:50 (50% G12C), 90:10 (10% G12C), 96:4 (4% G12C), 98:2 (2% G12C), 99:1 (1% G12C), and 99.5:0.5 (0.5% G12C), respectively, so that the total number of the wild-type KRAS gene target nucleic acid and the G12C mutated KRAS gene target nucleic acid is about 4,000 copies; or the wild-type KRAS gene target nucleic acid and the non-target mutations G12A (codon 12 is changed to GGT>GCT), G12D (codon KRAS gene target nucleic acids in which codon 12 has been changed from GGT to GAT), G12R (codon 12 has been changed from GGT to CGT), G12S (codon 12 has been changed from GGT to AGT), G12V (codon 12 has been changed from GGT to GTT), G13S (codon 12 has been changed from GGT to GTT), G13C (codon 13 has been changed from GGC to TGC), and G13D (codon 13 has been changed from GGC to GAC) have been mixed in a ratio of 90:10 (10% G12A, 10% G12D, 10% G12R, 10% G12S, 10% G12V, 10% G13C, 10% G13D) to form the intended amplification products under conditions of a total number of approximately 4,000 copies. A polymerase chain reaction composition was prepared containing pmoles of guide probe 4, 15 pmoles of guide probe 5, 25 pmoles of partial primer 2, 7 pmoles of partial primer 9, 3 pmoles of specific primer 2, 2 pmoles of specific primer 4, 3 pmoles of detection probe 2, and 3 pmoles of detection probe 4.

[0129] The polymerase chain reaction composition typically contains a nucleic acid polymerase (DNA polymerase) used in polymerase chain reaction, as well as other components such as a buffer, deoxyribonucleotide-5-tripolyphosphate (dNTP), potassium chloride (KCl), magnesium chloride (MgCl), and a detergent.

[0130] The temperature for the polymerase chain reaction was controlled using a conventional thermal cycler. After initial denaturation (95°C, 5 minutes), 15 cycles of the first step (denaturation (95°C, 20 seconds) - annealing (63°C, 20 seconds) - extension (72°C, 20 seconds)) were performed, followed by 35 cycles of the second step (denaturation (95°C, 10 seconds) - measurement (50°C, 10 seconds) - denaturation (95°C, 20 seconds) - annealing (58°C, 20 seconds) - extension (72°C, 20 seconds). Fluorescence values in the FAM and HEX channels were measured every cycle during the measurement step of the second step to derive an amplification curve.

[0131] The Ct value was calculated from the amplification curve derived from the FAM channel and called the "Target site amplification Ct," and the Ct value was calculated from the amplification curve derived from the HEX channel and called the "Control site amplification Ct." The ΔCt value was calculated by subtracting the Target site amplification Ct from the Control site amplification Ct, and a clear difference in ΔCt value was confirmed between the conditions in which the G12C mutant was injected and the conditions in which the wild type or non-target mutant was injected.

[0132] Specifically, a ΔCt value of -5 or greater was considered "positive for G12C mutation," while a ΔCt value of less than -5 was considered "negative for G12C mutation." All reactions containing 100% G12C, 50% G12C, 10% G12C, 4% G12C, 2% G12C, and 1% G12C were considered positive for G12C mutation, while six of eight reactions containing 0.5% G12C were considered positive for G12C mutation. Meanwhile, all reactions containing wild-type and non-targeted mutants were considered negative for G12C mutation. These results demonstrate the high sensitivity and specificity of this assay, enabling detection of approximately 20-40 copies of the target mutant nucleic acid per reaction (Figure 9). [Industrial Applicability]

[0133] The target nucleic acid amplification method according to the present invention utilizes a guide probe to bind to all hybridizable nucleic acids present in a sample, thereby assisting the binding of the partial primer to the target nucleic acid detection site, making it possible to amplify target nucleic acids present even at very low concentrations. Furthermore, due to the sequence specificity of the partial primer, differences in the amplification rate occur for nucleic acids other than the target nucleic acid, which has the advantage of allowing the target nucleic acid to be detected with high specificity. This makes the method useful for molecular diagnosis, prenatal diagnosis, early diagnosis, cancer diagnosis, genetic-related diagnosis, genotype diagnosis, infectious disease diagnosis, discrimination of drug-resistant bacteria, forensic medicine, and biological species discrimination.

[0134] Although specific portions of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is to be defined by the appended claims and their equivalents.

Claims

1. A method for amplifying a target nucleic acid comprising the steps of: (a) Nucleic acids isolated from the specimen i) a guide probe capable of hybridizing to a site other than the detection site of the target nucleic acid; ii) a partial primer including a sequence that binds to a site other than the target nucleic acid hybridization site of the guide probe and a sequence that binds to the target nucleic acid detection site; and iii) a specific primer that can pair with the partial primer to amplify the target nucleic acid; and performing a polymerase chain reaction (PCR); (b) determining the presence or absence of an amplification product;

2. 2. The method for amplifying a target nucleic acid according to claim 1, wherein the site of the guide probe that hybridizes with the target nucleic acid is located at the 5'-end or N-end, and the site of the guide probe that hybridizes with the partial primer is located at the 3'-end or C-end.

3. 2. The method of claim 1, wherein the guide probe further comprises a linker between the target nucleic acid hybridization site and the partial primer hybridization site.

4. 2. The method of claim 1, wherein the linker is at least one 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)), aminoethoxyethoxyacetic acid (2-[2-[2-[2-(amino)ethoxy]ethoxy]ethoxy]acetic acid (AEEEA), AEEEEA, DL15, and L35.

5. 2. The method for amplifying a target nucleic acid according to claim 1, wherein the guide probe, the partial primer, and the specific primer are composed of any one of an oligonucleotide, an LNA (locked nucleic acid), and a PNA (peptide nucleic acid), or a mixture thereof.

6. 6. The method for amplifying a target nucleic acid according to claim 5, wherein the guide probe is a PNA having a length of 10 to 500 bases.

7. 7. The method for amplifying a target nucleic acid according to claim 6, wherein the guide probe is a PNA having a length of 20 to 150 bases.

8. 2. The method for amplifying a target nucleic acid according to claim 1, wherein the guide probe hybridizes to the opposite strand of the target nucleic acid to which the specific primer hybridizes.

9. 2. The method of claim 1, wherein the partial primer further comprises a spacer, which is a single-stranded oligonucleotide having a length of 1 to 100 bases, between a sequence that binds to a site other than the target nucleic acid hybridization site of the guide probe and a sequence that binds to the target nucleic acid detection site.

10. 2. The method for amplifying a target nucleic acid according to claim 1, wherein the sequence that binds to the target nucleic acid detection site of the partial primer is a 3-15 base sequence.

11. 2. The method for amplifying a target nucleic acid according to claim 1, wherein the length of the amplification product is 50 bp to 1 kbp.

12. 2. The method of claim 1, wherein the step (b) of determining the presence or absence of the amplification product uses a nucleic acid-binding dye or a probe capable of binding to the amplification product.

13. 13. The method of claim 12, wherein the nucleic acid binding dye is selected from the group consisting of ethidium bromide, SYBR® Green I, SYBR® Gold, EvaGreen, YO-PRO-1, SYTO, BEBO, and BEXTO.

14. 13. The method for amplifying a target nucleic acid according to claim 12, wherein the probe capable of binding to the amplification product is selected from the group consisting of an oligonucleotide, an LNA, a PNA, and a mixture thereof.

15. 15. The method for amplifying a target nucleic acid according to claim 14, wherein the probe capable of binding to the amplification product has a reporter and a quencher attached to both ends.

16. 16. The method of claim 15, wherein the reporter is 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.

17. 16. The method of claim 15, wherein the quencher is 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.

18. i) a guide probe capable of hybridizing to a site other than the detection site of the target nucleic acid; ii) a partial primer including a sequence that binds to a site other than the target nucleic acid hybridization site of the guide probe and a sequence that binds to the target nucleic acid detection site; and iii) a specific primer that can pair with the partial primer to amplify the target nucleic acid; A PCR composition for amplifying a target nucleic acid comprising:

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