Detection kit and detection method for base sequence difference

Abstract

In a DNA-dependent DNA polymerase having 5 '-3' exonuclease activity, only a mutant DNA having a partial sequence different from the sequence of a target DNA cannot be specifically amplified. The purpose of the present invention is to provide a kit which is provided with a DNA polymerase that can be used in PCR and a single-stranded RNA and in which (1) the DNA polymerase is subjected to a temporary inactivation treatment and / or (2) the single-stranded RNA is resistant to degradation by an RNA degrading enzyme, the DNA polymerase has 5 '-3' exonuclease activity, and the single-stranded RNA has a base sequence capable of hybridizing with a target sequence in a target nucleic acid molecule.

Description

Technical Field

[0001] The present invention relates to a kit and a method for detecting base sequence differences.

Background Art

[0002] The polymerase chain reaction (PCR) method is a technique that uses a specific pair of primers and a DNA-dependent DNA polymerase to amplify a specific DNA sequence, and is widely used in the field of molecular biology. In the PCR reaction, in addition to the DNA sequence to be amplified, there are cases where primers and other annealed DNA sequences are amplified. As a technique for suppressing the amplification of such undesired DNA sequences, a blocking PCR method using blockers (nucleic acids such as DNA and RNA) has been developed. The blocker binds to a complementary sequence within the DNA sequence amplified by PCR and inhibits the extension of DNA polymerase and the annealing of primers, thereby suppressing PCR amplification.

[0003] Known blockers include long-chain RNA, 3'-modified DNA, artificial nucleic acids, and oligoribonucleotides (ORN).

[0004] Non-Patent Document 1 discloses a method for specifically suppressing a PCR-based amplification reaction using long-chain RNA (about 750 bases).

[0005] Non-Patent Documents 2 to 5 disclose a method of synthesizing 3'-end modified DNA, locked nucleic acid (LNA) and peptide nucleic acid (PNA) as artificial nucleic acids in a manner of hybridizing within the region amplified by PCR and adding them to the reaction system.

[0006] Patent Document 1, Patent Document 2, Non-Patent Document 6, and Non-Patent Document 7 disclose a method using ORN with a length of about 20 to 30 bases as a blocker (ORNi-PCR method).

[0007] In addition, Non-Patent Documents 2 and 4 to 7 disclose the following method: using blocking PCR using 3'-modified DNA, artificial nucleic acids or ORN as blockers to amplify only the mutant sequence from a state where the wild-type sequence and the mutant sequence coexist. In addition, Non-Patent Document 5 discloses that when using PNA as a blocker, a fluorescent dye-labeled probe complementary to the mutant sequence is simultaneously added to the PCR reaction (using Taq DNA polymerase), so that the presence of mutant DNA can be detected in real time by fluorescence emission accompanying probe degradation.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Non-Patent Document

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Summary of the Invention

Problems to be Solved by the Invention

[0010] In the method disclosed in Non-Patent Document 1, the operation of synthesizing long-chain RNA by in vitro transcription is rather complicated. In addition, since the amplification of the sequence in the long-chain RNA and a part of the complementary template DNA may also be inhibited, it is expected that the inhibition specificity will become low.

[0011] When using the methods disclosed in Non-Patent Documents 2 to 4, since there is a possibility that the 3'-side modification is removed and the inhibitory nucleotide functions as a primer, there is a problem that a DNA-dependent DNA polymerase having 3'-5' exonuclease activity cannot be used. In addition, it has been pointed out that when using the method disclosed in Non-Patent Document 5, PNA may be insoluble in an aqueous solution depending on the sequence (Non-Patent Document 8), and the sequence design also requires know-how.

[0012] The methods disclosed in Patent Document 1, Patent Document 2, Non-Patent Document 6, and Non-Patent Document 7, although the ORN has a simple design, the ORN is removed by a DNA-dependent DNA polymerase (such as Taq DNA polymerase) having 5'-3' exonuclease activity, so it is predicted that this DNA polymerase cannot be used.

[0013] Regarding Non-Patent Document 5 and Non-Patent Document 7, it has been pointed out that PNA may be insoluble in an aqueous solution depending on the sequence (Non-Patent Document 8), and the design of the sequence requires know-how.

Technical Solution for Solving the Problem

[0014] The inventors of the present invention found a combination of a DNA polymerase having 5'-3' exonuclease activity and ORN, which can specifically inhibit the amplification of target DNA having a sequence complementary to the sequence of ORN even in a nucleic acid amplification method using a combination of a DNA polymerase having 5'-3' exonuclease activity and ORN, and can specifically amplify only mutant DNA having a partial sequence different from the sequence of the target DNA, and completed the present invention.

[0015] An object of the present invention is to provide a kit, which includes a DNA polymerase that can be used for PCR and single-stranded RNA, In the above kit, (1) The above DNA polymerase is subjected to a temporary inactivation treatment, and / or (2) The above single-stranded RNA is resistant to degradation by an RNA degrading enzyme, The above DNA polymerase has 5'-3' exonuclease activity, The above single-stranded RNA has a base sequence that can hybridize with a target sequence in a target nucleic acid molecule.

[0016] By using this kit, even in a nucleic acid amplification method using a DNA polymerase having 5'-3' exonuclease, the amplification of target DNA having a sequence complementary to the sequence of ORN can be specifically inhibited, and only mutant DNA having a partial sequence different from the sequence of the target DNA can be specifically amplified.

[0017] The above kit may further include a nucleic acid probe labeled with a fluorescent dye. The base sequence of the above nucleic acid probe may be different from the above base sequence of the single-stranded RNA due to having at least one mutation.

[0018] The above single-stranded RNA that is resistant to degradation by the above RNA degrading enzyme may be a phosphorothioate type single-stranded RNA.

[0019] The above DNA polymerase having the above chemical modification may be a DNA polymerase modified with a dicarboxylic anhydride.

[0020] The above kit may further include dUTP.

[0021] In addition, another object of the present invention is to provide a method for specifically inhibiting nucleic acid amplification in a target region. The method includes: A providing step of providing a mixture containing a DNA polymerase usable for PCR, single-stranded RNA, a target nucleic acid molecule, a DNA primer pair, and dNTP; A denaturation step of maintaining the mixture at a temperature above the denaturation temperature; An annealing step of maintaining the mixture at the annealing temperature; A DNA extension step of maintaining the mixture at the DNA extension temperature; An amplification step of amplifying the DNA strand by sequentially repeating the denaturation step, annealing step, and DNA extension step; and A detection step of detecting an amplification product obtained by the amplification of the DNA strand; (1) The DNA polymerase is subjected to a temporary inactivation treatment, and / or (2) The single-stranded RNA is resistant to degradation by an RNA degrading enzyme, The DNA polymerase has a 5'-3' exonuclease, The single-stranded RNA has a base sequence capable of hybridizing with a target sequence in the target nucleic acid molecule.

[0022] In addition, another object of the present invention is to provide a method for specifically inhibiting nucleic acid amplification in a target region. The method includes: A providing step of providing a mixture containing a DNA polymerase usable for PCR, single-stranded RNA, a target nucleic acid molecule, a DNA primer, and dNTP; a denaturation step of maintaining the mixture at a temperature above the denaturation temperature; A maintaining step of maintaining the mixture at 45°C to 54°C; An amplification step of amplifying the DNA strand by sequentially repeating the denaturation step and the maintaining step; and A detection step of detecting an amplification product obtained by the amplification of the DNA strand; The DNA polymerase has a 5'-3' exonuclease, and the single-stranded RNA has a base sequence capable of hybridizing with a target sequence in the target nucleic acid molecule.

[0023] By using this method, even in a nucleic acid amplification method using a DNA polymerase having 5'-3' exonuclease activity, it is possible to specifically inhibit the amplification of a target DNA having a sequence complementary to the sequence of ORN, and it is possible to specifically amplify only a mutant DNA whose partial sequence is different from the sequence of the target DNA.

[0024] The above-mentioned mixture may further include a nucleic acid probe labeled with a fluorescent dye. The base sequence of the above-mentioned nucleic acid probe may be different from the base sequence of the above-mentioned single-stranded RNA due to having at least one mutation. The above-mentioned amplification product can be detected by the fluorescence of the above-mentioned fluorescent dye-labeled fluorescent.

[0025] The above-mentioned single-stranded RNA that is tolerant to degradation by the above-mentioned RNA degrading enzyme may be a phosphorothioate-type single-stranded RNA.

[0026] The above-mentioned DNA polymerase having the above-mentioned chemical modification may be a DNA polymerase modified with a dicarboxylic anhydride.

[0027] The above-mentioned dNTP may include dATP, dUTP, dGTP, dCTP, and optionally deoxythymidine triphosphate (dTTP) at a concentration lower than that of dUTP.

[0028] The above-mentioned method may further include a reverse transcription step of obtaining the above-mentioned target nucleic acid molecule from the target RNA using a reverse transcriptase.

Description of the Drawings

[0029] Figure 1 shows the base sequence of ORN_EGFR_L858 targeting L858 within the WT EGFR gene, the base sequence of WT EGFR corresponding to ORN_EGFR_L858, and the base sequence of its L858R mutation (L858R EGFR). Figure 2 shows an electrophoresis photograph of the nucleic acid after amplification using THUNDERBIRD Probe qPCR Mix and ORN_EGFR_L858. Figure 3 shows an electrophoresis photograph of the nucleic acid after amplification using GoTaq Probe qPCR Master Mix and ORN_EGFR_L858. Figure 4 shows an electrophoresis photograph of the nucleic acid after amplification using Premix Taq and ORN_EGFR_L858. Figure 5 shows the base sequence of ORN_EGFR_T790_18b targeting T790 within the WT EGFR gene, the base sequence of WT EGFR corresponding to ORN_EGFR_T790, and the base sequence of its T790M mutation (T790M EGFR). Figure 6 shows the electrophoresis photograph of the nucleic acid amplified using THUNDERBIRD Probe qPCR Mix and ORN_EGFR_T790_18b. Figure 7 shows the electrophoresis photograph of the nucleic acid amplified using GoTaq Probe qPCR Master Mix and ORN_EGFR_T790_18b. Figure 8 shows the electrophoresis photograph of the nucleic acid amplified using Premix Taq and ORN_EGFR_T790_18b. Figure 9 shows the base sequences of ORN_KRAS_G13 targeting the base sequence within WT KRAS gene at G13, the base sequence of WT KRAS corresponding to ORN_KRAS_G13, and the base sequence of its G13D mutation (G13D KRAS). Figure 10 shows the electrophoresis photograph of the nucleic acid amplified using THUNDERBIRD Probe qPCR Mix and ORN_KRAS_G13. Figure 11 shows the electrophoresis photograph of the nucleic acid amplified using GoTaq Probe qPCR Master Mix and ORN_KRAS_G13. Figure 12 shows the electrophoresis photograph of the nucleic acid amplified using Premix Taq and ORN_KRAS_G13. In Figure 13, Figure 13A schematically shows the amplification result using a DNA polymerase (α type) without 5'-3' exonuclease activity and ORN. Figure 13B schematically shows the amplification result using Taq DNA polymerase with 5'-3' exonuclease activity and ORN near the melting temperature (Tm) of ORN / DNA hybridization. Figure 13C schematically shows the amplification result using Taq DNA polymerase with 5'-3' exonuclease activity and ORN at a temperature lower than the melting temperature (Tm) of ORN / DNA hybridization. In Figure 14, Figure 14A shows the electrophoresis photograph of the nucleic acid amplified using GeneAce Taq and ORN_EGFR_L858. Figure 14B shows the result of DNA sequence analysis of the amplification product indicated by the arrow in Figure 14A. Figure 15 schematically shows the inhibition pattern of nucleic acid amplification in ORNi-PCR using GeneAce Taq near the Tm of ORN / DNA hybridization. Figure 16 shows the electrophoresis photograph of the nucleic acid amplified using GeneAce Taq and ORN_EGFR_T790_18b. Figure 17 shows the electrophoresis photograph of the nucleic acid amplified using GeneAce Taq and ORN_KRAS_G13. Figure 18 shows the base sequence of the EGFR gene to be amplified using the forward primer (hEGFR-Exon21-F8) and the reverse primer (hEGFR-Exon21-R8). Figure 19 shows the base sequence targeting L858 within the WT EGFR gene (ORN_EGFR_L858_17b), the base sequence of WT EGFR corresponding to ORN_EGFR_L858_17b, and the base sequence of its L858R mutation (L858R EGFR). In Figure 20, Figure 20A shows an electrophoresis photograph of the nucleic acid amplified using GeneAce Taq and ORN_EGFR_L858_17b. Figure 20B shows the result of DNA sequence analysis of the amplification product indicated by the arrow in Figure 20A. Figure 21 shows an electrophoresis photograph of the nucleic acid amplified using ORN_EGFR_L858 (56 °C in the annealing + extension step). Figure 22 shows an electrophoresis photograph of the nucleic acid amplified using ORN_EGFR_L858 (59 °C in the annealing + extension step). Figure 23 shows a schematic diagram of traditional real-time PCR using a dual-labeled fluorescent probe. Figure 24 shows a schematic diagram of real-time ORNi-PCR using a dual-labeled fluorescent probe. Figure 25 shows the base sequences of various dual-labeled fluorescent probes targeting the L858R EGFR mutation. Figure 26 shows the result of traditional real-time PCR using Probe_EGFR_L858R_15b_2. Figure 27 shows the result of traditional real-time PCR using Probe_EGFR_L858R_16b. Figure 28 shows the result of traditional real-time PCR using Probe_EGFR_L858R_18b. Figure 29 shows the result of traditional real-time PCR using Probe_EGFR_L858R_Sense_15b. In Figure 30, Figure 30A shows the result of real-time ORNi-PCR using ORN_EGFR_L858_17b and Probe_EGFR_L858R_18b when the L858R EGFR gene is 1% of the overall EGFR template. Figure 30B shows the result of DNA sequence analysis of the amplification product. In Figure 31, Figure 31A shows the results of real-time ORNi-PCR using ORN_EGFR_L858_17b and Probe_EGFR_L858R_18b when the L858R EGFR gene is 0.2% of the overall EGFR template. Figure 31B shows the results of DNA sequence analysis of the amplification product. In Figure 32, Figure 32A shows the results of conventional real-time PCR using Probe_EGFR_L858R_18b when the L858R EGFR gene is 1% of the overall EGFR template. Figure 32B shows the results of DNA sequence analysis of the amplification product. In Figure 33, Figure 33A shows the results of real-time ORNi-PCR using ORN_EGFR_L858 and Probe_EGFR_L858R_Sense_15b when the L858R EGFR gene is 1% of the overall EGFR template. Figure 33B shows the results of DNA sequence analysis of the amplification product. In Figure 34, Figure 34A shows the results of conventional real-time PCR using Probe_EGFR_L858R_Sense_15b when the L858R EGFR gene is 1% of the overall EGFR template. Figure 34B shows the results of DNA sequence analysis of the amplification product. In Figure 35, Figure 35A shows the results of real-time ORNi-PCR using Probe_EGFR_L858R_18b and ORN_EGFR_L858_17b when the L858R EGFR gene is 5% of the overall EGFR template. Figure 35B shows the results of DNA sequence analysis of the amplification product. In Figure 36, Figure 36A shows the results of conventional real-time PCR using Probe_EGFR_L858R_18b when the L858R EGFR gene is 5% of the overall EGFR template. Figure 36B shows the results of DNA sequence analysis of the amplification product. Figure 37 shows an electrophoresis photograph of the nucleic acid after amplification using Hot-Start Gene Taq NT and ORN_EGFR_L858_17b in the presence of dTTP or dUTP. Figure 38 shows an electrophoresis photograph of the nucleic acid after amplification using Hot-Start Gene Taq NT, ORN_EGFR_L858_17b, and genomic DNA from 293T cells or genomic DNA from NCI-H1975 cells in the presence of dUTP. Figure 39 shows the results of DNA sequence analysis of the amplification product indicated by the arrow in Figure 38. In Figure 40, Figure 40A shows an electrophoresis photograph of nucleic acids amplified using AmpliTaq Gold 360, ORN_EGFR_L858_17b, and genomic DNA from 293T cells or genomic DNA from NCI-H1975 cells in the presence of dTTP or dUTP. Figure 40B shows the result of DNA sequence analysis of the amplification product indicated by the arrow in Figure 40A. Figure 41 shows an electrophoresis photograph of nucleic acids amplified using unmodified Taq DNA polymerase (Gene Taq NT) and ORN_EGFR_L858_17b in the presence of dUTP. Figure 42 shows an electrophoresis photograph of nucleic acids amplified using unmodified Taq DNA polymerase (Gene Taq NT), ORN_EGFR_L858_17b, and the reaction buffer of Hot-Start Gene Taq NT in the presence of dUTP. Figure 43 shows an electrophoresis photograph of nucleic acids amplified using unmodified Taq DNA polymerase (Gene Taq NT) and ORN_EGFR_L858_17b in the presence of dUTP and under long-time heat treatment conditions. In Figure 44, Figure 44A shows an electrophoresis photograph of nucleic acids amplified using conventional Taq DNA polymerase (THUNDERBIRD Probe qPCR Mix), ORN_EGFR_L858_S_All, and genomic DNA from 293T cells or genomic DNA from NCI-H1975 cells. Figure 44B shows the result of DNA sequence analysis of the amplification product indicated by the arrow in Figure 44A. Figure 45 shows an electrophoresis photograph of nucleic acids amplified using conventional Taq DNA polymerase (THUNDERBIRD Probe qPCR Mix) and ORN (ORN_EGFR_L858_S_5) with partial modification of the 5 phosphodiester bonds at the 5' end. Figure 46 shows an electrophoresis photograph of nucleic acids amplified using conventional Taq DNA polymerase (GoTaq Probe qPCR Master Mix), fully phosphorothioated ORN (ORN_EGFR_L858_S_All), and genomic DNA from 293T cells or genomic DNA from NCI-H1975 cells. In Figure 47, Figure 47A shows an electrophoresis photograph of nucleic acids amplified using a conventional Taq DNA polymerase (THUNDERBIRD Probe qPCR Mix), a fully thiophosphorylated ORN (ORN_EGFR_L858_S_All), and genomic DNA from 293T cells or genomic DNA from NCI-H1975 cells. Figure 47B shows the result of DNA sequence analysis of the amplification product indicated by the arrow in Figure 47A. Figure 48 shows an electrophoresis photograph of nucleic acids amplified using a conventional Taq DNA polymerase (THUNDERBIRD Probe qPCR Mix) and a fully thiophosphorylated ORN (ORN_EGFR_L858_S_All) within the time of the annealing + extension step of 30 seconds or 2 minutes. In Figure 49, Figure 49A shows the result of real-time ORNi-PCR using ORN_EGFR_L858_S_All and Probe_EGFR_L858R_Sense_15b. Figure 49B shows the result of DNA sequence analysis of the amplification product. In Figure 50, Figure 50A shows the result of conventional real-time PCR using Probe_EGFR_L858R_Sense_15b. Figure 50B shows the result of DNA sequence analysis of the amplification product. In Figure 51, Figure 51A shows an electrophoresis photograph of nucleic acids amplified using 0.1, 0.2, and 1 μM of a fully thiophosphorylated ORN (ORN_EGFR_L858_S_All). Figure 51B shows the result of DNA sequence analysis of the amplification product indicated by the arrow in Figure 51A. Figure 52 shows an electrophoresis photograph of nucleic acids amplified using an ORN (ORN_EGFR_L858_S_5) with partial modification of the 5 phosphodiester bonds at the 5' end. In Figure 53, Figure 53A shows the result of real-time ORNi-PCR using ORN_EGFR_L858_S_All and Probe_EGFR_L858R_Sense_15b with the L858R EGFR mutation accounting for 1% of the overall EGFR template. Figure 53B shows the result of DNA sequence analysis of the amplification product. Figure 54 schematically shows the protocol of one-step RT-ORNi-PCR. Figure 55 schematically shows the positions of mutations, ORNs, probes, and primer pairs on the WT EGFR gene. In FIG. 56, FIG. 56A shows an electrophoresis photograph of nucleic acids amplified by one-step RT-ORNi-PCR (which uses ORN_EGFR_L858_S_All (0.5 μM ORN; annealing + extension step at 57.5° C.) for detecting the L858R EGFR mutation) using total RNA extracted from MRC-5 cells and NCI-H1975 cells having the WT EGFR gene. FIG. 56B shows the result of DNA sequence analysis of the amplification product indicated by the arrow in FIG. 56A. FIG. 57 shows an electrophoresis photograph of nucleic acids amplified by one-step RT-ORNi-PCR (which uses ORN_EGFR_L858_S_All (0.25, 0.5, and 1 μM ORN; annealing + extension step at 56° C. or 59° C.) for detecting the L858R EGFR mutation) using total RNA extracted from MRC-5 cells and NCI-H1975 cells. FIG. 58 shows an electrophoresis photograph of nucleic acids amplified by one-step RT-ORNi-PCR (which uses ORN_EGFR_L858 (0.5 and 6 μM ORN) as an unmodified ORN) using total RNA extracted from MRC-5 cells and NCI-H1975 cells. FIG. 59 schematically shows a protocol for one-step real-time RT-ORNi-PCR. In FIG. 60, FIG. 60A shows the result of one-step real-time RT-PCR using the QIAGEN OneStep RT-PCR Kit and Probe_EGFR_L858R_Sense_15b. FIG. 60B shows the result of DNA sequence analysis of the amplification product. In FIG. 61, FIG. 61A shows the result of one-step real-time RT-ORNi-PCR using the QIAGEN OneStep RT-PCR Kit, Probe_EGFR_L858R_Sense_15b, and ORN_EGFR_L858_S_All. FIG. 61B shows the result of DNA sequence analysis of the amplification product. FIG. 62 schematically shows a protocol for one-step real-time RT-ORNi-PCR using the iTaq Universal Probes One-Step Kit. In FIG. 63, FIG. 63A shows the result of one-step real-time RT-PCR using the iTaq Universal Probes One-Step Kit containing unmodified Taq DNA polymerase and Probe_EGFR_L858R_Sense_15b. FIG. 63B shows the result of DNA sequence analysis of the amplification product. In Figure 64, Figure 64A shows the results of one-step real-time RT-ORNi-PCR using the iTaq Universal Probes One-Step Kit, Probe_EGFR_L858R_Sense_15b, and ORN_EGFR_L858_S_All. Figure 64B shows the results of DNA sequence analysis of the amplification products. In Figure 65, Figure 65A shows an electrophoresis photograph of nucleic acids amplified by one-step RT-ORNi-PCR (which uses ORN_EGFR_L858_S_All (0.25 and 0.5 μM ORN)) using total RNA extracted from MRC-5 cells and NCI-H1975 cells. Figure 65B shows the results of DNA sequence analysis of the amplification products indicated by the arrows in Figure 65A. Figure 66 schematically shows the research stage of the one-step RT-ORNi-PCR reaction in which ORN_EGFR_L858R_S_All sequence-specifically inhibits DNA extension. In Figure 67, Figure 67A shows an electrophoresis photograph of ORN_EGFR_L858R_S_All sequence-specifically inhibiting the amplification of target DNA from cDNA in the PCR stage. Figure 67B shows the results of DNA sequence analysis of the amplification products indicated by the arrows in Figure 67A. In Figure 68, Figure 68A shows an electrophoresis photograph in which DNA amplification is non-specifically inhibited when PCR is performed after RT in the presence of ORN_EGFR_L858R_S_All. Figure 68B shows the results of DNA sequence analysis of the amplification products indicated by the arrows in Figure 68A. Figure 69 schematically shows the positions where phosphorothioated ORNs non-complementary to EGFR mRNA hybridize. Figure 70 shows the base sequence of ORN_EGFR_L858_Sense_S_All (which is a phosphorothioated ORN non-complementary to EGFR mRNA), and the base sequences of WT EGFR mRNA and L858R EGFR mRNA corresponding to this base sequence. In Figure 71, Figure 71A shows an electrophoresis photograph of nucleic acids amplified by one-step RT-ORNi-PCR (which uses ORN_EGFR_L858_Sense_S_All (0.1 and 0.25 μM ORN)) using total RNA extracted from MRC-5 cells and NCI-H1975 cells. Figure 71B shows the results of DNA sequence analysis of the amplification products indicated by the arrows in Figure 71A. In Figure 72, Figure 72A shows an electrophoresis photograph of the sequence-specific inhibition of the amplification of target DNA from cDNA during the PCR stage by ORN_EGFR_L858_Sense_S_All. Figure 72B shows the result of DNA sequence analysis of the amplification product indicated by the arrow in Figure 72A. In Figure 73, Figure 73A shows an electrophoresis photograph of the non-specific inhibition of DNA amplification when PCR is performed after RT in the presence of ORN_EGFR_L858_Sense_S_All. Figure 73B shows the result of DNA sequence analysis of the amplification product indicated by the arrow in Figure 73A.

Detailed implementation manners

[0030] Definitions For convenience, the specific terms used in this application are grouped here. Unless otherwise defined, all technical terms and scientific terms used in this application have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains. Unless otherwise stated in the context, the singular forms "a", "an", and "the" include plural referents.

[0031] The numerical ranges and parameters shown in the present invention are approximate values. However, the numerical values shown in specific embodiments are recorded as accurately as possible. However, any numerical value inherently includes specific errors inevitably caused by the standard deviation observable in each test measurement value. In addition, the term "about" used in this specification generally means within 10%, 5%, 1%, or 0.5% of the given numerical value or range. Alternatively, the term "about" means within the standard error acceptable when considered by those skilled in the art.

[0032] Hereinafter, the embodiments of the present invention will be described. The following embodiments are illustrative, and the scope of the present invention is not limited to the content shown in the following embodiments. In addition, for the sake of avoiding repetitive cumbersome, the description of similar content is omitted.

[0033] 1 Kit The kit of this embodiment includes a DNA polymerase and single-stranded RNA that can be used for PCR.

[0034] DNA polymerase DNA polymerase functions as an enzyme responsible for DNA synthesis within a cell. In living organisms, DNA polymerase is involved in DNA synthesis processes, including DNA replication, DNA repair, recombination, and gene amplification. The DNA polymerase that can be used in PCR refers to a DNA polymerase that does not lose its DNA polymerase activity even when exposed to the temperatures set in the PCR method, and is also called a thermostable DNA polymerase. In the present embodiment, the DNA polymerase has 5'-3' exonuclease activity. 5'-3' exonuclease activity refers to the activity of sequentially removing nucleotides from the 5' end of an oligonucleotide.

[0035] In certain embodiments, a temporary inactivation treatment is performed on the DNA polymerase. The DNA polymerase subjected to the temporary inactivation treatment exhibits DNA polymerase activity when it reaches a specific surrounding environment (such as temperature). Examples of the temporary inactivation treatment include a wax barrier (U.S. Patent Nos. 5,599,660 and 5,411,876), an antibody that temporarily inactivates the DNA polymerase, and chemical modification. In certain embodiments, the chemical modification is dicarboxylic anhydride modification. The DNA polymerase subjected to the temporary inactivation treatment can be a DNA polymerase modified with dicarboxylic anhydride or a DNA polymerase to which an antibody that temporarily inactivates the DNA polymerase is attached. In certain embodiments, examples of the dicarboxylic anhydride include citraconic anhydride, 2-methylmaleic anhydride, 2,3-dimethylmaleic anhydride, cis-aconitic anhydride, and tetrafluorosuccinic anhydride.

[0036] In certain embodiments, the DNA polymerase is a DNA polymerase capable of performing hot start PCR. Hot start PCR is a PCR method in which the DNA polymerase is separated from the sample DNA until a specific temperature.

[0037] Single-stranded RNA The single-stranded RNA has a base sequence capable of hybridizing with a target sequence (target region) in a target nucleic acid molecule. "Capable of hybridizing" means that in a nucleic acid amplification method such as PCR, a nucleic acid molecule and a single-stranded nucleic acid can bind complementarily.

[0038] The single-stranded RNA of the present embodiment can specifically inhibit nucleic acid amplification in the target region of the target nucleic acid molecule by the complementary binding of the target nucleic acid molecule and the single-stranded RNA in the target sequence of the target nucleic acid molecule.

[0039] In some embodiments, the single-stranded RNA can hybridize with a target sequence (target region) in one allele and does not hybridize with a mutant sequence (mutant region) corresponding to the target sequence (target region) in the other allele except under specific temperature conditions. Therefore, the mutant sequence (mutant region) can be described as the region where the single-stranded RNA hybridizes under specific temperature conditions. The target sequence is different from the mutant sequence due to at least one mutation. Thus, the nucleic acid molecule in this embodiment contains the target nucleic acid molecule. In some embodiments, the nucleic acid molecule includes the target nucleic acid molecule and a mutant nucleic acid molecule having a mutant sequence (mutant region).

[0040] The specific temperature condition is near the Tm value of the single-stranded RNA, and near the Tm value means within ±1°C, ±2°C, ±3°C or ±4°C of the Tm value.

[0041] The Tm value can be calculated according to known calculation methods such as the Nearest Neighbor Method, the GC% method, etc., and is more preferably calculated using the following formula. Tm = (a + u) * 2 + (g + c) * 4 In addition, a, u, g, and c respectively represent the number of bases of A, U, G, and C.

[0042] In some embodiments, when it is desired to hybridize the single-stranded RNA with the mutant sequence (mutant region) in the other allele, the single-stranded RNA and the target nucleic acid molecule can be hybridized under a temperature condition lower than the Tm value of the single-stranded RNA (for example, a temperature 1°C to 8°C lower than the Tm value and within the range between two values selected from the group consisting of temperatures 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, and 8°C lower than the Tm value). In another embodiment, when it is desired to hybridize the single-stranded RNA with the mutant sequence (mutant region) in the other allele, the single-stranded RNA and the target nucleic acid molecule can be hybridized within the range of 45°C to 56°C (for example, within the range between two values selected from the group consisting of 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, and 56°C).

[0043] In certain embodiments, the single-stranded RNA is resistant to degradation by RNA-degrading enzymes (such as ribonucleases). When the resistance of RNA to degradation by RNA-degrading enzymes is high, the antisense oligonucleotides can be stabilized. In certain embodiments, the single-stranded RNA that is resistant to degradation by RNA-degrading enzymes is a phosphorothioate-type single-stranded RNA. Phosphorothioate-type RNA means an RNA in which the oxygen atom of the phosphate group of an oligonucleotide having a phosphodiester bond is replaced by a sulfur atom. The phosphorothioate-type single-stranded RNA has a phosphorothioate modification on a part or all of the phosphodiester bonds. For a part of the phosphodiester bonds, the phosphorothioate modification is present on 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more and 100% or less of the phosphodiester bonds.

[0044] In the kit according to this embodiment, (i) the DNA polymerase has a chemical modification, (ii) the single-stranded RNA is resistant to degradation by RNA-degrading enzymes, or (iii) the DNA polymerase has a chemical modification and the single-stranded RNA is resistant to degradation by RNA-degrading enzymes.

[0045] To design a single-stranded RNA (single-stranded nucleic acid) that hybridizes to a target region, the base sequence information of the target region is necessary. When the base sequence of the template nucleic acid (target nucleic acid) and the position of the target region in the template nucleic acid are clear, the base sequence information of the target region can be obtained based on this information. When the base sequence information of the target region is unknown, the non-target amplification products amplified by the nucleic acid amplification reaction are separated by a known method such as agarose gel electrophoresis, and a known sequence analysis method is applied to it, whereby the base sequence information of the target region can be obtained. Based on the obtained base sequence information of the target region, a single-stranded nucleic acid that hybridizes to the target region can be designed. When the template nucleic acid is double-stranded (for example, double-stranded DNA composed of a sense strand and an antisense strand), the single-stranded nucleic acid can hybridize to either strand.

[0046] Ideally, the single-stranded RNA (single-stranded nucleic acid) should be completely complementary to the base sequence of the target region, but even a sequence that is not completely complementary can be used as long as it can specifically inhibit the nucleic acid amplification of the target region. As long as it can hybridize to the target region, one to several (for example, 2, 3, 4) mismatches are acceptable. The single-stranded nucleic acid that hybridizes to the target region includes a sequence that is 80% or more complementary to the target sequence, preferably 90% or more complementary, more preferably 95% or more complementary, and further preferably 98% or more complementary, and contains a single-stranded nucleic acid that can specifically inhibit the nucleic acid amplification of the target region.

[0047] The single-stranded nucleic acid hybridizing with the target region can be RNA or a chimera of RNA and other nucleic acids, but is preferably single-stranded RNA. Examples of other nucleic acids include DNA, modified DNA, modified RNA, etc. When the single-stranded nucleic acid is a chimera of RNA and other nucleic acids, the other nucleic acid is preferably 50% or less, more preferably 40% or less, further preferably 30% or less, further preferably 20% or less, still further preferably 10% or less, and even more preferably 5% or less of the total base length.

[0048] The length (base length) of the single-stranded nucleic acid hybridizing with the target region is not particularly limited, and is preferably 10 to 200 bases, more preferably 10 to 150 bases, more preferably 10 to 120 bases, more preferably 10 to 100 bases, more preferably 10 to 90 bases, further preferably 10 to 80 bases, further preferably 10 to 70 bases, further preferably 10 to 60 bases, further preferably 10 to 50 bases, further preferably 12 to 45 bases, further preferably 14 to 40 bases, further preferably 16 to 35 bases, further preferably 18 to 32 bases, further preferably 20 to 30 bases, further preferably 21 to 28 bases, further preferably 22 to 26 bases. Particularly preferably, it is 23 bases.

[0049] The single-stranded nucleic acid hybridizing with the target region can be modified at its 5'-end and / or 3'-end. For example, modified single-stranded nucleic acids modified at the 5'-end and / or 3'-end with phosphorylation, amination, biotinylation, thiolation, cholesterolation, DIG (digoxigenin) modification, quencher modification (BHQ-1, BHQ-3, etc.), fluorescence modification (DNP, Cy3, Cy5, TAMRA, 6-FAM, etc.) can be used.

[0050] As long as it can hybridize with the target region, the nucleotides (ribonucleotides, deoxyribonucleotides) in the single-stranded nucleic acid hybridizing with the target region can be nucleotides with chemically modified sugars, bases, and / or phosphates. As nucleotides with modified bases, for example, 5-position modified uridine or cytidine (e.g., 5-propynyluridine, 5-propynylcytidine, 5-methylcytidine, 5-methyluridine, 5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine, 5-methoxyuridine, etc.) can be cited; 8-position modified adenosine or guanosine (e.g., 8-bromoguanosine, etc.); deazapurines (e.g., 7-deazaadenosine, etc.); O- and N-alkylated nucleotides (e.g., N6-methyladenosine, etc.). In addition, as an example of a nucleotide with a modified sugar, for example, 2'-position sugar modification in which the 2'-OH of a ribonucleotide is replaced by H, OR, R, a halogen atom, SH, SR, NH2, NHR, NR2, or CN (wherein R represents an alkyl, alkenyl, or alkynyl group having 1 to 6 carbon atoms), etc.; and 5'-terminal phosphorylation modification in which the 5'-terminal is monophosphorylated. As an example of a nucleotide with a modified phosphate, a nucleotide in which the phosphate ester group connecting adjacent ribonucleotides is replaced by a phosphorothioate group can be cited.

[0051] The single-stranded nucleic acid hybridizing with the target region can be prepared by artificially chemically synthesizing it using a known method. In addition, it can be prepared from template DNA by in vitro transcription.

[0052] The kit of this embodiment may further include a nucleic acid probe labeled with a fluorescent dye. The nucleic acid probe labeled with a fluorescent dye of this embodiment is used in the real-time PCR method, particularly in the probe method. The nucleic acid probe labeled with a fluorescent dye for the probe method has a fluorescent substance (such as FAM, etc.) at the 5'-end and a quenching substance (such as TAMRA, etc.) at the 3'-end, or has a fluorescent substance at the 3'-end and a quenching substance at the 5'-end. In this specification, the nucleic acid probe is also simply referred to as a probe. The nucleic acid probe can also be a single-stranded DNA probe.

[0053] The base sequence of the nucleic acid probe is different from the above base sequence of the single-stranded RNA due to having at least one mutation. In some embodiments, the nucleic acid probe hybridizes with a mutant sequence (mutant region) in an allele (which is different from the allele with which the single-stranded RNA hybridizes). Therefore, the single-stranded RNA hybridizes with a target sequence (target region) in one allele, and the nucleic acid probe hybridizes with a mutant sequence (mutant region) in another allele. Therefore, the nucleic acid probe can bind to the mutant sequence in the target nucleic acid molecule in a manner that the target nucleic acid molecule and the nucleic acid probe are complementary.

[0054] The kit of this embodiment may further include dUTP. In addition, the kit of this embodiment may further include a primer pair.

[0055] 2 Method for specifically inhibiting nucleic acid amplification of a target region The method for specifically inhibiting nucleic acid amplification of a target region according to this embodiment includes a provision step, a denaturation step, an annealing step, a DNA extension step, an amplification step, and a detection step. This method can be implemented in the same reaction system. In addition, after this method, a further nucleic acid amplification step can be set.

[0056] 2-1 Provision step In the provision step, a mixture containing a DNA polymerase usable for PCR, single-stranded RNA, a nucleic acid molecule, a DNA primer pair, and dNTP is provided.

[0057] The concentration of the single-stranded RNA in the mixture is not particularly limited as long as it can specifically inhibit nucleic acid amplification of the target region. It is preferably determined through preliminary studies for each specific condition in the applicable nucleic acid amplification reaction and set appropriately. Specifically, for example, it is preferably 10 μM or less, more preferably 5 μM or less, further preferably 1 μM or less, further preferably 500 nM or less, further preferably 200 nM or less, further preferably 100 nM or less, further preferably 90 nM or less, further preferably 80 nM or less, further preferably 70 nM or less, further preferably 60 nM or less, further preferably 50 nM or less. There is no particular limitation on the lower limit, but it is preferably 10 nM or more, more preferably 20 nM or more, further preferably 30 nM or more, further preferably 40 nM or more.

[0058] In addition to the DNA polymerase, single-stranded RNA, nucleic acid molecule, DNA primer pair, and dNTP in the mixture, a buffer, salts, etc. may be added, and an enzyme protectant, a melting temperature (Tm) regulator, a surfactant, etc. may be added as needed. As the buffer, a buffer such as Tris-HCl having a buffering action from neutral to weakly alkaline is used. The pH is adjusted to near the optimal pH according to the DNA polymerase used. Salts are appropriately added to maintain the enzyme activity or the melting temperature (Tm) of the nucleic acid, and specifically, KCl, NaCl, MgCl2, MgSO4, (NH4)2SO4, etc. are used. As the enzyme protectant, bovine serum albumin or sugars are used. In addition, as the melting temperature (Tm) regulator, dimethyl sulfoxide (DMSO), formamide, betaine (N,N,N,-trimethylglycine), etc. are used. As the surfactant, Tween20, TritonX, etc. are used.

[0059] In a nucleic acid amplification method, a DNA primer pair can amplify a first amplification region including a target sequence (target region) in a target nucleic acid molecule contained in a nucleic acid molecule and a second amplification region including a mutant sequence (mutant region). In other words, the DNA primer pair includes a forward primer and a reverse primer. The forward primer is designed to be located upstream of the first amplification region and the second amplification region, and the reverse primer is designed to be located downstream of the first amplification region and the second amplification region.

[0060] dNTPs include deoxyadenosine triphosphate (dATP), deoxyuridine triphosphate (dUTP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and optionally deoxythymidine triphosphate (dTTP) at a concentration lower than that of dUTP.

[0061] In some embodiments, dNTPs do not include dTTP. In other embodiments, dNTPs contain dTTP, and the concentration of dTTP is lower than that of dUTP. The concentration of dTTP can be 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.1% or less, 0.01% or less of the concentration of dUTP.

[0062] 2-2 Denaturation step In this step, the mixture is maintained at a temperature above the denaturation temperature. The denaturation temperature is the temperature at which double-stranded nucleic acid (nucleic acid molecule) reversibly thermally denatures (dissociates) into single-stranded nucleic acid, generally 90°C or higher, 91°C or higher, 92°C or higher, 93°C or higher, 94°C or higher, 95°C or 98°C or higher. The denaturation step is carried out for 10 seconds to 60 seconds, but is not limited thereto.

[0063] 2-3 Annealing step In the annealing step, the mixture is maintained at the annealing temperature. In the annealing step, the nucleic acid molecule (template DNA) thermally denatured into single-stranded binds to single-stranded RNA and the primer pair to form a double-strand (priming reaction). The annealing temperature is 50°C to 72°C, and can be in the range between two values selected from the group consisting of 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, and 72°C. The annealing step is usually carried out for 10 seconds to 30 seconds, but is not limited thereto.

[0064] 2-4 DNA Extension Step In the DNA extension step, the above-mentioned mixture is maintained at the DNA extension temperature. The DNA extension step uses the primer as a starting point and extends the DNA through DNA polymerase. The DNA extension temperature depends on the DNA polymerase used, and a widely used temperature is around 72 °C. The DNA extension step can be changed according to the length of the amplified strand to be amplified. Usually, it is 1 kb of amplified strand length per minute, but it is not limited to this.

[0065] In addition, the temperatures of the annealing step and the DNA extension step can also be the same.

[0066] 2-5 Amplification Step In the amplification step, the DNA strand is amplified by sequentially repeating the above-mentioned denaturation step, annealing step, and DNA extension step. The number of repetitions (cycles) of the amplification step is usually 25 to 40 cycles, but it is not limited to this.

[0067] 2-6 Detection Step In the detection step, the amplification product obtained by the amplification of the DNA strand is detected. For example, by the amount of the amplification product of the target region in the mixture after the nucleic acid amplification reaction, it is possible to confirm the case where the nucleic acid amplification of the target region is specifically inhibited. The amount of the amplification product of the target region in the mixture after the nucleic acid amplification reaction can be confirmed, for example, by subjecting the reaction solution after the nucleic acid amplification reaction to agarose gel electrophoresis and according to the concentration of the band of the amplification product of the target region. Compared with the case where the band of the amplification product of the target region is not detected or no single-stranded nucleic acid is added to the reaction system, if the amount of the amplification product of the target region decreases, it can be determined that the nucleic acid amplification of the target region is specifically inhibited. Therefore, when implementing the nucleic acid amplification inhibition method of the present invention, it is preferable to use, in addition to the reaction system to which the single-stranded nucleic acid hybridizing with the target region is added, a reaction system without adding the single-stranded nucleic acid as a control at the same time. The amplification product is separated by a known method such as agarose gel electrophoresis, and a known sequence analysis method is applied thereto to obtain the base sequence information of the amplification product.

[0068] When detecting the amplification product by the real-time PCR method, the mixture contains a nucleic acid probe labeled with a fluorescent dye. In this case, the amplification product is detected by the fluorescence of the fluorescent dye labeling.

[0069] 2-6 Reverse Transcription Step In the reverse transcription step, the above-mentioned nucleic acid molecule is obtained from the target RNA using reverse transcriptase. The reverse transcription step is useful when it is necessary to confirm mutations in RNA. This is because this method can be applied by reverse transcribing the RNA into cDNA.

[0070] 3 Method for Specifically Inhibiting Nucleic Acid Amplification of Target Region The method for specifically inhibiting nucleic acid amplification of the target region in this embodiment includes a provision step, a denaturation step, a maintenance step, an amplification step, and a detection step.

[0071] 3-1 Maintenance Step In the maintenance step, the mixture is maintained at 45°C to 54°C.

Examples

[0072] Experimental Materials and Methods Oligonucleotides ODN (oligodeoxyribonucleotide) was chemically synthesized by Eurofins Genomics (Tokyo, Japan) and used as a primer. Dual-labeled fluorescent ODN was chemically synthesized by FASMAC (Tokyo, Japan). Chemically synthesized ORN (oligoribonucleotide) and phosphorothioate-modified ORN were purified using high-performance liquid chromatography (FASMAC).

[0073] Template DNA and RNA Genomic (g) DNA was extracted from 293T, NCI-H1975, and HCT116 cells using the standard phenol-chloroform extraction method. 293T cells harbor wild-type (WT) epidermal growth factor receptor (EGFR) and KRAS genes. NCI-H1975 has a mutant EGFR sequence equivalent to T790M and L858R in one allele. HCT116 has a mutant KRAS sequence equivalent to G13D in one allele. The gDNAs of 293T and NCI-H1975 were mixed to prepare a 10 ng gDNA mixture composed of WT and mutant EGFR genes (the ratio of mutant to WT was 5 - 0.2%). The copy number of a single-copy gene can be calculated from the concentration of the gDNA used as a template and the average mass of DNA in the cell. Generally, 10 ng of template gDNA contains 3,333 copies of a single-copy gene (such as EGFR), so a gene with a mutant-to-WT ratio of 1% corresponds to 33 copies. WT EGFR (HD141, Horizon Discovery, Cambridge, UK) or DNA was extracted from FFPE samples composed of human cells with WT and mutant EGFR genes (HD300, Horizon Discovery) using the Quick-DNA FFPE kit (Zymo Research, Irvine, CA, USA). RNA was extracted from MRC-5 and NCI-H1975 cells using Isogen II (NIPPON GENE CO., LTD., Tokyo, Japan). MRC-5 harbors the WT EGFR gene sequence.

[0074] ORNi-PCR and Real-Time ORNi-PCR Using Taq DNA Polymerase ORNi-PCR was performed using 10 ng of gDNA and THUNDERBIRD Probe qPCR Mix (Toyobo, Osaka, Japan), GoTaq Probe qPCR Master Mix (Promega, Madison, WI, USA), Premix Taq (TaKaRa Taq Version) (Takara Bio Inc., Shiga, Japan), GeneAce Probe qPCR Mix II (NIPPON GENE CO., LTD., Tokyo, Japan), or EmeraldAmp MAX PCR Master Mix (Takara Bio Inc.). The reaction mixture (10 μl) was prepared according to the manufacturer's operating instructions, and ORNi-PCR was performed using a Mastercycler nexus (Eppendorf, Hamburg, Germany) or LifeEco ver. 2.0 (Hangzhou Bioer Technologies, Hangzhou, China).

[0075] In real-time ORNi-PCR, a dual-labeled fluorescent probe (0.2 μM) was added to the reaction mixture, and real-time ORNi-PCR was performed using a CFXConnect real-time PCR detection system (CFX Connect Real-Time PCR Detection System, Bio-Rad Laboratories, Hercules, CA, USA). The ORNi-PCR products were electrophoresed on a 2-3% agarose gel. Each product was subjected to DNA sequence analysis (Eurofins Genomics) as needed. DNA sequence analysis was performed using Applied Biosystems Sequence Scanner Software v2.0 (ThermoFisher Scientific, Waltham, MA, USA).

[0076] The ORN-mediated blocking assay using chemically modified Taq DNA polymerase was performed using Hot-Start Gene Taq NT (NIPPON GENE CO., LTD.) or AmpliTaq Gold 360 DNA Polymerase (ThermoFisher Scientific, Vilnius, Lithuania) containing 10 ng of gDNA and chemically modified Taq DNA polymerase, or Gene Taq NT (NIPPON GENE CO., LTD.) containing unmodified (conventional) Taq DNA polymerase for ORNi-PCR. Hot-Start Gene Taq NT is the Taq DNA polymerase used in GeneAce Probe qPCR Mix II (NIPPON GENE CO., LTD.). The reaction mixture (10 μl) was prepared using a dNTP mixture containing dTTP (component of the kit) or dUTP (NTP-501, Toyobo), and ORNi-PCR was performed using Mastercycler nexus (Eppendorf) or LifeEcover.2.0 (Hangzhou Bioer Technologies).

[0077] One-step RT-ORNi-PCR and one-step real-time RT-ORNi-PCR One-step RT-ORNi-PCR was performed using 25 ng of RNA and QIAGEN OneStep RT-PCR Kit (Qiagen, Hilden, Germany). The reaction mixture (10 μl) was prepared using a dNTP mixture containing dUTP (Toyobo) (instead of dTTP (component of the kit)), and one-step RT-ORNi-PCR was performed using LifeEco ver.2.0 (Hangzhou Bioer Technologies). Alternatively, one-step RT-ORNi-PCR was performed using 25 ng of RNA and iTaq Universal Probes One-Step Kit (Bio-Rad Laboratories). The reaction mixture (10 μl) was prepared according to the manufacturer's instructions, and one-step RT-ORNi-PCR was performed using LifeEco ver.2.0 (Hangzhou Bioer Technologies).

[0078] In one-step real-time RT-ORNi-PCR, a dual-labeled fluorescent probe (0.2 μM) was added to the reaction mixture before amplification using the CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories).

[0079] Study using the ORN-induced blocking mechanism of one-step RT-ORNi-PCR To investigate the RT inhibitory effect of ORN, a reaction mixture (10 μl) containing ORN, 25 ng of RNA, and reagents of the iTaq Universal Probes One-Step Kit (Bio-Rad Laboratories) was prepared according to the manufacturer's operating instructions. The RT reaction was carried out at 50 °C, and the cDNA was purified using the FastGene Gel / PCR Extraction Kit (FastGene Gel / PCR Extraction Kit, NIPPON GENE CO., LTD., Tokyo, Japan). Subsequently, a reaction mixture (10 μl) containing the purified cDNA and THUNDERBIRD Probe qPCR Mix (Toyobo) was prepared according to the manufacturer's operating instructions. PCR was performed using the Mastercycler nexus (Eppendorf). To investigate the blocking effect of ORN in the PCR step, a reaction mixture (10 μl) containing 25 ng of RNA and reagents of the iTaq Universal Probes One-Step Kit (Bio-Rad Laboratories) was prepared according to the manufacturer's operating instructions. After the RT reaction was carried out at 50 °C, ORN was added, and the PCR step was performed.

[0080] Example 1 ORNi-PCR using Taq DNA polymerase Experimental Example 1-1 Using the gDNA and ORN extracted from 293T cells and NCI-H1975 cells, three Taq DNA polymerases (THUNDERBIRD Probe qPCR Mix (Toyobo), GoTaq Probe qPCR Master Mix (Promega), Premix Taq (TaKaRa Taq Version) (Takara Bio Inc.)) were tested for their suitability for ORNi-PCR. 293T cells have WT EGFR, and one allele of the EGFR gene in NCI-H1975 cells has an L858R mutation (hereinafter referred to as "L858R EGFR") (Figure 1). The sequence of ORN is a sequence targeting the WT EGFR sequence corresponding to L858 (ORN_EGFR_L858, (Sequence ID No. (SEQ ID NO:) 1)) (Figure 1).

[0081] ORNi-PCR was performed using a Taq DNA polymerase with 5'-3' exonuclease activity. The primer pairs used were hEGFR-Exon21-F (Sequence ID No. 2) and hEGFR-Exon21-R (Sequence ID No. 3). Table 1 shows the PCR conditions in THUNDERBIRD Probe qPCR Mix and GoTaq Probe qPCR Master Mix, and Table 2 shows the PCR conditions in Premix Taq.

[0082]

Table 1

[0083]

Table 2

[0084] Figures 2 to 4 respectively show the electrophoresis photographs of the nucleic acids amplified using THUNDERBIRD Probe qPCR Mix, GoTaq Probe qPCR Master Mix, and Premix Taq.

[0085] In our previous study, we found that under optimized experimental conditions (i.e., 0.5 - 2 μM ORN; 56 to 59 °C in the annealing + extension step), when using ORN_EGFR_L858 targeting the WT EGFR sequence corresponding to L858 and KOD DNA polymerase without 5'-3' exonuclease activity, the amplification of the L858R EGFR sequence was effectively inhibited (not shown), rather than that of WT.

[0086] As shown in FIGS. 2 to 4, in ORNi-PCR using Taq DNA polymerase with 5'-3' exonuclease activity, even when the concentration of the ORN was increased and the annealing + extension step was carried out at 56°C, this inhibitory effect was not observed (FIGS. 2 to 4). This indicates that the hybridized ORN is removed during DNA extension.

[0087] On the other hand, when the annealing + extension step was carried out at 50°C, the ORN specifically inhibited the amplification of the WT EGFR gene (FIGS. 2 to 4).

[0088] Experimental Example 1-2 ORNi-PCR was carried out using an ORN (ORN_EGFR_T790_18b, (SEQ ID NO: 4)) targeting another position (corresponding to T790) within the WT EGFR gene (FIG. 5). Using NCI-H1975 cells with a T790M mutation (hereinafter referred to as "T790M EGFR") in one allele of the EGFR gene, ORN_EGFR_T790_18b, and the hEGFR-Exon20 primer pair (hEGFR-Exon20-F3 (SEQ ID NO: 32) and hEGFR-Exon20-R3 (SEQ ID NO: 33)), except for this, ORNi-PCR was carried out by the method of Experimental Example 1-1. In any of the Taq DNA polymerases, DNA amplification at 50°C (annealing + extension step) was sequence-specifically inhibited (FIGS. 6 to 8).

[0089] Experimental Example 1-3 ORNi-PCR was carried out using an ORN (ORN_KRAS_G13 (SEQ ID NO: 5)) targeting the wild-type KRAS (WT KRAS) gene (corresponding to G13) (FIG. 9). Using HCT116 cells with a G13D mutation (hereinafter referred to as "G13D KRAS") in one allele of the KRAS gene, ORN_KRAS_G13, and the hKRAS primer pair (hKRAS-F4 (SEQ ID NO: 6) and hKRAS-R2 (SEQ ID NO: 7)), except for this, ORNi-PCR was carried out by the method of Experimental Example 1-1. In any of the Taq DNA polymerases, DNA amplification at 50°C (annealing + extension step) was sequence-specifically inhibited (FIGS. 10 to 12).

[0090] Conclusion of Example 1 These results indicate that Taq DNA polymerase with 5'-3' exonuclease activity is suitable for ORNi-PCR with an annealing + extension step at a low temperature (around 50°C).

[0091] Unlike DNA polymerase (α type) without 5'-3' exonuclease activity, Taq DNA polymerase with 5'-3' exonuclease activity can actively remove hybridized (Figure 13A and Figure 13B) during DNA extension near the melting temperature (Tm) of ORN / DNA hybridization (for example, 56 °C in ORN_EGFR_L858).

[0092] At temperatures below Tm (around 50 °C), the removal of hybridized ORN can be inhibited. At such temperatures, Taq DNA polymerase cannot actively remove ORN hybridized during DNA extension (Figure 13C). In this case, ORN that does not match the target sequence is removed more effectively than fully hybridized ORN, and the efficiency of DNA amplification is different (Figure 13C).

[0093] Example 2 Inhibition of sequence-specific DNA amplification by ORNi-PCR using GeneAce qPCR Master Mix II Example 2-1 Evaluate GeneAce qPCR Master Mix II (hereinafter referred to as GeneAce Taq), which is different from the Taq DNA polymerase used in Example 1. Except for the use of GeneAce Taq and PCR conditions, the method of Experiment Example 1-1 was followed for ORNi-PCR. Table 3 shows the PCR conditions.

[0094]

Table 3

[0095] Figure 14A shows an electrophoresis photograph of the nucleic acid amplified using GeneAce Taq. Figure 14B shows the result of DNA sequence analysis of the amplification product indicated by the arrow in Figure 14A. As shown in Figure 14A and Figure 14B, the amplification of the WT EGFR gene was sequence-specifically inhibited under specific experimental conditions (i.e., 2 μM of ORN_EGFR_L858; annealing + extension step at 59 °C).

[0096] These experimental conditions are the same as the optimal conditions for ORNi-PCR using KOD DNA polymerase, so it is considered that the inhibition mode is the same (Figure 13A and Figure 15).

[0097] Example 2-2 ORNi-PCR was performed using ORN (ORN_EGFR_T790_18b, (SEQ ID NO: 4)) targeting another position within the WT EGFR gene (equivalent to T790). Except for the use of GeneAce Taq and the PCR conditions, ORNi-PCR was performed by the method of Experimental Example 1-2. Table 4 shows the PCR conditions.

[0098]

Table 4

[0099] In GeneAce Taq, DNA amplification at 59 °C (annealing + extension step) was also inhibited sequence-specifically (Figure 16). Experimental Example 2-3 ORNi-PCR was performed using ORN (ORN_KRAS_G13 (SEQ ID NO: 5)) targeting the WT KRAS gene (equivalent to G13). Except for the use of GeneAce Taq and the PCR conditions, the rest was carried out by the method of Experimental Example 1-3. Table 5 shows the PCR conditions.

[0100]

Table 5

[0101] In GeneAce Taq, DNA amplification at 59 °C (annealing + extension step) was also inhibited sequence-specifically (Figure 17).

[0102] Experimental Example 2-4 GeneAce Taq is a reagent optimized for real-time PCR using dual-labeled fluorescent probes. To investigate its application in real-time ORNi-PCR, the primer positions were optimized to amplify approximately 0.2 kbp of the EGFR gene. Figure 18 shows the base sequence of the nucleic acid to be amplified by the optimized primer pair (SEQ ID NO: 8). The box shown upstream (5' side) of the base sequence indicates the position of the forward primer (hEGFR-Exon21-F8, SEQ ID NO: 9), and the box shown downstream (3' side) of the base sequence indicates the position of the reverse primer (hEGFR-Exon21-R8, SEQ ID NO: 10). The underlined part indicates the position corresponding to L858.

[0103] To specifically inhibit the WT EGFR sequence, the concentrations of ORN_EGFR_L858 were set to 0 μM, 3 μM, and 6 μM. In addition, another ORN (ORN_EGFR_L858_17b (SEQ ID NO: 11), Figure 19) targeting the WT EGFR sequence corresponding to L858 was also tested. Table 6 shows the PCR conditions.

[0104]

Table 6

[0105] As shown in FIGS. 20A and 20B, the ORN also specifically inhibited the amplification of the WT EGFR sequence under the conditions of the annealing + extension step at 6 μM and 56°C.

[0106] FIGS. 21 and 22 show the electrophoresis photographs of ORN_EGFR_L858 (annealing + extension step at 56°C) and ORN_EGFR_L858 (annealing + extension step at 59°C), respectively. These ORNs also specifically inhibited the amplification of the WT EGFR sequence under the conditions of the annealing + extension step at 6 μM and 56°C.

[0107] Thus, it can be seen that GeneAce Taq can be applied to real-time ORNi-PCR using dual-labeled fluorescent probes.

[0108] Example 3 Perform real-time ORNi-PCR using a combination of GeneAce Taq and dual-labeled fluorescent probes Experimental Example 3-1 Perform real-time ORNi-PCR using GeneAce Taq with dual-labeled fluorescent probes. FIG. 23 shows a schematic diagram of conventional real-time PCR using dual-labeled fluorescent probes, and FIG. 24 shows a schematic diagram of real-time ORNi-PCR using dual-labeled fluorescent probes. Use hEGFR-Exon21-F8 and hEGFR-Exon21-R8 as primers.

[0109] Prepare various dual-labeled fluorescent probes targeting the L858R EGFR mutation (FIGS. 25 and Table 7).

[0110]

Table 7

[0111] Table 8 shows the PCR conditions.

[0112]

Table 8

[0113] The results of conventional real-time PCR are shown in FIGS. 26 to 29. As shown in FIGS. 28 and 29, Probe_EGFR_L858R_18b and Probe_EGFR_L858R_Sense_15b detected the target mutation more specifically and effectively in the conventional real-time PCR using GeneAce Taq.

[0114] The results of real-time ORNi-PCR and real-time PCR are shown in FIGS. 30A, 30B to 32A, and 32B. In the real-time ORNi-PCR of ORN_EGFR_L858_17b and Probe_EGFR_L858R_18b, strong fluorescence was detected even when the L858R EGFR gene accounted for only 0.2 to 1% of the total EGFR template (6 to 33 copies of L858R EGFR) (FIGS. 30A and 30B, 31A and 31B).

[0115] By DNA sequence analysis, the amplification of the mutant sequence was confirmed in the real-time ORNi-PCR amplicons, and due to incomplete inhibition, the WT EGFR sequence was also amplified (FIGS. 30B and 31B). The same results were obtained in the real-time ORNi-PCR using ORN_EGFR_L858 and Probe_EGFR_L858R_Sense_15b (FIGS. 33A and 33B). On the other hand, in the conventional real-time PCR using Probe_EGFR_L858R_18b or Probe_EGFR_L858R_Sense_15b, limited amplification of fluorescence intensity was shown when the mutation accounted for 1% of the total EGFR template (FIGS. 32A and 34A). The presence of the mutation was not confirmed in the DNA sequence analysis (FIGS. 32B and 34B).

[0116] Therefore, real-time ORNi-PCR preferably amplifies a specific sequence (e.g., the mutant sequence) and can detect it with a dual-labeled fluorescent probe. On the other hand, the results of conventional real-time PCR may have false positives.

[0117] Experimental Example 3-2 DNA extracted from formalin-fixed paraffin-embedded (FFPE) specimens is used in the medical field for cancer diagnosis and the like. Therefore, DNA extracted from commercially available FFPE human specimens simulating patient specimens was used in real-time ORNi-PCR.

[0118] As shown in FIGS. 35A to 35B and 36A to 36B, real-time ORNi-PCR using Probe_EGFR_L858R_18b and ORN_EGFR_L858_17b can specifically and stably amplify L858R EGFR in the extracted DNA compared to conventional real-time PCR.

[0119] Conclusion of Example 3 From the above, we successfully performed real-time ORNi-PCR using double-labeled fluorescent probes with GeneAce Taq and confirmed that its sensitivity and specificity in the detection of single-base differences were superior to those of traditional real-time PCR. When necessary, a definitive diagnosis can be made by DNA sequence analysis based on the amplicons generated by real-time ORNi-PCR.

[0120] Example 4 Mechanism of DNA extension inhibition mediated by ORN using GeneAce Taq Experimental Example 4-1 To investigate the effect of chemically modified Taq DNA polymerase / dUTP on the inhibition of target DNA amplification by ORN, a chemically modified Taq DNA polymerase (Hot-Start Gene Taq NT) and an unmodified version (Gene Taq NT) used in GeneAce Taq were purchased from the same company.

[0121] Except for the use of chemically modified Taq DNA polymerase (Hot-Start Gene Taq NT), primer pairs (hEGFR-Exon21-F8 (SEQ ID NO: 9) and hEGFR-Exon21-R8 (SEQ ID NO: 10)), and PCR conditions, ORNi-PCR was performed according to the method of Experimental Example 1-1. Table 9 shows the PCR conditions.

[0122]

Table 9

[0123] As shown in FIGS. 37 to 39, ORN_EGFR_L858_17b specifically inhibited the amplification of WT EGFR in the case of adding dUTP (without dTTP) to the chemically modified Taq DNA polymerase.

[0124] Experimental Example 4-2 In addition, experiments were performed using another chemically modified Taq DNA polymerase (AmpliTaq Gold 360). Except for the use of AmpliTaq Gold 360 and PCR conditions, ORNi-PCR was performed according to the method of Experimental Example 4-1. Table 10 shows the PCR conditions.

[0125]

Table 10

[0126] As shown in FIGS. 40A to 40B, the same results were obtained with AmpliTaq Gold 360.

[0127] Comparative Example 4-1 Experiments were conducted using unmodified Taq DNA polymerase (Gene Taq NT). Except for Gene Taq NT, ORNi-PCR was performed according to the method of Experimental Example 4-1 for the rest.

[0128] On the other hand, even under the conditions of the presence of dUTP, the reaction buffer of Hot-Start Gene Taq NT, and the long-term heat treatment (5 minutes) required for the activation of chemically modified Taq DNA polymerase, DNA amplification caused by unmodified Taq DNA polymerase (Gene Taq NT) was not inhibited (Figs. 41 to 43).

[0129] Results of Example 4 These results clearly show that in order to inhibit target DNA amplification caused by ORN, it is important to use chemically modified Taq DNA polymerase and dUTP.

[0130] Example 5 Inhibition of DNA extension reaction caused by Taq DNA polymerase by phosphorothioate-modified ORN Experimental Example 5-1 Phosphorothioate modification can improve the resistance of RNA to degradation caused by ribonuclease. Utilizing this property, antisense oligonucleotides can be stabilized and used as oligonucleotide therapeutic agents.

[0131] It was investigated whether the resistance of ORN to degradation caused by the 5'-3' exonuclease activity of Taq DNA polymerase would be enhanced by phosphorothioate modification.

[0132] ORN_EGFR_L858 (ORN_EGFR_L858_S_All) with phosphorothioate modification of all phosphodiester bonds and ORN_EGFR_L858 (ORN_EGFR_L858_S_5) with partial modification of 5 phosphodiester bonds at the 5' end were synthesized (Table 11).

[0133]

Table 11

[0134] Except for the PCR conditions, ORNi-PCR was performed according to the method of Experimental Example 1-1 for the rest. Table 12 shows the PCR conditions.

[0135]

Table 12

[0136] ORN_EGFR_L858_S_All (0.5 μM) sequence-specifically inhibited DNA extension by conventional Taq DNA polymerase (THUNDERBIRD Probe qPCR Mix) under the conditions of the annealing + extension step at 56 °C (Figures 44A to 44B).

[0137] On the other hand, ORN_EGFR_L858_S_5 did not show such inhibitory effect even at high concentrations (Figure 45).

[0138] ORN_EGFR_L858_S_All also showed sequence-specific inhibition of DNA amplification when using another conventional Taq DNA polymerase (GoTaq Probe qPCR Master Mix) (Figures 46A - 46B).

[0139] Thus, fully phosphorothioated ORN (ORN_EGFR_L858_S_All) can sequence-specifically inhibit DNA extension by conventional Taq DNA polymerase, and thus can identify single-base mutations.

[0140] Experimental Example 5-2 The experimental conditions of ORN_EGFR_L858_S_All were optimized for application to real-time ORNi-PCR. Except for the use of THUNDERBIRD Probe qPCR Mix and PCR conditions, ORNi-PCR was performed according to the method of Experimental Example 2-4. Table 13 shows the PCR conditions.

[0141]

Table 13

[0142] As shown in Figures 47A to 47B, ORN_EGFR_L858_S_All also showed sequence-specific inhibition of DNA amplification even under these conditions.

[0143] Next, experiments were conducted by extending the time of the annealing + extension step (from 30 seconds to 2 minutes). In this case, the inhibitory efficiency of ORN_EGFR_L858_S_All decreased (Figure 48).

[0144] This indicates that fully phosphorothioated ORN is removed from the target DNA hybridized via Taq DNA polymerase when using a longer extension step. Therefore, the control of DNA extension time is important.

[0145] Experimental Example 5-3 ORN_EGFR_L858_S_All and Probe_EGFR_L858R_Sense_15b were combined and used for real-time ORNi-PCR using traditional Taq DNA polymerase (THUNDERBIRD Probe qPCR Mix) and traditional real-time PCR using Probe_EGFR_L858R_Sense_15b. Except for the PCR conditions, real-time ORNi-PCR and traditional real-time PCR were performed according to the method of Experimental Example 5-2. Table 14 shows the PCR conditions.

[0146]

Table 14

[0147] In the real-time ORNi-PCR based on the combination of ORN_EGFR_L858_S_All and Probe_EGFR_L858R_Sense_15b, when the L858R EGFR mutation accounted for 1% of the overall EGFR template, significant amplification occurred (Figs. 49A to 49B).

[0148] In contrast, in the traditional real-time PCR using Probe_EGFR_L858R_Sense_15b, the detection of the mutation at the same WT-to-mutation ratio was not clear (Figs. 50A - 50B).

[0149] Thus, phosphorothioate ORN is interchangeable with traditional Taq DNA polymerase for real-time ORNi-PCR and can enhance the specificity for the target mutation.

[0150] Experimental Example 5-4 In addition, real-time ORNi-PCR using phosphorothioate ORN was also tested for GeneAce Taq DNA polymerase. Except for the use of GeneAce Taq DNA polymerase and the PCR conditions, ORNi-PCR was performed according to the method of Experimental Example 2-4. Table 15 shows the PCR conditions.

[0151]

Table 15

[0152] As shown in Figs. 51A to 51B, compared with unmodified ORN, fully phosphorothioated ORN blocked target DNA amplification at lower concentrations (0.1 - 0.2 μM) and non-specifically inhibited DNA amplification at higher concentrations (1 μM). Partially modified ORN (ORN_EGFR_L858_S_5) did not inhibit DNA extension (Fig. 52).

[0153] When the L858R EGFR mutation accounted for 1% of the overall EGFR mold, real-time ORNi-PCR showed strong amplification (Figures 53A to 53B).

[0154] Conclusion of Example 5 Thus, phosphorothioated ORN can also effectively block DNA elongation caused by chemically modified Taq DNA polymerase.

[0155] Example 6 One-step real-time reverse transcription-ORNi-PCR (RT-ORNi-PCR) method using phosphorothioated ORN Experimental Example 6-1 To detect single-base mutations in RNA, a one-step RT-ORNi-PCR was attempted using a one-step RT-PCR reagent (QIAGEN OneStep RT-PCR Kit) containing reverse transcriptase and chemically modified Taq DNA polymerase. The protocol for one-step RT-ORNi-PCR is schematically shown in Figure 54. The positions of mutations, ORN, probes, and primer pairs on the WT EGFR gene are schematically shown in Figure 55.

[0156] ORN_EGFR_L858_S_All, which is a fully phosphorothioated ORN, was used, and ORN_EGFR_L858, which is an unmodified ORN, was used. The primer pair used was hEGFR-Exon21-F8 (SEQ ID NO: 9) as the forward primer and hEGFR-T790M_L858R_cDNA-R5 (SEQ ID NO: 19) as the reverse primer. Table 16 shows the PCR conditions.

[0157]

Table 16

[0158] The results are shown in Figures 56A, 56B, and 57. Using total RNA extracted from MRC-5 cells and NCI-H1975 cells with the WT EGFR gene, a one-step RT-ORNi-PCR using ORN_EGFR_L858_S_All (0.5 μM ORN; annealing + extension step at 57.5 °C) for detecting the L858R EGFR mutation was successfully performed.

[0159] ORN_EGFR_L858, an unmodified ORN, did not show complete inhibition of target DNA amplification even at a higher concentration (6 μM) (Figure 58). This indicates the possibility that unmodified ORN is degraded by the reverse transcriptase and DNA polymerase in this reagent.

[0160] Therefore, one-step RT-ORNi-PCR using phosphorothioated ORN can identify single-base mutations in RNA.

[0161] Experimental Example 6-2 Although the QIAGEN OneStep RT-PCR Kit is not designed for real-time detection of DNA amplification, an attempt was made to perform one-step real-time RT-ORNi-PCR using the QIAGEN OneStep RT-PCR Kit and a dual-labeled fluorescent probe. FIG. 59 schematically shows the protocol for one-step real-time RT-ORNi-PCR.

[0162] The L858R EGFR mutation in the EGFR mRNA of NCI-H1975 cells was clearly detected by Probe_EGFR_L858R_Sense_15b (FIG. 60A), and real-time detection was possible by the combination of this reagent (QIAGEN OneStep RT-PCR Kit) and the probe.

[0163] However, one-step real-time RT-PCR failed to detect the L858R EGFR mutation in the mixed RNA (which mixed 25 ng of total RNA of MRC-5 and 0.25 ng of NCI-H1975 so that the proportion of NCI-H1975 RNA in the total RNA was 1%) (FIGS. 60A to 60B).

[0164] In contrast, one-step real-time RT-ORNi-PCR normally detected the L858R EGFR mutation in the mixed total RNA (FIGS. 61A to 61B), showing significant technical advantages in the detection of single-base differences in RNA.

[0165] Experimental Example 6-3 Next, the usefulness of one-step real-time RT-ORNi-PCR was confirmed using the iTaq Universal Probes One-Step Kit containing unmodified Taq DNA polymerase. FIG. 62 schematically shows the protocol for one-step real-time RT-ORNi-PCR using the iTaq Universal Probes One-Step Kit.

[0166] ORN_EGFR_L858_S_All, which is a fully phosphorothioated ORN, was used. As the primer pair used, hEGFR-Exon21-F8 (SEQ ID NO: 9) was used as the forward primer, and hEGFR-T790M_L858R_cDNA-R5 (SEQ ID NO: 19) was used as the reverse primer. Table 17 shows the PCR conditions.

[0167]

Table 17

[0168] Different from one-step real-time RT-PCR (Figs. 63A to 63B), one-step real-time RT-ORNi-PCR can detect the L858R EGFR mutation under optimal experimental conditions even when the mixed total RNA contains 1 to 0.2% of NCI-H1975 total RNA (Figs. 64A to 64B and Figs. 65A to 65B).

[0169] Thus, in the detection of single-base mutations in RNA, one-step real-time RT-ORNi-PCR is superior to traditional one-step real-time RT-PCR in terms of sensitivity and specificity.

[0170] Experimental Example 6-4 When using 25 ng of NCI-H1975 total RNA, one-step real-time ORNi-RT-PCR showed a lower quantification cycle (Cq) than one-step real-time RT-PCR (compare Figs. 61A and 60A, and Figs. 64 and 63).

[0171] Since ORN_EGFR_L858R_S_All is complementary to EGFR mRNA (Fig. 55), it binds to mRNA even in the RT step of the one-step RT-ORNi-PCR reaction, thus inhibiting cDNA synthesis by reverse transcriptase.

[0172] Then, the stage of the one-step RT-ORNi-PCR reaction in which this ORN sequence-specifically inhibits DNA extension was investigated. Fig. 66 schematically shows the steps of this study. In this study, except for the addition timing of ORN_EGFR_L858R_S_All, the other procedures were carried out according to the method of Experimental Example 6-3 for RT-ORNi-PCR.

[0173] As shown in Figs. 67A to 67B, this ORN sequence-specifically inhibited the amplification of target DNA from cDNA in the PCR stage.

[0174] When cDNA was purified after RT in the presence of this ORN and then PCR was carried out, DNA amplification was non-specifically inhibited (Figs. 68A to 68B).

[0175] Experimental Example 6-5 Designed as an phosphorothioated ORN non-complementary to EGFR mRNA, ORN_EGFR_L858_Sense_S_All (SEQ ID NO: 18) (FIGS. 69 and 70). Except for the use of ORN_EGFR_L858_Sense_S_All and the PCR conditions, the one-step RT-ORNi-PCR was carried out according to the method of Experimental Example 6-3. Table 18 shows the PCR conditions.

[0176]

Table 18

[0177] As shown in FIGS. 71A to 71B, it was shown that ORN_EGFR_L858_Sense_S_All also inhibited the DNA synthesis of WT EGFR in the one-step RT-ORNi-PCR reaction.

[0178] Experimental Example 6-6 Then, the stage of the one-step RT-ORNi-PCR reaction in which the ORN sequence specifically inhibits DNA extension was studied. The steps of this study were the same as those shown in FIG. 66 except for the use of ORN_EGFR_L858_Sense_S_All. In addition, in this study, RT-ORNi-PCR was carried out according to the method of Experimental Example 6-5 except for the timing of addition of the ORN.

[0179] As shown in FIGS. 72A-72B, the ORN sequence specifically inhibited the amplification of the target DNA from cDNA in the PCR stage. When cDNA was purified after RT in the presence of the ORN and then PCR was carried out, DNA amplification was non-specifically inhibited, indicating that the ORN caused non-specific inhibition in the RT step (FIGS. 73A-73B).

[0180] Thus, although the discrimination of single-base differences is carried out in the PCR step of one-step RT-ORNi-PCR and it was found that phosphorothioated ORN non-specifically inhibits the activity of reverse transcriptase, it has been clarified that this does not affect the discrimination of single-base differences in one-step RT-ORNi-PCR.

[0181] Therefore, phosphorothioated ORNs complementary and non-complementary to the target RNA can be used in one-step RT-ORNi-PCR without restricting the degree of freedom in ORN design.

Claims

1. A kit, which comprises a DNA polymerase that can be used for PCR and single-stranded RNA. In the kit, (1) The DNA polymerase is subjected to a temporary inactivation treatment, and / or (2) The single-stranded RNA is resistant to degradation by RNA degrading enzymes. The DNA polymerase has 5'-3' exonuclease activity. The single-stranded RNA has a base sequence that can hybridize with a target sequence in a target nucleic acid molecule.

2. The kit according to claim 1, which further comprises a nucleic acid probe labeled with a fluorescent pigment. The base sequence of the nucleic acid probe is different from the base sequence of the single-stranded RNA due to at least one mutation.

3. The kit according to claim 1, wherein the single-stranded RNA that is resistant to degradation by the RNA degrading enzymes is a phosphorothioate-type single-stranded RNA.

4. The kit according to claim 1, wherein the DNA polymerase with chemical modification is a DNA polymerase modified with a dicarboxylic anhydride.

5. The kit according to claim 1, which further comprises dUTP.

6. A method for specifically inhibiting nucleic acid amplification in a target region. The method includes: A providing step of providing a mixture containing a DNA polymerase that can be used for PCR, single-stranded RNA, a target nucleic acid molecule, a DNA primer pair, and dNTP. A denaturation step of maintaining the mixture at a temperature above the denaturation temperature. An annealing step of maintaining the mixture at the annealing temperature. A DNA extension step of maintaining the mixture at the DNA extension temperature. An amplification step of amplifying a DNA strand by sequentially repeating the denaturation step, annealing step, and DNA extension step. And A detection step of detecting an amplification product obtained by the amplification of the DNA strand. (1) The DNA polymerase is subjected to a temporary inactivation treatment. and / or (2) The single-stranded RNA is resistant to degradation by RNA degrading enzymes. The DNA polymerase has 5'-3' exonuclease. The single-stranded RNA has a base sequence that can hybridize with a target sequence in the target nucleic acid molecule.

7. The method according to claim 6, wherein the mixture further comprises a nucleic acid probe labeled with a fluorescent pigment. The base sequence of the nucleic acid probe is different from the base sequence of the single-stranded RNA due to at least one mutation. The amplification product is detected by the fluorescence of the fluorescent pigment label.

8. The method according to claim 6, wherein the single-stranded RNA that is resistant to degradation by the RNA degrading enzymes is a phosphorothioate-type single-stranded RNA.

9. The method according to claim 6, wherein the DNA polymerase with chemical modification is a DNA polymerase modified with a dicarboxylic anhydride.

10. The method according to claim 9, wherein the dNTP includes dATP, dUTP, dGTP, dCTP, and optionally deoxythymidine triphosphate (dTTP) at a concentration lower than dUTP.

11. The method according to claim 6 further comprises a reverse transcription step of obtaining the target nucleic acid molecule from the target RNA using reverse transcriptase.

12. A method for specifically inhibiting nucleic acid amplification of a target region, the method comprising: a providing step of providing a mixture containing a DNA polymerase usable for PCR, single-stranded RNA, a target nucleic acid molecule, a DNA primer, and dNTP; a denaturation step of maintaining the mixture at a temperature above the denaturation temperature; a maintenance step of maintaining the mixture at 45°C to 54°C; an amplification step of amplifying the DNA strand by sequentially repeating the denaturation step and the maintenance step; and a detection step of detecting the amplification product obtained by the amplification of the DNA strand; the DNA polymerase has 5'-3' exonuclease, the single-stranded RNA has a base sequence capable of hybridizing with the target sequence in the target nucleic acid molecule.

13. The method according to claim 12, wherein the mixture further comprises a nucleic acid probe labeled with a fluorescent pigment, the base sequence of the nucleic acid probe is different from the base sequence of the single-stranded RNA due to at least one mutation, and the amplification product is detected by the fluorescence of the fluorescent pigment label.

14. The method according to claim 12 further comprises a reverse transcription step of obtaining the target nucleic acid molecule from the target RNA using reverse transcriptase.

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