High-sensitivity PCR

Abstract

To solve the problem that both the accuracy and detection sensitivity for identifying single nucleotide mutations are low in conventional identification of single nucleotide mutations in genes using TaqMan probes since the difference in binding affinity (melting temperature) between genes and probes is used for identification and unnecessary genes are also amplified.SOLUTION: In a highly sensitive PCR method using a DNA polymerase having proofreading activity and a chemically modified primer, in which an artificial nucleic acid that inhibits polymerase activity is placed at the second position from the 3'-end and natural DNA is placed at the 3'-end, if the 3'-end does not match the target base, extremely accurate detection becomes possible since the 3'-end is removed, the artificial nucleic acid is exposed, and polymerase activity is stopped.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a highly sensitive PCR method capable of detecting a single nucleotide variant with high precision.

Background Art

[0002] When discriminating a single nucleotide mutation of a gene, a method using a TaqMan probe is often used.

[0003] In Patent Document 1, in addition to the TaqMan probe, a WT (wild type) blocker nucleic acid fragment that is not decomposed by the 5'-exonuclease activity of polymerase is bound to the region containing the mutation point, and then primers are bound to perform PCR. If there is a mutation point in the DNA, the WT blocker nucleic acid fragment binds weakly to the mutant DNA, and PCR is performed, but it binds strongly to the wild type DNA without a mutation point and interferes with the wild type PCR. On the other hand, the TaqMan probe, conversely, binds weakly to the wild type DNA, binds more strongly to the mutant DNA, is decomposed by the 5'-exonuclease activity of polymerase, and fluorescence is enhanced as PCR progresses.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] The method using TaqMan probes identifies single nucleotide mutations in genes by taking advantage of the difference in binding affinity (melting temperature) between the gene and the probe. Therefore, it is theoretically impossible to set a temperature at which the TaqMan probe or the WT blocker binds 100% to either the mutant DNA or the wild-type DNA and does not bind at all to the other. To the extent that the TaqMan probe binds to the unintended wild-type DNA and the WT blocker binds to the unintended mutant DNA, the detection sensitivity will decrease. Therefore, it has not been possible to quantitatively analyze the mRNA derived from the wild-type gene and its single nucleotide mutant gene expressed in the same cell by real-time PCR, and there has been no means to quantitatively analyze the effect of a drug targeting the single nucleotide mutant gene at the gene level.

[0006] Therefore, first, the detection limit is a sample with a detection sensitivity of 50 - 100 copies or more. That is, it does not have high sensitivity such that amplification occurs with a smaller number of samples. Also, the mutation discrimination accuracy (the ratio of mutant type mixed in the wild type) was limited to about 1 - 5%. In addition, precise primer and probe design and reaction temperature setting are required for each individual evaluation system, and variation in mutation discrimination accuracy and detection accuracy is likely to occur for each test. Also, TaqMan probes are expensive.

Means for Solving the Problems

[0007] The high-sensitivity PCR method according to the present invention was conceived in view of the above problems. Nucleotides that inhibit polymerase activity can be amplified if they are not at the 3'-end, but when they are exposed at the 3'-end, the polymerase activity is inhibited. By utilizing this property, single nucleotide mutations are detected.

[0008] More specifically, the high-sensitivity PCR method according to the present invention uses a DNA polymerase having proofreading activity, and a chemically modified primer in which an artificial nucleic acid that inhibits polymerase activity is arranged second from the 3'-end and natural-type DNA is arranged at the 3'-end. 、the artificial nucleic acid is either of the formulas (3) or (4) It is characterized by this.

Effects of the Invention

[0009] The present invention is a PCR technique using the Cyber Green method in combination with a novel chemically modified primer and a DNA polymerase having proofreading activity. Since detection based on the difference in melting temperature is not performed, it can be said that it is a method for identifying single nucleotide mutations based on a principle different from the conventional one.

[0010] Therefore, even a target gene with a very small copy number (even possible from 10 copies) can be amplified, and even a contamination amount of 0.01% can be clearly detected.

[0011] In addition, since the mutation points can be specified with both forward and reverse primers, only the specimens that match at two positions simultaneously can be amplified, enabling very sensitive detection.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Figure 3

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Figure 8

Figure 9

Figure 10

Figure 11

Embodiments for Carrying Out the Invention

[0013] The highly sensitive PCR method according to the present invention will be described below with reference to the drawings and examples. It should be noted that the following description illustrates one embodiment and one example of the present invention, and the present invention is not limited to the following description. The following description can be modified without departing from the gist of the present invention. Also, embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention. In addition, all the documents described in this specification are incorporated herein by reference.

[0014] The present invention inserts a nucleotide that intentionally inhibits polymerase activity into the primer, utilizes the 3'-exonuclease activity to remove the base at the 3'-end when the 3'-end of the primer is different from the gene, exposes the nucleotide that inhibits polymerase activity at the 3'-end, and prevents further extension from the primer, thereby detecting single-base mutations.

[0015] In the high-sensitivity PCR method according to the present invention, a DNA polymerase having proofreading activity and an artificial nucleic acid that inhibits polymerase activity at the second position from the 3'-end are arranged, and a chemically modified primer in which natural DNA is arranged at the 3'-end is used.

[0016] The DNA polymerase having proofreading activity refers to a DNA polymerase having a function of removing a base that is incorrect between the template strand. It suffices to have 3'-exonuclease activity. Such a DNA polymerase is also called a HiFi polymerase and can be obtained through commercial channels. For example, TaKaRa EX Taq polymerase (Takara Bio Inc.), PfuUltra II Fusion DNA polymerase (Agilent Technologies), Phusion Plus DAN polymerase (Thermo Fisher Scientific), etc. can be preferably used.

[0017] Fig. 1 illustrates the primer used in the present invention. The primer has a structure in which nucleotides 20 from the 5'-end are linked by phosphodiester bonds. Nucleotide 20 is one in which a phosphate diester moiety 16 is bonded to a sugar 14 to which 18 nucleobases are linked. What is formed by linking nucleobase 18 and sugar 14 is called a nucleoside. Therefore, it can be said that nucleosides are linked by a phosphate diester moiety 16.

[0018] In the primer 10 used in the present invention, the chemical modification at the 2'-position of the sugar 14 of the second nucleotide 20b from the 3'-end may be a modification that inhibits the polymerase activity of the polymerase having proofreading activity. Note that the nucleotide 20a at the 3'-end is a natural-type nucleotide. Note that the natural type refers to deoxyribose or ribose, which is naturally present and does not inhibit polymerase activity.

[0019] As a specific example of the second nucleotide 20b from the 3'-end, the general formula is 2'-OR 1 RNA (the meaning of RNA in which the 2'-position of sugar 14 is ether-bonded to R 1 ), and 2'-O-modified RNA derivatives are exemplified. The general formula is shown in formula (1). Here, the base part is described as adenine, but it is for illustration and other bases may also be used (the same applies to the examples of the following structural formulas).

[0020]

Chemical formula

[0021] Here, R 1 can preferably use functional groups such as methyl group, ethyl group, allyl group, propargyl group, and methoxyethyl group. Formula (2) shows the case where R 1 is a methyl group (2'-O-methyladenosine), and formula (3) shows the case where R 1 is a methoxyethyl group (2'-O-(methoxyethyl)adenosine). In formula (3), a methoxyethyl group is (ether) -bonded to the 2'-O of ribose.

[0022]

Chemical formula

[0023] In addition, the nucleoside itself of the second nucleotide 20b from the 3'-end may be 2',4'-Locked RNA or BNA NC (Me). Formula (4) shows BNA NC (Me) (2'-O,4'-C-(N-methylaminomethylene)adenosine). In formula (4), the 2'-O and 4'-C of ribose are cross-linked by an N-methylaminomethylene group. This is because if these nucleotides are at the 3'-end, the polymerase activity will be inhibited.

[0024]

Chemical formula

[0025] The 20 nucleotides from the third nucleotide from the 3'-end to the 5'-end of primer 10 may contain any modified nucleic acid as long as it does not inhibit the reaction of DNA polymerase such as DNA, RNA, 2'-OMeRNA, LNA, PNA, morpholino nucleic acid, etc. It may also contain a nucleic acid conjugate with a fluorescent dye or a functional molecule, etc.

[0026] Also, the phosphodiester moiety 16a of the 3'-terminal nucleotide 20a may be a phosphorothioate bond. For example, the oxygen double-bonded to phosphorus may be replaced by sulfur. Even if the phosphodiester moiety 16a is modified in this way and is at the 3'-end, when the base at the 3'-end is deleted by exonuclease activity, the polymerase activity can be inhibited.

[0027] Those having at least either a nucleotide chemically modified on the sugar 14 or the phosphodiester moiety 16a are called artificial nucleotides. In the present invention, the artificial nucleotide inhibits polymerase activity. It may also be called an artificial nucleic acid. Also, the artificial nucleotide may include those having other shapes that inhibit polymerase activity when present at the 3'-end. A primer containing an artificial nucleotide is also called a chemically modified primer.

[0028] Also, in the highly sensitive PCR method according to the present invention, it is preferable to use a dye that specifically binds to the DNA forming a double helix. Especially when performing PCR, it is necessary. SYBR Green, which is an asymmetric cyanine-based dye used for nucleic acid staining, can be preferably used. Note that as long as it is a dye that specifically binds to the DNA forming a double helix, it is not limited to SYBR Green and may be a derivative of SYBR Green.

[0029] <Principle of operation> The operating principle of the PCR method according to the present invention is shown in FIG. 2. FIG. 2(a) shows the target DNA and primer 10. One strand of the target DNA is defined as the main strand 30, and its complementary strand is defined as the complementary strand 32. Now, assume that the base sequence of a predetermined portion of the main strand 30 is "gat". Note that the bases a, t, g, and c in the base sequence represent adenine, thymine, guanine, and cytosine, respectively. The corresponding portion of the complementary strand 32 has the sequence "atc" from the 5'-end.

[0030] The adenine (a) marked with an inverted triangle in this main strand 30 is used as the base to be detected. In this case, the 3'-end of the primer 10 is a natural nucleotide to which adenine (a) is bound, and the second nucleotide from the 3'-end is an artificial nucleotide to which guanine (g) is bound.

[0031] When PCR is performed with this primer 10, as shown in FIG. 2(b), after the main strand 30 and the complementary strand 32 are separated by annealing, the primer 10 binds to the complementary strand 32, and the primer 10 is extended continuously from the 3'-end of the primer 10. That is, normal PCR is performed and amplification occurs.

[0032] On the other hand, FIG. 2(c) shows DNA in which the adenine (a) of the main strand 30 has been displaced by guanine (g). PCR is performed using the same primer 10 as that used in FIG. 2(a). At this time, a polymerase having proofreading activity is used.

[0033] In the annealing step, the target portion of the complementary strand 32 separated from the main strand 30 is cytosine (c) instead of thymine (t) that binds to adenine (a) (FIG. 2(d)). Therefore, the polymerase having proofreading activity removes the adenine (a) of the primer 10 by exonuclease activity (FIG. 2(e)). Then, the polymerase attempts to perform extension subsequently.

[0034] However, the 3'-end of primer 10 is an artificial nucleotide, and the polymerase cannot exhibit polymerase activity, and nucleic acid elongation is not performed (Fig. 2(f)). As a result, due to the difference in a single base in the main chain 30, amplification by PCR is not carried out. In addition, although the reverse primer set at an appropriate position performs a polymerase reaction with the complementary strand 32, since only one pair of double-stranded DNA can be generated from one pair of double-stranded DNA, no matter how many cycles are repeated, the overall copy number does not increase. As described above, the highly sensitive PCR method according to the present invention can surely detect a difference in a single base.

[0035] <Mode of use> In the highly sensitive PCR method according to the present invention, as shown in the examples described later, since the mutation points of specific proteins can be detected with high sensitivity, a master mix for single nucleotide polymorphism detection can be configured. Specifically, it contains the primer according to the present invention, a polymerase having proofreading activity, a dye that specifically binds to DNA, dNTP, MgCl2, a buffer, etc. in optimal amounts.

[0036] Here, the primer has a target base (or its complementary base) at the 3'-end, and an artificial nucleotide that inhibits polymerase activity is bound to the second position from the 3'-end.

Example

[0037] (Example 1) The highly sensitive PCR method according to the present invention will be described below with reference to examples.

[0038] <Explanation of amplification target> The target DNA was the KRAS gene, which is a cancer gene. The KRAS gene is known to have a mutation point at the 35th position in the sequence starting from atg (start codon) in the KRAS protein coding region (191..760) (protein information sequence) out of the full-length 5430 bp of its mRNA.

[0039] The 96 bases from position 205 in the protein coding region of KRAS were used as a template. The sequence of 96 bases from position 205 of wild-type KRAS is shown in Table 1 as SEQ ID NO: 1. SEQ ID NO: 1 is referred to as "KRAS wild type: KRASwt(35G), or Tg(35G)".

[0040] Also, as mutants, the one with guanine mutated to adenine "KRASG12D(35G>A), or Ta(35A)" was designated as SEQ ID NO: 2, the one with guanine mutated to cytosine "KRASG12A(35G>C), or Tc(35C)" was designated as SEQ ID NO: 3, and the one with guanine mutated to thymine "KRASG12V(35G>T), or Tt(35T)" was prepared as SEQ ID NO: 4. Note that the amino acids G, D, A, and V represent glycine, aspartic acid, alanine, and valine, respectively. The mutation point at the 35th position from the start codon corresponds to the 21st position from the 5'-end in SEQ ID NOs: 1, 2, 3, and 4.

[0041] Also, as a template for the confirmation experiment, the one with five thymines attached to the 5'-end of the complementary strand of 21 bases from the 5'-end of Ta(35A) was designated as Ta-t5 and SEQ ID NO: 5.

[0042]

Table 1

[0043] In contrast, referring to Table 2, the forward primers with guanine, adenine, cytosine, and thymine at the 3'-end were designated as POM2g, POM2a, POM2c, and POM2t, respectively, and SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9. These primers were designed to bind to the 1st to 21st positions from the 5'-end of SEQ ID NOs: 1 to 4 indicated by the underline in Table 1.

[0044] Also, as shown by formula (2), for these primers, the second nucleotide from the 3'-end has "-OCH at the 2'-position of sugar 14b" 3」(which can also be described as 「-OMe」) is a modified artificial nucleotide. That is, the second nucleoside from the 3'-end is 2'-O-methylguanosine. This is represented as 「G m 」. The second nucleotide 20b from the 3'-end is a 「modified nucleotide: artificial nucleotide」.

[0045] Also, the primer with the 3'-end of these forward primers removed was designated as POM2-1 and is shown in SEQ ID NO: 10.

[0046] Also, primers with the phosphodiester bond 16a connecting the 3'-end and the second nucleoside from the 3'-end of POM2g, POM2a, POM2c, and POM2t replaced with phosphorothioate were designated as PSM2^g, PSM2^a, PSM2^c, and PSM2^t, and are shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14.

[0047] Phosphorothioate is obtained by replacing the oxygen double-bonded to the phosphorus in the phosphodiester bond 16a with sulfur. In Table 2, it is represented by attaching a 「^」 symbol to the left shoulder of the base symbol at the 3'-end to indicate the presence of a phosphorothioate bond.

[0048] Also, the reverse primer for wild-type and mutant KRAS was designated as PRev and is shown in SEQ ID NO: 15. This is 22 bases from the 3'-end of the amplified portion in Table 1. The reverse primer PRev binds to the underlined portion from the 3'-end of the sequence in Table 1.

[0049]

Table 2

[0050] For PCR, the Premix Taq standard protocol was followed. Also, as the DNA polymerase having proofreading activity, 「TaKaRa EX Taq polymerase TMIt used "". The execution cycle was to hold at 98°C for 30 seconds (thermal denaturation), then hold at 55°C for 30 seconds (annealing), and hold at 72°C for 60 seconds (polymerase reaction) for 30 to 50 cycles. Finally, it was held at 98°C for 30 seconds. Table 3 shows the final concentrations of each component before the start of the reaction. Note that the "mastermix" does not contain primers. The above-mentioned primers were used for the primers. Also, "dsGreen" is a dye that specifically binds to DNA with a double helix. This protocol is simply called the "standard protocol". The following examples performed PCR according to the standard protocol unless otherwise noted. Note that the PCR apparatus used was the AriaMx Real-Time PCR System manufactured by Agilent Technologies.

[0051]

Table 3

[0052] <The first preliminary experiment> Figure 3 shows the results when primer POM2-1 was used. Figure 3(a) shows the amplification curve of the results used for KRASwt(35G), and Figure 3(b) shows the amplification curve of the results used for KRASG12D(35A). The number of broken lines is the result for each number of trials. In both graphs, the horizontal axis is the number of cycles, and the vertical axis represents the fluorescence intensity (ΔRn). The threshold line is marked with a triangle. In both graphs, the threshold line is the top line. In both graphs, even when the PCR cycle was repeated, it did not exceed the threshold line and was not amplified.

[0053] <3'-exonuclease activity> Next, template Ta-t5 (0.2 μM) (SEQ ID NO: 5) and primer POM2c (0.2 μM) (SEQ ID NO: 8) were mixed at a ratio of 1:1, Takara Ex Taq DNA polymerase (1 U) was added, and the reaction was carried out at 72°C for 1 minute. The results of examining the solution by liquid chromatography are shown in Figure 4. Referring to Figure 4, the horizontal axis represents the retention time (minutes), and the vertical axis represents the intensity (μV). As a result, the peak of POM2c used as the primer was not observed, and only POM2-1 lacking the 3'-end and template Ta-t5 were observed.

[0054] As described above, when the base at the corresponding position (here, the 21st position) of the template at the 3'-end does not match the 3'-end of the primer, it was confirmed that the base at the 3'-end of the primer is removed by the 3'-exonuclease activity of the polymerase having proofreading activity.

[0055] <Verification of single-base detection> Next, using Tg(35G) (SEQ ID NO: 1), Ta(35A) (SEQ ID NO: 2), Tc(35C) (SEQ ID NO: 3), Tt(35T) (SEQ ID NO: 4) as templates, real-time PCR of the standard protocol was performed using primers POM2a (SEQ ID NO: 7), POM2c (SEQ ID NO: 8), POM2t (SEQ ID NO: 9), PSM2^g (SEQ ID NO: 11), PSM2^a (SEQ ID NO: 12), PSM2^c (SEQ ID NO: 13), PSM2^t (SEQ ID NO: 14).

[0056] The results when primer POM2a was used are illustrated in Figures 5 and 6. Figure 5(a) shows the results of performing real-time PCR with KRASG12D(35A), Figure 5(b) shows the results for KRASwt(35G), Figure 6(c) shows the results for KRASG12A(35C), and Figure 6(d) shows the results for KRASG12V(35T).

[0057] For all of these figures, the horizontal axis represents the number of cycles, and the vertical axis represents the fluorescence intensity (ΔRn). Also, the threshold line is the line represented by the triangle mark. In addition, each real-time PCR was repeated three times under the same conditions, and they were plotted. From Figures 5 and 6, it was found that only when POM2a was used against KRASG12D(35A), the threshold line was exceeded and amplification was observed as the number of cycles was overlaid. For other combinations, no amplification was observed even when the number of cycles was overlaid. The state where no amplification was observed is called "not detected".

[0058] The results of other combinations are also shown in Table 4 together. Referring to Table 4, the leftmost column shows the names of the primers, and the upper row shows the Ct values for each template. Referring to Table 4, the Ct value can be measured only when the base at the 3'-end of the primer is the same as the 21st base of the template. In other cases, the result was "not detected".

[0059]

Table 4

[0060] This can be concluded because the DNA polymerase having proofreading activity loses its polymerase activity due to the combination of the 3'-end of the primer and the 21st base of the template not matching, and the 3'-end of the primer is deleted and the second modified nucleotide 20b from the 3'-end. That is, with the highly sensitive PCR method of the present invention, a single-base difference at a specific position could be detected.

[0061] <Detection sensitivity of POM2> Next, the detection sensitivity of the present invention was examined. Ta(35A) (SEQ ID NO: 2) was used as the template, and POM2a (SEQ ID NO: 7) was used as the primer. The initial concentration of the primer POM2 was 2.0×10 -1 μM, and the concentration of the template Ta(35A) was 1-fold (8.3×10 -9Five types with concentrations of 1 μM, 1 / 10-fold, 1 / 100-fold, 1 / 1000-fold, and 1 / 10000-fold were prepared. The results are shown in Fig. 7 and Table 5. Referring to Fig. 7, the horizontal axis represents the number of cycles, and the vertical axis represents the fluorescence intensity (ΔRn). Table 5 shows the cycle threshold Ct values and the values of ΔCt, which is the difference between the Ct values.

[0062] Referring to Table 5 as well, theoretically, when the concentration is reduced to one-tenth, the ΔCt value should increase by 3.32 each time. The measured values in Table 5, including experimental errors, are approximately the same as this difference, indicating that the quantitativeness was confirmed. Also, it can be seen that amplification was possible even from as few as 10 copies (10 molecules) in one sample.

[0063]

Table 5

[0064] <Detection of rare mutations> In the highly sensitive PCR method according to the present invention, even when the sample is very small as described above, reliable amplification can be achieved. Furthermore, since this highly sensitive PCR method does not amplify if the base at the detection point is different, it can detect even when a small amount of mutant type is mixed in the sample. Therefore, it was investigated whether it was possible to detect and quantify a sample in which 0.01% of the mutant type KRASG12D (G35>A) was mixed in the wild type (KRASwt(35G)) (Tg(35G) / Ta(35A)=1 / 0.0001).

[0065] Tg(35G) (SEQ ID NO: 1) was used as a template at 8.3×10 -9 μM, and Ta(35A) (SEQ ID NO: 2) was prepared at 8.3×10 -13 μM.

[0066] Also, POM2g (SEQ ID NO: 6) and POM2a (SEQ ID NO: 7) were prepared as primers at 2.0×10 -1 μM each. Using these, RT-PCR was performed according to the protocol in Table 3. The results are shown in Fig. 8 and Table 6.

[0067] Referring to FIG. 8, the horizontal axis represents the number of cycles, and the vertical axis represents the fluorescence intensity (ΔRn). Also, FIG. 8 is a superposition of the results obtained using primer POM2a and the results obtained using primer POM2g, and real-time PCR was performed for each primer.

[0068] From these results, the Ct value was determined, and the results are shown in Table 6. Referring to Table 6, when primer POM2g was used, the Ct value for template Tg(35G) was 31.48 ± 0.33, and for template Ta(35A), the Ct value was 42.12 ± 0.44, and ΔCt was 10.64 ± 0.36. This result means that primer POM2g could detect template Tg(35G) 2^10.64 times, that is, about 1596 times more sensitively than template Ta(35A).

[0069]

Table 6

[0070] <Detection of SARS-CoV-2 Variants> The SARS-CoV-2 virus is currently raging around the world. By using the highly sensitive PCT method according to the present invention, even with a small amount of sample, detection can be performed with very high accuracy. For the identification of this virus, mutations in the spike protein part are used for discrimination. Table 7 shows the mutations at positions 1355 and 1486 of the wild type "Wu", Omicron BA1 type "o-BA.1", Omicron BA2 type "BA.2", and Omicron BA5 type "BA.5". In the wild strain, the amino acids containing these mutation points are leucine "L" at position 452 and glycine "G" at position 496.

[0071]

Table 7

[0072] From the combination of two mutations at positions 1355 and 1486 of the spike protein mRNA, the o-BA1 strain, BA2 strain, and BA5 strain can be identified. The detection principle is shown in Fig. 9.

[0073] Referring to Fig. 9, a forward primer Mx of a certain length is provided from position 1355 of the spike protein to the 5'-terminal side. Also, a reverse primer yM of a certain length is provided from position 1486 to the 3'-terminal side. As already shown, in the highly sensitive PCR method according to the present invention, in such a case, amplification does not occur unless elongation occurs with both the forward and reverse primers. Therefore, by using positions 1355 and 1486 in Table 7 as the 3'-terminals and modifying the nucleotide one before them, these types can be detected. Specifically, the primers were set as shown in Table 8.

[0074]

Table 8

[0075] Referring to Table 8, each forward primer has the initial letter "F", and the reverse primer has the initial letter "R". The wild-type forward primer F1355w / o has thymine "T" at position 1355 at the 3'-terminal, and cytosine "C" that binds to guanine "G" at position 1486 is arranged at the 3'-terminal of the reverse primer R1486w / BA2. Similarly, for the BA1 type strain, BA2 strain, and BA5 strain, the base at position 1355 is arranged at the 3'-terminal of the forward primer, and the complementary base of the base at position 1486 is arranged at the 3'-terminal of the reverse primer.

[0076] And for both the forward primer and the reverse primer, the second base from the 3'-terminal was modified with -OMe at the 2' of the sugar moiety. Since the second base from the 3'-terminal of any primer was cytosine "C", it became 2'-O-methylcytidine as a nucleoside. In Table 8, it is represented as "(Cm)".

[0077] As templates, the spike proteins of each mutant strain were used, and the PCR protocol followed the standard protocol. The results of real-time PCR are shown in Table 9.

[0078]

Table 9

[0079] Referring to Table 9, only the spike proteins with matching application types were detected by the primers for each virus type. Also, quantitative detection is possible from the Ct values.

[0080] This indicates that the primers for each virus can be used as primers for detecting their respective viruses. That is, the primer combination applied to the o-BA.5 strain is the primer for the o-BA.5 strain, the primer combination applied to the o-BA.1 strain is the primer for the o-BA.1 strain, and the primer combination applied to the o-BA.2 strain is the primer for the o-BA.2 strain.

[0081] Figure 10 shows examples of the use of these primers. The saliva sample is divided into three parts, and real-time PCR is performed using each dedicated primer. Then, it can be determined that it is negative for the virus mutant strain corresponding to the primer that did not amplify. And when amplification occurs, it can be determined that it is positive for the virus strain corresponding to that primer. In Figure 10, amplification can be confirmed only in the case of the primer for the o-BA.2 strain, and the saliva sample can be determined to be positive for the o-BA.2 strain. Also, quantitative analysis is possible from the Ct at this time.

[0082] <Other polymerase> In the above examples, Takara Ex Taq polymerase (Takara Bio Inc.) was used, but as long as it has proofreading activity, other polymerases can of course implement the highly sensitive PCR method according to the present invention.

[0083] The same experiment for identifying KRAS mutations was conducted using the primers POM2 shown in Table 2, PfuUltra II Fusion DNA Polymerase (Agilent Technologies), and Phusion Plus DNA Polymerase (Thermo Fisher Scientific). The standard protocol was used. The results are shown in Tables 10 and 11.

[0084]

Table 10

[0085]

Table 11

[0086] As can be seen from Tables 10 and 11, amplification occurred only when the 3'-ends of the primers matched, and it was found that regardless of the type of polymerase, if it had proofreading activity.

[0087] <Primers with other configurations> The modified primer POM2 has a structure where the second nucleoside from the 3'-end has the structure of formula (2), and being at the 3'-end inhibits polymerase activity. Experiments were carried out on nucleosides using ribose of formula (3) and ribose of formula (4) as primers with other structures that inhibit polymerase activity. The primer using the nucleoside of ribose of formula (3) is called POME, and the primer using the nucleoside of ribose of formula (4) is called POB.

[0088] Using these primers with these nucleosides, RT-PCR was performed according to the standard protocol to detect KRAS. The primer sequences are shown in Table 12.

[0089]

Table 12

[0090] The second nucleotide from the 3'-end of primer POME was represented as (GME). It is the ribose structure of formula (3). The second nucleotide from the 3'-end of primer POB was represented as (GB). It is the ribose of the structure of formula (4). The protocol was the same as that for Takara Ex Taq polymerase. The results are shown in Table 13. Note that both GME and GB are modified forms of guanine.

[0091]

Table 13

[0092] Referring to Table 13, amplification occurred only when the 3'-ends of both primer POME and primer POB were identical. It can be seen that in the case where artificial nucleotides were exposed due to the exonuclease activity of the polymerase having proofreading activity, the polymerase activity was inhibited.

[0093] <Detection sensitivity of POB2> POB2 is one in which the second nucleoside from the 3'-end has the structure of formula (4). The detection sensitivity of this primer was also examined. The experimental conditions were the same as <Detection sensitivity of POM2>. That is, the initial concentration of primer POB2g was 2.0×10 -1 μM, and five concentrations of the template Tg(35G) were prepared: 1-fold (8.3×10 -9 μM), 1 / 10-fold, 1 / 100-fold, 1 / 1000-fold, and 1 / 10000-fold.

[0094] The results are shown in Figure 11 and Table 14. Referring to Figure 11, the horizontal axis represents the number of cycles, and the vertical axis represents the fluorescence intensity (ΔRn). Referring to Table 14, theoretically, when the concentration becomes 1 / 10, the ΔCt value should increase by 3.32 each time. The measured values in Table 14, including experimental errors, are approximately the same as this difference, and the quantitativeness was confirmed. Also, it can be seen that amplification could be achieved even from as few as 10 copies (10 molecules) in one sample.

[0095]

Table 14

[0096] Also, the same experiment was conducted for primer POB2a. In this case, the target template is Ta(35A) (see Table 1). The same results as in Fig. 11 were obtained in this case as well. The Ct values and ΔCt values are shown in Table 15.

[0097]

Table 15

[0098] <When the target substances are different> As an example of identifying single nucleotide polymorphisms in other target base sequences, the T790M mutation (2630T) of the EGFR gene WT(2630C) was investigated. In the mutant type, the 2630th position from the 5'-terminal side has mutated from cytosine (c) to thymine (t). Table 15 shows the base sequences of the EGFR gene WT and the part used as the template in the mutant type. They were designated as SEQ ID NO: 32 and SEQ ID NO: 33, respectively.

[0099]

Table 16

[0100] In T-EGFR-2630c / WT, the amplification target was 23 bases from the mutation point towards the 5'-terminal. The primers for this are shown in Table 17. 2'-OMe ((2) formula) was introduced at the second position from the 3'-terminal of the forward primer. This is designated as (Am). This is a modified alanine (2'-O-methyladenosine). In the wild type, the 3'-terminal of the forward primer is cytosine (c), and in the mutant type, the 3'-terminal of the forward primer is thymine (t). The respective forward primers and reverse primers were designated as SEQ ID NO: 34, 35 and SEQ ID NO: 36, 37.

[0101]

Table 17

[0102] Real-time PCR of the standard protocol was performed on wild-type T-EGFR-2630c / wt and mutant T-EGFR-2630t / T790M using the primer sets of SEQ ID NO: 34 and 35 and the primer sets of SEQ ID NO: 36 and 37. The Ct values and ΔCt values of the results are shown in Table 18.

[0103]

Table 18

[0104] Referring to Table 18, when wild-type primers were used, the Ct value for wild-type (T-EGFR-2630c / wt) was about 20.1, but for mutant (T-EGFR-2630t / T790M), it was 33.1 and ΔCt was 13.0 ± 0.1. That is, when the wild-type primer F-EGFR / 2630M2c was used, the wild-type template was detected 2^13.0 times, about 8192 times more sensitively than the mutant template.

[0105] On the other hand, when the mutant primer F-EGFR / 2630M2t was used, the Ct value for the wild-type template was 33.4 ± 0.21, but for the mutant template, the Ct value was 18.9 ± 0.13 and ΔCt was 14.5 ± 0.1. This result indicates that the mutant primer could detect the mutant template 2^14.5 times, 23170 times more sensitively than the wild-type template. In the analysis of cell-free DNA by liquid biopsy in cancer gene mutation diagnosis, the detection of 0.01% (1 / 10000) mutant genes for wild-type genes is raised as the target value. Therefore, the value of 23170 times shows a detection sensitivity exceeding that.

[0106] Using the mutant primers (SEQ ID NO: 36, 37), the concentration dependence for the detection of mutant EGFR was examined. The experimental conditions were the same as <detection sensitivity>. That is, the initial concentration of the wild-type primers was 2.0×10 -1 μM for both the forward primer and the reverse primer, and the concentrations of the template mutant EGFR were prepared in five types: 1-fold (8.3×10 -9 μM), 1 / 10-fold, 1 / 100-fold, 1 / 1000-fold, and 1 / 10000-fold. The results of the Ct values and ΔCt values are shown in Table 19.

[0107]

Table 19

[0108] Referring to Table 19, theoretically, when the concentration becomes one-tenth, the ΔCt value should increase by 3.32 each time. Considering the measurement values in Table 19 and adding the experimental error, it is almost the same difference, and the quantification ability was confirmed. Also, it can be seen that amplification was possible even from only 10 copies (10 molecules) in one sample.

[0109] Next, the effect of the highly sensitive PCR method according to the present invention on the delE746-A750 deletion mutation of the EGFR gene WT (2630C) was examined. The target was 100 bases from 2461 to 2560 of the EGFR gene. The base sequences of the wild type and the delE746-A750 deletion mutant are shown in Table 20. Each base sequence was set as SEQ ID NO: 38 and SEQ ID NO: 39.

[0110]

Table 20

[0111] In the delE746-A750 deletion mutant (SEQ ID NO: 39), 15 bases from the 37th to the 51st from the 5' end are deleted compared to the wild type (SEQ ID NO: 38). In SEQ ID NO: 39, the guanine (g) immediately before the deletion and the adenine (a) immediately after the deletion are underlined. The primers for discriminating these are shown in Table 21.

[0112]

Table 21

[0113] Referring to Table 21, F-EGFR2461-2560 (SEQ ID NO: 40) is a forward primer for 30 bases from the 5'-end of wild-type T-EGFR2461-2560 / WT. R-EGFR2461-2560 (SEQ ID NO: 41) is a reverse primer for 30 bases from the 3'-end of wild-type T-EGFR2461-2560 / WT.

[0114] F-EGFR-del746-A750 2497M2g (SEQ ID NO: 42) and F-EGFR-del746-A750 2497M2a (SEQ ID NO: 44) are forward primers for the delE746-A750 deletion mutant (SEQ ID NO: 39). The second nucleotide from their 3'-ends is an artificial nucleotide with a "-OCH3" modification at the 2'-position of sugar 14b. That is, the second nucleoside from the 3'-end is 2'-O-methylguanosine. In Table 21, it is represented as "Gm".

[0115] Also, F-EGFR-delE746-A750 2497M2a (SEQ ID NO: 44) and R-EGFR-delE746-A750 2511M2c (SEQ ID NO: 45) are reverse primers for the delE746-A750 deletion mutant (SEQ ID NO: 39). The second nucleotide from their 3'-ends is an artificial nucleotide with a "-OCH3" modification at the 2'-position of sugar 14b. That is, the second nucleoside from the 3'-end is 2'-O-methyluridine. In Table 21, it is represented as "Um".

[0116] Discrimination studies were conducted by combining these primers. The primer combinations and the results of real-time PCR using their standard protocols are shown in Table 22.

[0117]

Table 22

[0118] Referring to Table 22, in Experiment A, when using the primer combination F-EGFR-delE746-A750 / 2497M2g and R-EGFR2461-2560 for detecting the wild type, the Ct value for the wild type template T-EGFRwt2461-2560 was 21.86 ± 0.09, the Ct value for the deletion type template T-EGFR-delE746-A750 was 32.78 ± 0.28, and the ΔCt was 10.92. That is, with this primer combination, the wild type template could be detected 2 to the power of 10.92 times, approximately 1938 times more sensitively than the mutant template.

[0119] In Experiment B, when using the primer combination F-EGFR-delE746-A750 2497M2a and R-EGFR2461-2560 for detecting the deletion type, the Ct value for the wild type template T-EGFRwt2461-2560 was 30.40 ± 0.43, the Ct value for the deletion type template T-EGFR-delE746-A750 was 16.74 ± 0.05, and the ΔCt value was 13.66. That is, with this primer combination, the wild type template could be detected 2 to the power of 13.66 times, approximately 12944 times more sensitively than the mutant template. As described above, in the analysis of cell-free DNA by liquid biopsy in cancer gene mutation diagnosis, the detection of mutant genes at 0.01% (1 / 10000) of the wild type gene is raised as the target value. Therefore, the value of 12944 times indicates a detection sensitivity that exceeds this.

[0120] In Experiment U, when using the primer combination F-EGFR2461-2560 and R-EGFR-delE746-A750 2511M2t for detecting the wild type, the Ct value for the wild type template T-EGFRwt2461-2560 was 30.94±0.09, the Ct value for the deletion type template T-EGFR-delE746-A750 was >45.0 (not detected), and the ΔCt was >14.06. That is, with this primer combination, the wild type template could be detected more than 2 to the power of 14.06 times, more than 17080 times more sensitively than the mutant template.

[0121] In Experiment E, when using the primer combination F-EGFR2461-2560 and R-EGFR-delE746-A750 2511M2c for detecting the deletion type, the Ct value for the wild type template T-EGFRwt2461-2560 was 31.49±0.02, the Ct value for the deletion type template T-EGFR-delE746-A750 was 18.40±0.08, and the ΔCt value was 13.09. That is, with this primer combination, the wild type template could be detected 2 to the power of 13.09 times, about 8719 times more sensitively than the mutant template.

[0122] As described above, in the cell-free DNA analysis by liquid biopsy in cancer gene mutation diagnosis, the detection of a mutant gene at 0.01% (1 / 10000) of the wild type gene is raised as the target value. Therefore, the value of 8719 times shows a detection sensitivity almost close to that.

[0123]

Table 23

[0124] Referring to Table 23, theoretically, when the concentration of the EGFR deletion mutation template T-EGFR-delE746-A750 is the initial concentration of 8.3×10 -9The ΔCt value should increase by 3.32 each time it becomes one-tenth from μM, and the measured values in Table 23 are also 3.56 to 3.81, which is almost the same difference within the experimental error range, and the quantitativeness was confirmed. Also, it can be seen that amplification was possible even from only 10 copies (10 molecules) in one sample.

[0125] From the above, it is considered that this highly sensitive PCR method can be widely applied to PCR methods using general chemically modified primers containing a 2'-position modified nucleic acid at the second position from the 3'-end and general DNA polymerases having proofreading activity.

Industrial Applicability

[0126] The highly sensitive PCR method according to the present invention can be suitably used as a PCR method that can accurately identify and quantitatively analyze single nucleotide mutations in DNA and RNA, such as an intracellular mRNA quantitative analysis kit in molecular biology research, a virus mutant detection kit in infectious disease prevention, and a cell-free DNA analysis kit by liquid biopsy in cancer gene mutation companion diagnosis.

Explanation of Symbols

[0127] 10 Primer 14 Sugar 14b (Sugar of the second nucleotide from the 3'-end) 16 Phosphodiester moiety 16a (Phosphodiester moiety of the nucleotide 20a at the 3'-end) 18 Nucleobase 20 Nucleotide 20a Nucleotide at the 3'-end 20b Second nucleotide from the 3'-end 30 Main chain 32 Complementary strand

Claims

1. a DNA polymerase having proofreading activity; A highly sensitive PCR method using a chemically modified primer in which an artificial nucleic acid that inhibits polymerase activity is arranged at the second position from the 3'-end and natural DNA is arranged at the 3'-end, the artificial nucleic acid being either of formula (3) or (4). 【Chemistry 100】

2. The highly sensitive PCR method according to claim 1, further comprising SYBR Green.

3. 3. The highly sensitive PCR method according to claim 1, wherein the backbone between each of the bases of the primer containing the artificial nucleic acid is either a phosphodiester bond or a phosphorothioate bond.

4. 3. The highly sensitive PCR method according to claim 1, wherein the third to the 5'-end from the 3'-end of the chemically modified primer are nucleotides that do not inhibit polymerase activity.

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