JP2024035173A - High sensitivity PCR method - Google Patents

Abstract

[Problem] Conventionally, single nucleotide mutations in genes have been identified using TaqMan probes, but since the identification uses the difference in binding affinity (melting temperature) between the gene and the probe, unnecessary Things are also amplified. Therefore, there was a problem that both the accuracy and detection sensitivity for identifying single nucleotide mutations were low. [Solution] A chemically modified primer in which a DNA polymerase having proofreading activity, an artificial nucleic acid that inhibits polymerase activity is placed at the second position from the 3'-end, and a natural type of DNA is placed at the 3'-end. In the high-sensitivity PCR method that uses It becomes possible. [Selection diagram] Figure 1

Description

There is an application for exception to loss of novelty.

The present invention relates to a highly sensitive PCR method that can detect single nucleotide variants with high accuracy.

When identifying single nucleotide mutations in genes, a method using TaqMan probes is often used.

Patent Document 1 discloses that in addition to a TaqMan probe, a WT (wild type) blocker nucleic acid fragment that is not degraded by the 5'-exonuclease activity of polymerase is bound to a region containing a mutation point, and then primers are bound and PCR is performed. . If the DNA has a mutation point, the WT blocker nucleic acid fragment binds weakly to the mutant DNA and performs PCR, but strongly binds to the wild-type DNA without the mutation point and interferes with wild-type PCR. On the other hand, it has been disclosed that TaqMan probes, on the contrary, weakly bind to wild-type DNA, but bind more strongly to mutant DNA, are degraded by the 5'-exonuclease activity of polymerase, and fluorescence increases as PCR progresses. .

International Publication No. 2016/093333

The method using TaqMan probes uses the difference in binding affinity (melting temperature) between genes and probes to identify single nucleotide mutations in genes. In principle, it is impossible to set a temperature that allows 100% binding to one side and no binding to the other. The TaqMan probe binds to a certain extent to unintended wild-type DNA, and the WT blocker binds to unintended mutant DNA to some extent, reducing detection sensitivity. Therefore, it is not 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 quantitative analysis of the effect of drugs targeting single nucleotide mutant genes at the gene level is not possible. There was no way to do it.

Therefore, first, a specimen with a detection sensitivity of 50-100 copies or more becomes the detection limit. In other words, it does not have high sensitivity enough to amplify samples with a smaller number of samples. Furthermore, the mutation identification accuracy (ratio of mutant types mixed in the wild type) was limited to about 1-5%. Furthermore, precise primer, probe design, and reaction temperature settings are required for each evaluation system, which tends to cause variation in mutation identification accuracy and detection accuracy for each test. Additionally, TaqMan probes are expensive.

The high-sensitivity PCR method of the present invention was conceived in view of the above-mentioned problems, and amplification is possible if the nucleotide that inhibits polymerase activity is not at the 3'-end, but if it is exposed at the 3'-end, , single nucleotide mutations are detected by utilizing the property that polymerase activity is inhibited.

More specifically, the high-sensitivity PCR method according to the present invention includes:
a DNA polymerase having proofreading activity;
It is characterized by using 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 a natural type of DNA is placed at the 3'-end.

The present invention is a PCR technology that utilizes the cybergreen method, which combines a novel chemically modified primer and a DNA polymerase with proofreading activity. Since detection is not based on differences in melting temperature, this method can be said to be a method for identifying single nucleotide mutations based on a different principle from conventional methods.

Therefore, even a gene to be detected with a very small number of copies (even 10 copies is possible) can be amplified, and even a contaminating amount of 0.01% can be clearly detected.

Furthermore, since the mutation point can be specified using both the forward and reverse primers, only specimens that match at the two sites can be amplified at the same time, allowing extremely sensitive detection.

FIG. 2 is a diagram showing the configuration of primers used in the present invention. FIG. 3 is a diagram illustrating the operating principle of the PCR method according to the present invention. (a) Results of using primer POM2-1 for KRASwt (35G), and (b) graphs showing amplification curves of results of using primer POM2-1 for KRASG12D (35A). 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 polymer (1 U) was added, and 72 This is a graph showing the results of a reaction at ℃ for 1 minute and a liquid chromatography examination of the resulting solution. (a) An amplification curve using primer POM2a for KRASG12D (35A), and (b) an amplification curve using KRASwt (35G). (c) An amplification curve using the primer POM2a for KRASG12A (35C), and (d) an amplification curve using the primer POM2a for KRASG12V (35T). This is an amplification curve when PCR was performed using primer POM2a and Ta (35A) at different concentrations. This is an amplification curve showing detection of 8.3×10 ⁻¹³ μM Ta (35A) contained in 8.3×10 ⁻⁹ μM Tg (35G). FIG. 2 is a diagram illustrating the principle of identifying δ strain (o-BA.5), BA1 strain, and BA2 strain among SARS-CoV-2 mutant types. FIG. 3 is a diagram illustrating how to use primer pairs. This is an amplification curve obtained when PCR was performed on Tg (35G) at varying concentrations from 8.3×10 ⁻⁹ μM to 8.3×10 ⁻¹³ μM using primer POB2g.

The high-sensitivity PCR method according to the present invention will be explained below with reference to drawings and examples. In addition, 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 spirit of the present invention. Furthermore, embodiments and examples obtained by appropriately combining technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention. Additionally, all documents mentioned herein are incorporated by reference herein.

The present invention intentionally inserts a nucleotide that inhibits polymerase activity into the primer, and utilizes 3'-exonuclease activity that removes the base at the 3'-end when the 3'-end of the primer differs from the gene. Single nucleotide mutations are detected by exposing a nucleotide that inhibits polymerase activity at the 3'-end and preventing further extension from the primer.

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 are placed at the second position from the 3'-end, and natural DNA is placed at the 3'-end. Utilizes chemically modified primers that have been placed.

A DNA polymerase having proofreading activity refers to a DNA polymerase that has the function of removing an incorrect base from the template strand if it occurs. It suffices if it has 3'-exonuclease activity. Such DNA polymerase is also called HiFi polymerase and can be obtained commercially. 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 suitably used.

FIG. 1 illustrates the primers used in the present invention. The primer has a structure in which 20 nucleotides are linked from the 5'-end through a phosphodiester bond. The nucleotide 20 has a phosphodiester moiety 16 bonded to a sugar 14 to which a nucleobase 18 is linked. A linkage of nucleobase 18 and sugar 14 is called a nucleoside. Therefore, it can be said that the nucleosides are bonded to each other through the phosphodiester moieties 16.

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 a polymerase having proofreading activity. Note that the 3'-terminal nucleotide 20a is a natural nucleotide. Note that the natural type refers to deoxyribose or ribose, which exists naturally and does not inhibit polymerase activity.

A specific example of the second nucleotide 20b from the 3'-end is 2'-O expressed by the general formula 2'-OR ₁ RNA (meaning RNA in which the 2' position of sugar 14 is ether bonded to R ₁ ). Examples include modified RNA derivatives. The general formula is shown in formula (1). Note that although adenine is described as the base portion here, this is just an example, and other bases may be used (the same applies to the examples of the structural formulas below).

Here, R ₁ is preferably a functional group such as a methyl group, ethyl group, allyl group, propargyl group, or 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 (ether) is bonded to 2'-O of ribose.

Furthermore, the nucleoside 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 crosslinked with an N-methylaminomethylene group. This is because if these nucleotides are at the 3'-end, polymerase activity is inhibited.

Nucleotide 20 from the third nucleotide from the 3'-end to the 5'-end of primer 10 may be any type of nucleotide that does not inhibit the reaction of DNA polymerase, such as DNA, RNA, 2'-OMeRNA, LNA, PNA, and morpholino nucleic acid. It may contain modified nucleic acids. It may also include nucleic acid conjugates with fluorescent dyes, functional molecules, and the like.

Further, the phosphodiester portion 16a of the 3'-terminal nucleotide 20a may be a phosphorothioate bond. For example, oxygen double bonded to phosphoric acid may be replaced with sulfur. This is because even if the phosphodiester portion 16a is modified in this way, if it is located at the 3'-end, polymerase activity can be inhibited if the base at the 3'-end is deleted by exonuclease activity.

A nucleotide having at least either a chemically modified nucleotide or a phosphodiester portion 16a in the sugar 14 is called an artificial nucleotide. In the present invention, the artificial nucleotide inhibits polymerase activity. It may also be called an artificial nucleic acid. Artificial nucleotides may also include other shapes that inhibit polymerase activity when present at the 3'-end. Primers containing artificial nucleotides are also called chemically modified primers.

Furthermore, in the high-sensitivity PCR method according to the present invention, it is preferable to use a dye that specifically binds to DNA in a double helix. This is especially necessary when performing PCR. Cybergreen, which is an asymmetrical cyanine dye used for staining nucleic acids, can be suitably used. Note that the dye is not limited to Cybergreen as long as it specifically binds to DNA forming a double helix, and may be a derivative of Cybergreen.

<Operating principle>
The operating principle of the PCR method according to the present invention is shown in FIG. FIG. 2(a) shows the target DNA and the primer 10. One of the target DNAs is designated as a main strand 30, and its target strand is designated as a complementary strand 32. Assume now that the base sequence of a predetermined portion of the main chain 30 is "gat". Note that a, t, g, and c in the base sequence represent adenine, thymine, guanine, and cytosine, respectively. The corresponding portion of complementary strand 32 has an "atc" sequence from the 5'-end.

The adenine (a) marked with an inverted triangle in the main chain 30 is 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 from the 3'-end is an artificial nucleotide to which guanine (g) is bound.

When PCR is performed using this primer 10, as shown in FIG. Primer 10 is extended continuously from the end. That is, normal PCR is performed and amplification is performed.

On the other hand, FIG. 2(c) shows DNA in which adenine (a) in the main chain 30 has been displaced to guanine (g). PCR is then performed using the same primer 10 used in FIG. 2(a). In this case, a polymerase having proofreading activity is used.

The target site of the complementary strand 32 separated from the main chain 30 in the annealing process is not thymine (t), which binds to adenine (a), but cytosine (c) (Figure 2(d)), so proofreading activity is improved. The polymerase having this removes adenine (a) of primer 10 by exonuclease activity (FIG. 2(e)). The polymerase then attempts to extend.

However, the 3'-end of primer 10 is an artificial nucleotide, and the polymerase is unable to exhibit polymerase activity, and the nucleic acid is not elongated (FIG. 2(f)). As a result, due to a single base difference in the main chain 30, amplification by PCR is not performed. In addition, the reverse primer set at an appropriate position performs a polymerase reaction with the complementary strand 32, but since only one pair of double-stranded DNA can be generated from one pair of double-stranded DNA, the number of cycles is required. Even if repeated, the overall number of copies does not increase. As described above, the high-sensitivity PCR method according to the present invention can reliably detect a single base difference.

<Form of use>
The high-sensitivity PCR method according to the present invention can detect mutation points in specific proteins with high sensitivity, as shown in the examples described later, and therefore can constitute a master mix for detecting single nucleotide mutations. . Specifically, the optimal materials include the primer according to the present invention, a polymerase having proofreading activity, a dye that specifically binds to DNA, dNTP, MgCl2, a buffer, and the like.

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

(Example 1)
The high-sensitivity PCR method according to the present invention will be explained below with reference to Examples.

<Explanation of amplification target>
The target DNA was the KRAS gene, which is considered to be an oncogene. The KRAS gene is known to have a mutation point at position 35 in the sequence starting from atg (start codon) of the KRAS protein coding region (191..760) (protein information sequence) out of its total mRNA length of 5430 bp. .

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)."

In addition, as a mutant species, "KRASG12D (35G>A), or Ta (35A)" in which guanine is mutated to adenine, is SEQ ID NO: 2, and "KRASG12A (35G>C), or Tc, in which guanine is mutated to cytosine (35C)'' was prepared as SEQ ID NO: 3, and ``KRASG12V (35G>T) or Tt (35T)'' in which guanine was mutated to thymine was prepared as SEQ ID NO: 4. Note that amino acids G, D, A, and V represent glycine, aspartic acid, alanine, and valine, respectively. The 35th mutation point from the start codon corresponds to the 21st mutation point from the 5'-end in SEQ ID NOs: 1, 2, 3, and 4.

Furthermore, as a template for confirmation experiments, five thymines were bonded to the 5'-end of a complementary strand of 21 bases from the 5'-end of Ta (35A), which was designated as Ta-t5 and SEQ ID NO: 5.

On the other hand, referring to Table 2, forward primers with guanine, adenine, cytosine, and thymine at the 3'-end are designated as POM2g, POM2a, POM2c, and POM2t, respectively, and SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. , SEQ ID NO: 9. These primers were designed to bind to the 5'-terminal positions 1 to 21 of SEQ ID NOS: 1 to 4, which are underlined in Table 1.

Furthermore, as shown in formula (2), these primers have the second nucleotide from the 3'-end modified with "-OCH ₃ " (which can also be written as "-OMe") at the 2' position of sugar 14b. It is an artificial type of nucleotide that has been That is, the second nucleoside from the 3'-end is 2'-O-methylguanosine. This was expressed as "G _m ". The second nucleotide 20b from the 3'-end is a "modified nucleotide: artificial nucleotide."

Further, a primer obtained by removing the 3'-end of these forward primers was designated as POM2-1 and shown as SEQ ID NO: 10.

In addition, primers in which the phosphodiester bond 16a that binds the 3'-end and the second nucleoside from the 3'-end of POM2g, POM2a, POM2c, and POM2t are phosphorothioate are PSM2^g, PSM2^a, and PSM2^c. , PSM2^t, and are shown as SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14.

Phosphorothioate is obtained by replacing the oxygen forming a double bond with phosphoric acid in the phosphodiester bonding portion 16a with sulfur. In Table 2, the symbol "^" is added to the left of the base symbol at the 3'-terminus to indicate that it has a phosphorothioate bond.

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

The Premix Taq standard protocol was followed for PCR. Furthermore, "TaKaRa EX Taq Polymerase ^™ " was used as a DNA polymerase having proofreading activity. The execution cycle consisted of 30 to 50 cycles of holding at 98°C for 30 seconds (thermal denaturation), then holding at 55°C for 30 seconds (annealing), and holding at 72°C for 60 seconds (polymerase reaction). Finally, it was held at 98°C for 30 seconds. Table 3 shows the final concentrations of each component before starting the reaction. Note that "mastermix" does not include primers. The primers mentioned above are used. Furthermore, "dsGreen" is a dye that specifically binds to double-helix DNA. This protocol is simply referred to as the "standard protocol." In the following examples, PCR was performed according to standard protocols unless otherwise specified. The PCR device used was AriaMx Real-Time PCR System manufactured by Agilent Technologies.

<First preliminary experiment>
Figure 3 shows the results when primer POM2-1 was used. Figure 3(a) shows the resulting amplification curve used for KRASwt (35G), and Figure 3(b) shows the resulting amplification curve used for KRASG12D (35A). The number of broken lines is the result for each number of trials. In both graphs, the horizontal axis represents the cycle number, 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. Both graphs did not exceed the threshold line and were not amplified even after repeated PCR cycles.

<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, and Takara Ex Taq DNA polymer (1U) was added. The reaction was carried out at 72° C. for 1 minute, and the solution was examined by liquid chromatography. The results are shown in FIG. Referring to FIG. 4, the horizontal axis is residence time (minutes), and the vertical axis is intensity (μV). As a result, no peak of POM2c, which was inserted as a primer, was observed, and only POM2-1 lacking the 3'-end and Ta-t5 as a template were observed.

As mentioned above, if the 3'-end of the base at the corresponding location on the template (here, the 21st base) does not match the 3'-end of the primer, the 3'-exonuclease of the polymerase with proofreading activity It was confirmed that the 3'-terminal base of the primer was removed by the activity.

<Proof of single base detection>
Next, as a template, primers were used for Tg (35G) (SEQ ID NO: 1), Ta (35A) (SEQ ID NO: 2), Tc (35C) (SEQ ID NO: 3), and Tt (35T) (SEQ ID NO: 4). 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^ Real-time PCR according to the standard protocol was performed using t (SEQ ID NO: 14).

FIG. 5 and FIG. 6 illustrate the results when primer POM2a was used. Figure 5 (a) shows the results of real-time PCR performed with KRASG12D (35A), Figure 5 (b) shows KRASwt (35G), Figure 6 (c) shows KRASG12A (35C), and Figure 6 (d) shows KRASG12V. (35T).

For all these figures, the horizontal axis represents the cycle number and the vertical axis represents the fluorescence intensity (ΔRn). Further, the threshold line is a line represented by a triangle mark. In addition, each real-time PCR was repeated three times under the same conditions, and the results were plotted. From FIG. 5 and FIG. 6, it was only when POM2a was used for KRASG12D (35A) that the threshold line was exceeded and amplification was observed as the number of cycles increased. With other combinations, no amplification was observed even after repeated cycles. A state in which no amplification was observed is called "not detected."

The results are shown in Table 4, including other combinations. Referring to Table 4, the leftmost column shows the names of the primers, and the top row shows the Ct value for each template. Referring to Table 4, the Ct value can be measured only when the 3'-end base of the primer and the 21st base of the template are the same, and in other cases, the result is "not detected". .

This is because the 3'-end of the primer and the 21st base of the template do not match, so DNA polymerase with proofreading activity deletes the 3'-end of the primer and inserts the 21st base from the 3'-end. It can be concluded that this is due to the loss of polymerase activity due to the modified nucleotide 20b. In other words, the highly sensitive PCR method of the present invention was able to detect a single base difference at a specific position.

<Detection sensitivity of POM2>
Next, the detection sensitivity of the present invention was investigated. Ta(35A) (SEQ ID NO: 2) was used as a template, and POM2a (SEQ ID NO: 7) was used as a primer. The initial concentration of primer POM2 was 2.0×10 ⁻¹ μM, and the concentration of template Ta (35A) was 1× (8.3×10 ⁻⁹ μM), 1/10×, 1/100×, and 1/1000×. , 5 types of 1/10000 times were prepared. The results are shown in FIG. 7 and Table 5. Referring to FIG. 7, the horizontal axis is the cycle number, and the vertical axis is the fluorescence intensity (ΔRn). Table 5 lists the cycle threshold Ct value and the value of ΔCt, which is the difference between the Ct values.

Also referring to Table 5, the ΔCt value should theoretically increase by 3.32 as the concentration decreases to one-tenth, but the measured values in Table 5 are also approximately the same difference due to experimental errors. , quantitative properties were confirmed. It is also seen that it was possible to amplify even just 10 copies (10 molecules) in one sample.

<Detection of rare mutations>
With the high-sensitivity PCR method according to the present invention, even when the number of samples is very small as described above, it is possible to reliably amplify the sample. Furthermore, in this highly sensitive PCR method, if the bases at the detection point are different, they will not be amplified, so even if a small amount of mutant types is present in the sample, it can be detected. Therefore, we detected and quantified a sample containing 0.01% of the mutant KRASG12D (G35>A) in the wild type (KRASwt(35G)) (Tg(35G)/Ta(35A)=1/0. 0001) is possible.

As templates, Tg(35G) (SEQ ID NO: 1) was prepared at 8.3×10 ⁻⁹ μM and Ta(35A) (SEQ ID NO: 2) was prepared at 8.3×10 ⁻¹³ μM.

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

Referring to FIG. 8, the horizontal axis is the cycle number, and the vertical axis is the fluorescence intensity (ΔRn). Moreover, FIG. 8 shows the results obtained when primer POM2a was used and the results obtained when primer POM2g was used, and real-time PCR was performed for each primer.

The Ct value was determined from this result, and the results are shown in Table 6. Referring to Table 6, when primer POM2g is used, the Ct value is 31.48±0.33 for template Tg (35G), and the Ct value is 42 for template Ta (35A). .12±0.44, and ΔCt was 10.64±0.36. This result means that primer POM2g was able to detect template Tg (35G) more sensitively than template Ta (35A) by 2 to the power of 10.64, that is, about 1596 times more sensitively.

<Detection of SARS-CoV-2 mutant types>
The SARS-CoV-2 virus is currently rampant all over the world, but by using the highly sensitive PCT method of the present invention, it is possible to detect it with extremely high accuracy even if the amount of sample is small. be. This virus is identified by mutations in the spike protein. Table 7 shows mutations at positions 1355 and 1486 of 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.

From Table 7, the o-BA1 strain, BA2 strain, and BA5 strain can be identified by the combination of the two mutations 1355 and 1486 in the spike protein mRNA. Figure 9 shows the detection principle.

Referring to FIG. 9, a forward primer Mx of a certain length is provided on the 5'-end side from 1355 of the spike protein. Furthermore, a fixed length reverse primer yM is provided on the 3'-end side from 1486. As already shown, in the high-sensitivity PCR method according to the present invention, in such a case, amplification will not occur unless elongation occurs with both the forward and reverse primers. Therefore, these types can be detected by setting the 1355th and 1486th nucleotides in Table 7 as the 3'-terminus, and setting the nucleotide immediately before the 3'-end as a modified nucleotide. Specifically, primers were set as shown in Table 8.

Referring to Table 8, each forward primer has an initial "F" and each reverse primer has an initial "R." The wild-type forward primer F1355w/o has a thymine "T" at position 1355 at the 3'-end, and the cytosine "C" that binds to guanine "G" at position 1486 at the 3'-end of reverse primer R1486w/BA2. is located. Hereinafter, for the BA1 type strain, BA2 strain, and BA5 strain, the 1355th base was placed at the 3'-end of the forward primer, and the counterpart base of the 1486th base was placed at the 3'-end of the reverse primer.

The second base from the 3'-end of both the forward primer and the reverse primer was modified with -OMe at the 2' of the sugar part. In both primers, the second base from the 3'-end was cytosine "C", so the nucleoside was 2'-O-methylcytidine. In Table 8, it is expressed as "(Cm)".

The spike protein of each mutant strain was used as a template, and the PCR protocol followed a standard protocol. The results of real-time PCR are shown in Table 9.

Referring to Table 9, only spike proteins matching the applied type were detected using the primers for each virus type. Furthermore, quantitative detection is also possible based on the Ct value.

This indicates that primers for each virus can be used as primers for detecting each virus. In other words, o-BA. The primer combinations applied to the 5 strains were o-BA. Primer for 5 strains, o-BA. The combination of primers applied to one strain is o-BA. Primer for one strain, o-BA. The combination of primers applied to the two strains was o-BA. It can be said to be a primer for two stocks.

FIG. 10 shows an example of the use of these primers. The saliva sample is divided into three parts and real-time PCR is performed using dedicated primers for each part. Then, it can be determined that the result is negative for the virus mutant strain corresponding to the primer that did not amplify. When amplified, it can be determined that the virus is positive for the virus strain corresponding to that primer. In FIG. 10, o-BA. Amplification was confirmed only in the case of primers for 2 strains, and the saliva specimen was o-BA. It can be determined that it is positive for 2 strains. Furthermore, quantitative analysis is also possible from the Ct at this time.

<Other polymerases>
In the above example, Takara Ex Taq polymerase (Takara Bio Inc.) was used, but the high-sensitivity PCR method according to the present invention can of course be performed with other polymerases as long as they have proofreading activity. can.

The same experiment as for KRAS mutation identification was performed using the primer POM2 shown in Table 2 and the polymerases PfuUltra II Fusion DNA Polymerase (Agilent Technologies) and Phusion Plus DNA Polymerase (Thermo Fisher Scientific). A standard protocol was used. The results are shown in Tables 10 and 11.

As can be seen from Tables 10 and 11, amplification was achieved only when the 3'-ends of the primers matched, and it can be seen that it does not depend on the type of polymerase as long as it has proofreading activity.

<Primers with other configurations>
The modified primer POM2 has a structure in which the second nucleoside from the 3'-end has the structure of formula (2), and when present at the 3'-end, inhibits polymerase activity. Experiments were conducted using nucleosides using ribose of formula (3) and ribose of formula (4) as primers with other structures that inhibit polymerase activity. A primer using a ribose nucleoside of formula (3) is called POME, and a primer using a ribose nucleoside of formula (4) is called POB.

Using primers using these nucleosides, RT-PCR was performed according to a standard protocol to detect KRAS. The sequences of the primers are shown in Table 12.

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

Referring to Table 13, amplification is performed only when the 3'-ends of both primer POME and primer POB match, and in cases where artificial nucleotides are exposed by the exonuclease activity of a polymerase with proofreading activity, the polymerase It can be seen that the activity is inhibited.

<Detection sensitivity of POB2>
POB2 has a structure in which the second nucleoside from the 3'-end has the structure of formula (4). The detection sensitivity of this primer was also investigated. The experimental conditions are the same as <Detection sensitivity of POM2>. That is, the initial concentration of primer POB2g is 2.0×10 ⁻¹ μM, and the concentration of template Tg (35G) is 1× (8.3×10 ⁻⁹ μM), 1/10×, 1/100×, and 1/1×. Five types were prepared: 1000x and 1/10000x.

The results are shown in FIG. 11 and Table 14. Referring to FIG. 11, the horizontal axis is the cycle number, and the vertical axis is the fluorescence intensity (ΔRn). Referring to Table 14, the ΔCt value should theoretically increase by 3.32 as the concentration decreases to one-tenth, but the measured values in Table 14 are also approximately the same, including experimental errors. , quantitative properties were confirmed. It is also seen that it was possible to amplify even just 10 copies (10 molecules) in one sample.

Furthermore, similar experiments were conducted with primer POB2a. In this case, the targeted template is Ta(35A) (see Table 1). In this case as well, results similar to those in FIG. 11 were obtained. Table 15 shows the Ct values and ΔCt values.

<When target substances are different>
As an example of identifying single nucleotide mutations in other target nucleotide sequences, the T790M mutation (2630T) of the EGFR gene WT (2630C) was investigated. In the mutant type, the 2630th position from the 5' end is mutated from cytosine (c) to thymine (t). Table 15 shows the base sequences of the EGFR gene WT and the portion used as a template in the mutant type. SEQ ID NOs: 32 and 33, respectively.

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

Real-time PCR according to the standard protocol was performed on wild type T-EGFR-2630c/wt and mutant T-EGFR-2630t/T790M using the primer set of SEQ ID NOs: 34 and 35 and the primer set of SEQ ID NOs: 36 and 37. . The resulting Ct values and ΔCt values are shown in Table 18.

Referring to Table 18, when wild type primers were used, the Ct value was about 20.1 for wild type (T-EGFR-2630c/wt), but for mutant type (T-EGFR- 2630t/T790M) 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 more sensitively than the mutant template by a factor of 2 to the power of 13.0, approximately 8192 times.

On the other hand, when the mutant primer F-EGFR/2630M2t was used, the Ct value was 33.4±0.21 for the wild type template, but the Ct value for the mutant template was 18. 9±0.13, and ΔCt was 14.5±0.1. This result shows that the mutant primers were able to detect the mutant template more sensitively than the wild type template, 2^14.5 times, or 23,170 times more sensitive. The target value for cell-free DNA analysis using liquid biopsy in cancer gene mutation diagnosis is to detect 0.01% (1/10,000) of the mutant gene compared to the wild-type gene, so the value of 23,170 times is higher than that. Detection sensitivity exceeds .

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

Referring to Table 19, the ΔCt value should theoretically increase by 3.32 as the concentration decreases to one-tenth, but the measured values in Table 19 are almost the same difference when experimental errors are taken into account. , quantitative properties were confirmed. It is also seen that it was possible to amplify even just 10 copies (10 molecules) in one sample.

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 investigated. The target was 100 bases from EGFR gene 2461 to 2560. Table 20 shows the nucleotide sequences of the wild type and delE746-A750 deletion mutant. The respective base sequences were SEQ ID NO: 38 and SEQ ID NO: 39.

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

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.

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 the 3'-end is an artificial nucleotide modified with "-OCH3" 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 expressed as "Gm".

Furthermore, 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 the 3'-end is an artificial nucleotide modified with "-OCH3" at the 2' position of sugar 14b. That is, the second nucleoside from the 3'-terminus is 2'-O-methyluridine. In Table 21, it is expressed as "Um".

We conducted a discrimination study using a combination of these primers. Table 22 shows the combinations of primers and the results of real-time PCR using the standard protocols.

Referring to Table 22, in Experiment A, when the combination of primers F-EGFR-delE746-A750/2497M2g and R-EGFR2461-2560 to detect the wild type was used, the Ct value for the wild type template T-EGFRwt2461-2560 was 21.86±0.09, Ct value was 32.78±0.28, and ΔCt was 10.92 with respect to the deletion type template T-EGFR-delE746-A750. That is, with this combination of primers, it was possible to detect the wild type template more sensitively than the mutant template by 2 to the power of 10.92, or about 1938 times.

In experiment A, when the primer combination F-EGFR-delE746-A750 2497M2a and R-EGFR2461-2560 was used to detect the deletion type, the Ct value was 30.40 ± 0.43 with respect to the wild type template T-EGFRwt2461-2560. The Ct value was 16.74±0.05 and the ΔCt value was 13.66 for the deletion template T-EGFR-delE746-A750. That is, with this combination of primers, the wild-type template could be detected 2 to the power of 13.66, approximately 12,944 times more sensitively than the mutant template. As mentioned above, the target value for cell-free DNA analysis using liquid biopsy in cancer gene mutation diagnosis is to detect 0.01% (1/10,000) of the mutant gene compared to the wild-type gene, which is 12,944 times more effective. This value indicates a detection sensitivity that exceeds that value.

In experiment C, when the combination of primers F-EGFR2461-2560 and R-EGFR-delE746-A750 2511M2t for wild type detection was used, the Ct value was 30.94 ± 0.09 with respect to the wild type template T-EGFRwt2461-2560. For the deletion template T-EGFR-delE746-A750, the Ct value was >45.0 (not detected) and the ΔCt was >14.06. That is, with this combination of primers, the wild-type template could be detected more sensitively than the mutant template by 2^14.06 times or more, or 17,080 times more sensitively.

In experiment D, when using the primer combination F-EGFR2461-2560 and R-EGFR-delE746-A750 2511M2c to detect the deletion type, the Ct value was 31.49 ± 0.02 with respect to the wild type template T-EGFRwt2461-2560. The Ct value was 18.40±0.08 and the ΔCt value was 13.09 for the deletion template T-EGFR-delE746-A750. That is, with this combination of primers, it was possible to detect the wild-type template more sensitively than the mutant template by 2^13.09 times, or about 8719 times more sensitively.

As mentioned above, the target value for cell-free DNA analysis using liquid biopsy in cancer gene mutation diagnosis is to detect 0.01% (1/10,000) of the mutant gene compared to the wild-type gene, which is 8719 times more effective. This value indicates a detection sensitivity close to that value.

Referring to Table 23, theoretically, every time the concentration of the EGFR deletion mutant template T-EGFR-delE746-A750 becomes one-tenth from the initial concentration of 8.3×10 ⁻⁹ μM, the ΔCt value will be 3.32. However, the measured values in Table 23 also range from 3.56 to 3.81, which is approximately the same difference within the experimental error range, confirming quantitative performance. It is also seen that it was possible to amplify even just 10 copies (10 molecules) in one sample.

Based on the above, it is thought that this highly sensitive PCR method can be widely applied to PCR methods that use all chemically modified primers that contain a 2'-modified nucleic acid at the second position from the 3'-end and all DNA polymerases that have proofreading activity. .

The high-sensitivity PCR method according to the present invention is a PCR method that can identify and quantitatively analyze single nucleotide mutations in DNA and RNA with high precision. It can also be suitably used as a mutant strain detection kit, a cell-free DNA analysis kit using liquid biopsy in cancer gene mutation companion diagnosis, and the like.

10 Primer 14 Sugar 14b Sugar 16 (of the second nucleotide from the 3'-end) Phosphodiester portion 16a (of the nucleotide 20a of the 3'-end) 18 Nucleobase 20 Nucleotide 20a Nucleotide of the 3'-end 20b 2nd nucleotide from 3'-end 30 Main chain 32 Complementary strand

Claims (5)

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 placed at the second position from the 3'-end, and a natural type of DNA is placed at the 3'-end. The high-sensitivity PCR method according to claim 1, further comprising Cybergreen. 3. The high-sensitivity PCR method according to claim 1, wherein the artificial nucleic acid is an RNA derivative in which the 2' position of the sugar is substituted with 2'-OMe or 2'-OCH ₂ CH ₂ OMe. 3. The high-sensitivity PCR method according to claim 1, wherein the backbone between each base of the primer containing the artificial nucleic acid is either a phosphodiester bond or a phosphorothioate bond. 3. The high-sensitivity PCR method according to claim 1, wherein nucleotides from the 3'-end to the 5'-end of the chemically modified primer are nucleotides that do not inhibit polymerase activity.