JP7371405B2 - Nucleic acid sample quality evaluation method - Google Patents

Description

The present invention relates to a method for evaluating the quality of nucleic acids contained in a sample. In particular, the present invention relates to a method for evaluating the quality of a nucleic acid sample using a nucleic acid amplification reaction.

In recent years, research has been underway to analyze individual cells that make up body fluids such as blood and tissues such as organs, and to apply this to basic research, clinical diagnosis, and treatment. For example, morphological analysis, tissue type analysis, and genetic analysis are performed on tumor cells (Circulating Tumor Cells, hereinafter referred to as CTCs) circulating in the blood of cancer patients, and the results obtained from these analyzes and analyzes are Based on the findings, research is underway to determine treatment policies and determine changes in pathological conditions during treatment.

On the other hand, it is known that even cells of the same type exhibit heterogeneity in the amount and quality of biomolecules such as proteins and nucleic acids. In particular, CTCs are known to undergo apoptosis at a high frequency, and there is concern that in CTCs with advanced apoptosis, biomolecules such as proteins and nucleic acids are degraded. Therefore, when analyzing CTCs individually, it is expected that the success rate of analysis will differ depending on the CTC to be analyzed.

In addition, when analyzing target cells contained in a sample individually, generally, the target cells are collected individually using a flow cytometer, or a group of cells containing the target cells is placed on a substrate such as a slide glass. After arranging the cells, they are collected individually using a microscope and micromanipulator, and then analyzed. When collecting target cells individually, it is necessary to collect them separately from contaminant cells, so usually, the target cells are labeled with biomolecules such as proteins and nucleic acids that are present within the target cells, and then collected. . For example, to recover CTCs, fluorescently labeled antibodies against cytokeratin, an intracellular protein characteristic of epithelial cells such as CTCs, or DAPI (4' , 6-diamidino-2-phenylindole), the CTCs are fluorescently labeled and collected.

However, when labeling biomolecules within target cells, fixation treatment and membrane permeabilization treatment of the cells are required, and there is a concern that biomolecules such as proteins and nucleic acids may be denatured and/or eluted due to these treatments. Therefore, when analyzing biomolecules from cells collected individually using the cell collection method described above, the quality and quantity of the biomolecules decreases compared to the case without treatment, resulting in a deterioration in the success rate and accuracy of analysis. obtain.

In particular, when analyzing a small number of cells such as a single cell, the amount of biomolecules contained in the cell is small, so obtaining good analysis results depends on the quality of the cell and the biomolecules contained in the cell. The amount of is important. In particular, when performing the above-mentioned analysis using base sequence analysis of intracellular nucleic acids using a next-generation sequencer, the cost required for analysis is still high. It is preferable to subject only the sample to analysis.

As a method for evaluating the quality of a nucleic acid sample, a method is known in which a target nucleic acid contained in a sample is subjected to a nucleic acid amplification reaction such as a PCR method, and the evaluation is performed based on the presence or absence of a nucleic acid amplification product or its amount (Patent Document 1, Patent Documents 1 to 2). However, in nucleic acid amplification reactions, the combination of oligonucleotides that can hybridize with the target nucleic acid, that is, the combination of forward primer (first primer) and reverse primer (second primer) (hereinafter also referred to as primer set) is optimal. It is necessary to Furthermore, in evaluating the quality of a nucleic acid sample, it is preferable that there be a plurality of regions (target regions) on the target nucleic acid to be amplified. When performing quality evaluation using multiple target regions, prepare a primer set that can hybridize with the target region for each target region, and simultaneously target multiple target regions with a nucleic acid amplification reagent containing the multiple prepared primer sets. Preferably, multiplex nucleic acid amplification is performed. However, in multiplex nucleic acid amplification, nonspecific amplification and a decrease in amplification efficiency are likely to occur due to interference between primers, and optimization of primer sets is a challenge.

Special table 2016-538872 publication

EH van Beers, et al. , British J. Cancer, 94, 333-337 (2006) Rodolphe Marie, et al. , Lab on a Chip, 18, 1891-1902 (2018)

An object of the present invention is to provide a method for evaluating the quality of nucleic acids contained in a sample when performing base sequence analysis using a next-generation sequencer.

In order to solve the above problems, the present inventors have made extensive studies and have arrived at the present invention.

That is, the present invention
a step of reacting the nucleic acid contained in the sample with a nucleic acid amplification reagent containing an oligonucleotide primer set that can hybridize with the nucleic acid;
and a step of evaluating the amount of the nucleic acid amplification product obtained in the reaction step.
A method for evaluating the quality of nucleic acids contained in a sample, the method comprising:
In the method, the nucleic acid is a human cell-derived nucleic acid, and the oligonucleotide primer set is at least one of the following (a) to (d).
(a) A set of an oligonucleotide primer containing at least the sequence set forth in SEQ ID NO: 1 and an oligonucleotide primer containing at least the sequence set forth in SEQ ID NO: 2. (b) An oligonucleotide primer containing at least the sequence set forth in SEQ ID NO: 3. and a set of oligonucleotide primers containing at least the sequence set forth in SEQ ID NO: 4 (c) an oligonucleotide primer containing at least the sequence set forth in SEQ ID NO: 5, and an oligonucleotide primer containing at least the sequence set forth in SEQ ID NO: 6. (d) A set of an oligonucleotide primer containing at least the sequence set forth in SEQ ID NO: 7 and an oligonucleotide primer containing at least the sequence set forth in SEQ ID NO: 8.

The present invention will be explained in detail below.

In the present invention, the sample containing nucleic acid is not particularly limited as long as it contains human cell-derived nucleic acid. Examples include human samples such as urine, whole blood, diluted blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, ascites, amniotic fluid, and human samples such as cell aggregates, tumors, lymph nodes, and arteries. Examples include samples derived from human organs or tissues, and culture samples such as human-derived cell and tissue cultures and their culture solutions. Furthermore, as long as the sample can contain the target nucleic acid to be amplified by the oligonucleotide primer set described below, a sample not derived from biomolecules may be used, such as an aqueous solution containing the target nucleic acid that has been artificially synthesized. Furthermore, the sample containing the nucleic acid can be subjected to cell membrane permeabilization treatment with ethanol etc., cell fixation treatment with formaldehyde etc., as long as it maintains a state in which the target nucleic acid can be amplified by a nucleic acid amplification reagent containing an oligonucleotide primer set described below. The sample may be subjected to various treatments such as formalin fixation, paraffin embedding, or staining with an antibody or fluorescent labeling substance.

In the present invention, there are no particular limitations on the method for obtaining nucleic acids from a sample. A sample such as a human-derived biological sample or cultured cells may be directly reacted with a nucleic acid amplification reagent, or nucleic acids may be extracted from the sample using an organic solvent, a surfactant, or the like. Further, the extracted nucleic acid may be purified, and further pre-treatment such as whole genome amplification may be performed. In particular, when the amount of nucleic acid contained in a sample is extremely small, such as when targeting a single cell or a small number of cells, performing an amplification operation on the nucleic acid sample such as whole genome amplification in advance can reduce the amount of nucleic acid contained in the sample. This is preferable because the amount of the nucleic acid sample increases and a portion of the nucleic acid sample can be subjected to the quality evaluation process.

In the present invention, the oligonucleotide primer set refers to a combination of oligonucleotides that can hybridize with a nucleic acid contained in a sample, and specifically refers to a sequence of the nucleic acids that can hybridize with a complementary strand of a target nucleic acid. A combination of a forward primer (first primer) that is an oligonucleotide that includes at least a sequence of the nucleic acid, and a reverse primer (second primer) that is an oligonucleotide that includes at least a sequence that can hybridize with a homologous strand of the target nucleic acid among the nucleic acids. It is.

The present invention is characterized in that at least one of the following (a) to (d) is used as the oligonucleotide primer set.

(a) An oligonucleotide primer (forward primer) containing at least the base sequence from 91850th to 91869th nucleotide sequence (sequence set forth in SEQ ID NO: 1) of the SMAD4 gene (GenBank No. NG_013013), and 91929th to 91948th nucleotide sequence of the same gene. A combination with an oligonucleotide primer (reverse primer) containing at least a complementary sequence (sequence set forth in SEQ ID NO: 2) of the base sequence up to.

(b) An oligonucleotide primer (forward primer) containing at least the nucleotide sequence from 191258th to 191276th (sequence set forth in SEQ ID NO: 3) of the EGFR (Epidermal Growth Factor Receptor) gene (GenBank No. NG_007726), and the same gene A combination with an oligonucleotide primer (reverse primer) containing at least a complementary sequence of the base sequence from 191390th to 191409th (sequence set forth in SEQ ID NO: 4).

(c) An oligonucleotide primer (forward primer) containing at least the nucleotide sequence from 45324th to 45346th nucleotide sequence (sequence set forth in SEQ ID NO: 5) of the KIT gene (GenBank No. NG_007456), and 45502nd to 45522nd nucleotide sequence of the same gene. A combination with an oligonucleotide primer (reverse primer) containing at least a complementary sequence (sequence set forth in SEQ ID NO: 6) of the base sequence up to.

(d) An oligonucleotide primer (forward primer) containing at least the nucleotide sequence from 16294th to 16318th (sequence set forth in SEQ ID NO: 7) of the TP53 (Tumor Protein 53) gene (GenBank No. NG_017013), and A combination with an oligonucleotide primer (reverse primer) containing at least a complementary sequence of the base sequence from position 16520 to position 16539 (sequence set forth in SEQ ID NO: 8).

In the present invention, a nucleic acid amplification reagent containing at least one of the oligonucleotide primer sets shown in (a) to (d) above is reacted with a nucleic acid contained in a sample, and the nucleic acid amplification obtained by the reaction is However, if the nucleic acid amplification reagent contains two or more of the oligonucleotide primer sets shown in (a) to (d) above, the quality of the nucleic acids contained in the sample can be evaluated accurately. The oligonucleotides shown in (a) to (d) above are preferable in that they can be used, and it is more preferable to include any three or more in the nucleic acid amplification reagent because the quality of the nucleic acid contained in the sample can be evaluated more accurately. It is even more preferable to include all primer sets in the nucleic acid amplification reagent, since the quality of the nucleic acid contained in the sample can be evaluated even more accurately. Furthermore, as long as the nucleic acid amplification reagent contains at least one of the oligonucleotide primer sets shown in (a) to (d) above, other oligonucleotide primer sets capable of hybridizing with the nucleic acids contained in the sample can be used as a nucleic acid amplification reagent. The quality of nucleic acids contained in a sample may be evaluated by including them in a sample.

There is no particular limitation on the nucleic acid amplification method (method of reacting a nucleic acid contained in a sample with a nucleic acid amplification reagent) carried out in the present invention, and any nucleic acid amplification method commonly used by those skilled in the art, such as PCR method, may be used. In addition, a sequence that can form a loop structure, an RNA polymerase promoter sequence, etc. is added to the forward primer or reverse primer in advance, and then LAMP (Loop-Mediated Isothermal Amplification) method, NASBA (Nucleic Acid Sequence-Based Amplification) method, etc. ation) law, TMA It may also be used in isothermal nucleic acid amplification methods such as (Transcription Mediated Amplification) method and TRC method (Transcription-Reverse Transcription Concerted).

The reagent composition, reaction temperature, reaction time, etc. for nucleic acid amplification may be appropriately determined depending on the nucleic acid amplification method, nucleic acid amplification enzyme, target nucleic acid sample, etc. used. Furthermore, when reacting the nucleic acid contained in the sample with the nucleic acid amplification reagent, the entire amount of the nucleic acid sample may be reacted, or only a portion of the nucleic acid sample may be reacted. When performing various analyzes on the nucleic acid sample after quality evaluation of the nucleic acid sample, it is preferable to subject only a part of the nucleic acid sample to the quality evaluation process, since the remainder of the nucleic acid sample can be used for various analyses. .

The amount of target nucleic acid amplified by the method described above can be evaluated by conventionally known nucleic acid detection methods. Examples of the nucleic acid detection method include the methods shown in (A) to (C) below;
(A) Method using electrophoresis or liquid chromatography,
(B) a hybridization method using a nucleic acid probe labeled with a detectable label;
(C) A method using a fluorescent dye-labeled probe designed to change its fluorescent properties by hybridizing with a part of the base sequence of the amplification product.

Examples of the fluorescent dye labeled probe (C) include fluorescent labeled probes using FRET (fluorescence resonance energy transfer) and nucleic acid probes labeled with intercalating fluorescent dyes. Note that the detection of the target nucleic acid may be performed separately from the amplification reaction of the target nucleic acid, and the probe of (B) or (C) above is included in the nucleic acid amplification reagent in advance, and then the detection of the target nucleic acid is carried out separately from the amplification reaction of the target nucleic acid. Target nucleic acids may be detected simultaneously (eg, real-time PCR method or real-time TRC method).

In the present invention, the quality of nucleic acids contained in a sample is evaluated by evaluating the amount of nucleic acid amplification products detected by the method described above. The evaluation criteria may be determined as appropriate depending on the purpose and situation. For example, it may be evaluated simply by the presence or absence of detection of nucleic acid amplification products (i.e., qualitatively), or by setting a certain threshold for the amount (or concentration) of nucleic acid amplification products and determining whether the amount (or concentration) of the nucleic acid amplification products is exceeded (i.e., quantitative evaluation). may be evaluated). Furthermore, if the nucleic acids contained in the sample are amplified by a multiplex nucleic acid amplification method using multiple oligonucleotide primer sets, the number (type) of products amplified by each primer set, their amount or ratio, or the amplification The evaluation may be based on a profile or the like.

Nucleic acids evaluated to be of good quality according to the present invention can then be subjected to further analysis and processing. There are no particular limitations on the analysis or processing, and examples include analysis and processing targeting nucleic acids, such as base sequence analysis, hybridization analysis, and gene cloning. For the nucleic acid sample to be subjected to the analysis or processing, the solution after reaction with the nucleic acid amplification reagent may be used, or if a part of the sample containing the nucleic acid has been subjected to quality evaluation, the remainder of the sample may be used. Also good. Note that it is preferable to use the remainder of the nucleic acid sample in terms of eliminating the influence of the nucleic acid amplification reagent.

There are no particular limitations on the quality evaluation criteria for nucleic acids contained in samples, and they may be determined as appropriate depending on the situation and purpose, such as the total number of samples, quality trends, and throughput of analysis and processing. That is, only samples of very good quality may be selected, or a relatively wide range of samples may be selected. Note that when nucleic acids contained in a sample are amplified by a multiplex nucleic acid amplification method using multiple oligonucleotide primer sets and the quality of the nucleic acids is evaluated by the number of types of products amplified by the primer sets, it is possible to amplify the nucleic acids. Only nucleic acid samples in which all amplification products have been detected may be selected, nucleic acid samples in which multiple amplification products have been detected may be selected, or samples in which one or more amplification products have been detected may be selected. It's okay.

In evaluating the quality of a nucleic acid contained in a sample, the present invention involves reacting it with a nucleic acid amplification reagent containing an oligonucleotide primer set that can hybridize with the nucleic acid, and determining the amount of the amplified product of the nucleic acid obtained by the reaction. A method for evaluation based on the method, wherein the nucleic acid is a nucleic acid derived from human cells, and the oligonucleotide primer set includes at least one gene selected from the SMAD4 gene, the EGFR gene, the KIT gene, and the TP53 gene. It is characterized by using a combination of an oligonucleotide primer that can hybridize with a complementary strand and an oligonucleotide primer that can hybridize with a homologous strand of the same gene as the primer.

INDUSTRIAL APPLICATION This invention can evaluate the quality of the nucleic acid contained in a sample quickly and easily. Therefore, by selecting a high-quality nucleic acid sample using the present invention, the success rate and accuracy of subsequent analysis can be improved and efficient analysis can be performed.

FIG. 2 is an exploded view showing a cell holding device 100 used in Example 1 and capable of individually detecting and recovering target cells. FIG. 2 is a front view of the device shown in FIG. 1; FIG. 3 is a diagram illustrating recovery of cells held in the apparatus shown in FIGS. 1 and 2 by a recovery means 400. FIG. FIG. 7 is a diagram showing the results of Example 3.

Hereinafter, the present invention will be explained in more detail based on Examples, but the present invention is not limited to these Examples.

Example 1 Preparation of Nucleic Acid Sample A nucleic acid sample was prepared from a human cancer cell line by the method shown below.

(1) A human lung cancer cell line (H1975 strain) cultured in a 24-well microplate was detached with trypsin and collected in a microtube.

(2) After centrifuging at 600 x g for 5 minutes and removing the supernatant, the cell pellet was mixed with a solution containing PEG-BSA (0.1% (w/v) as BSA) and 280 mM xylitol (hereinafter referred to as "PEG-BSA"). It was washed by suspending it in "BSA xylitol solution"). The washing operation was repeated three times.

(3) The cancer cell line suspension obtained in (2) was introduced into the cell holding device 100 shown in FIGS. 1 and 2. By applying an AC voltage (1 MHz, 20 Vp-p) from the signal generator 50 to the electrode substrates 31 and 32 for 10 minutes, cancer cell lines (target cells 70) are transferred to the holding part 60 of the device by the dielectrophoretic force 300. was held. Note that the cell holding device 100 shown in FIGS. 1 and 2 includes an insulator 12 having a plurality of through holes 12a with a diameter of 30 μm, a light-shielding chrome film (light-shielding member 11) having a plurality of through-holes 11a with a diameter of 30 μm, and an electrode. The substrate 31 is placed in close contact with the insulator 12 - the light shielding member 11 - the electrode substrate 31 from above in this order, and further has a sample inlet 21, an outlet 22 and a through part 23 on the upper surface of the insulator 12, and has a thickness of 1 mm. This is a device in which a spacer 20 and an electrode substrate 32 are provided in close contact with the upper surface of the spacer 20, respectively. The through holes 11a/12a and the electrode substrate 31 form approximately 300,000 holding portions 60 each having a diameter of 30 μm and a depth of 40 μm and capable of holding the target cells 70.

(4) Introduce a 280 mM xylitol aqueous solution containing 0.01% (w/v) poly-L-lysine from the inlet 21 while applying the AC voltage, and after leaving it for 3 minutes, apply the AC voltage. was stopped, and the aqueous solution was removed by suction from the outlet 22.

(5) PBS containing 1% (w/v) formaldehyde (hereinafter also referred to as "cell fixation reagent") was introduced from the introduction port 21, and the cells were fixed by standing for 10 minutes. Thereafter, the cell fixation reagent was removed by suction from the outlet 22, and PBS-T (Phosphate buffered saline with Tween 20) was introduced from the introduction port 21 to wash away the remaining cell fixation reagent.

(6) An aqueous solution containing 95% (w/v) ethanol (hereinafter referred to as a membrane permeation reagent) was introduced from the inlet 21 and allowed to stand for 10 minutes to permeabilize the cells. Thereafter, the cell fixation reagent was removed by suction from the outlet 22, and PBS-T was introduced from the inlet 21 to wash away the remaining membrane permeation reagent.

(7) 850 μL of a cell staining reagent mixed with DAPI (4',6-DiAmidino-2-PhenylIndole) (manufactured by Dojindo Laboratories) was introduced from the introduction port 21, and the cells were labeled by allowing it to stand for 20 minutes. . Thereafter, the cell staining reagent was removed by suction from the outlet 22, and PBS-T was introduced from the inlet 21 to wash away the remaining cell staining reagent.

(8) After detecting the DAPI-positive cells (cancer cell line 70) held in the holding part 60 using a fluorescence microscope 200 (IX71 manufactured by Olympus), as shown in FIG. 400 was used to suction the cancer cell line 70 held in the holding part 60 of the cell holding device 100 from which the electrode substrate 32 on the upper surface was removed. After discharging the aspirated cancer cell line 70 into a 0.2 mL microtube, a nucleic acid sample was prepared by performing whole genome amplification using the Ampli1 WGA kit (manufactured by Silicon Biosystems).

In this example, the cancer cell line aspiration and whole genome amplification operations performed in (8) were performed on a total of 11 cancer cell lines, and the nucleic acid samples were named nucleic acid samples a to k, respectively.

Example 2 Quality Evaluation of Nucleic Acid Sample The quality of the nucleic acid sample prepared in Example 1 was evaluated by the method shown below.

(1) Mix 2.5 μL each of primer solutions (concentration: 100 μM) consisting of the base sequences listed in Table 1 (4 pairs of Forward/Reverse primers, 8 types in total) and 30 μL of RNase-free water, and prepare a primer mix solution. were prepared (concentration: 5 μM each).

(2) Using the primer mix solution prepared in (1), the target nucleic acid on the nucleic acid sample (template DNA) prepared in Example 1 was amplified by PCR. The reaction solution composition and reaction temperature in the PCR method are shown below.
(Reaction liquid composition)
HotStarTaq Master Mix (manufactured by Qiagen) 12.5μL
RNase-free water 10μL
5μM primer mix solution 1.5μL
Nucleic acid sample (Example 1) 1 μL
(reaction temperature)
95°C for 15 minutes: 1 cycle 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 60 seconds: 40 cycles 72°C for 10 minutes: 1 cycle (3) Agilent 2100 Bioanalyzer and Agilent DNA1000 Kit (both Amplification of the DNA was confirmed using Agilent Technologies (Agilent Technologies, Inc.).

Table 2 shows the amount of nucleic acid amplification products amplified by each primer set. It can be seen that the amount and type of nucleic acid amplification products (PCR products) differ for each nucleic acid sample.

Example 3 Gene mutation analysis of a nucleic acid sample Gene mutation analysis of a nucleic acid sample (mutation analysis using target sequences of cancer-related genes) was performed using the method shown below.

The nucleic acid sample prepared in Example 1 was purified using Agencourt AMPureXP (manufactured by Beckman Coulter). The purified sample was subjected to library preparation using Ion AmpliSeq Cancer Hotspot Panel v2 (hereinafter referred to as CHPv2) and Ion AmpliSeq Library Kit Plus (both manufactured by Thermo Fisher Scientific). Ion S5 (manufactured by Thermo Fisher Scientific) ) was used to conduct gene mutation analysis.

FIG. 4 shows the relationship between the detection rate of gene mutation analysis and the quality evaluation results of nucleic acid samples (Example 2). Here, the detection rate refers to the percentage of sites whose base sequences have been decoded among HotSpots (approximately 2,700 sites) within the CHPv2 detection target range. It can be seen that the larger the amount and number (types) of nucleic acid amplification products detected in Example 2, the higher the detection rate of gene mutation analysis. That is, when evaluating the quality of a nucleic acid derived from human cells, an oligonucleotide primer set that can hybridize with the nucleic acid (in this example, an oligonucleotide primer containing at least the sequence set forth in SEQ ID NO: 1 and an oligonucleotide primer set described in SEQ ID NO: 2) is used. A combination of an oligonucleotide primer containing at least the sequence set forth in SEQ ID NO: 3 and an oligonucleotide primer containing at least the sequence set forth in SEQ ID NO: 4, a combination of an oligonucleotide primer containing at least the sequence set forth in SEQ ID NO: 4, and a combination of an oligonucleotide primer containing at least the sequence set forth in SEQ ID NO: 4; A combination of an oligonucleotide primer containing at least the sequence set forth in SEQ ID NO: 6 and an oligonucleotide primer containing at least the sequence set forth in SEQ ID NO: 7, and an oligonucleotide primer containing at least the sequence set forth in SEQ ID NO: 7 and an oligonucleotide primer containing at least the sequence set forth in SEQ ID NO: 8. The nucleic acids contained in the sample are evaluated based on the amount of nucleic acid amplification products (including the number (type), the same applies hereinafter) obtained in the reaction, and the nucleic acids are By selecting nucleic acids with a large amount of amplification products, analysis of nucleic acid samples can be carried out efficiently.

100: Cell holding device 11: Light shielding member 12: Insulator 11a/12a: Through hole 20: Spacer 21: Inlet 22: Outlet 23: Penetrating part 31/32: Electrode substrate 40: Conductive wire 50: Signal generator 60: Holding part 70: Target cell (cancer cell line)
200: Fluorescence microscope 300: Dielectrophoretic force 400: Micromanipulator

Claims (2)

a step of reacting the nucleic acid contained in the sample with a nucleic acid amplification reagent containing an oligonucleotide primer set that can hybridize with the nucleic acid;
and a step of evaluating the amount of the nucleic acid amplification product obtained in the reaction step.
A method for evaluating the quality of nucleic acids contained in a sample before subjecting the nucleic acids to analysis or processing, the method comprising:
The method, wherein the nucleic acid is a human cell-derived nucleic acid, and the oligonucleotide primer set is at least one of the following (a) to (d):
(a) A set of an oligonucleotide primer consisting of the sequence set forth in SEQ ID NO: 1 and an oligonucleotide primer consisting of the sequence set forth in SEQ ID NO: 2. (b) A set of an oligonucleotide primer consisting of the sequence set forth in SEQ ID NO: 3, and an oligonucleotide primer consisting of the sequence set forth in SEQ ID NO: 3. (c) A set of an oligonucleotide primer consisting of the sequence set forth in SEQ ID NO: 5 and an oligonucleotide primer consisting of the sequence set forth in SEQ ID NO: 6. (d) Sequence A set of an oligonucleotide primer consisting of the sequence set forth in SEQ ID NO: 7 and an oligonucleotide primer consisting of the sequence set forth in SEQ ID NO: 8. The method according to claim 1, wherein the analysis or processing is base sequence analysis, hybridization analysis, or gene cloning.