JP4650420B2 - Base determination method and base determination kit - Google Patents

Description

  The present invention relates to a method for detecting or determining mutations, substitutions, polymorphisms, insertions or deletions, and base determination using oligonucleotides that amplify regions containing mutations, DNA polymerases, and oligonucleotides that specifically amplify mutant nucleic acid sequences. It relates to a kit for use. The present invention is particularly useful for diagnosis of genetic diseases and examination of diseases caused by point mutations. Mutations include both acquired gene mutations and SNPs.

  In the present invention, the nucleic acid sequence mutation means having a base sequence different from the wild type. It is known that gene mutations can cause disease or show resistance to drugs. Therefore, elucidation of variations in these nucleic acid sequences is clinically important, and routine phenotyping is particularly recommended for psychiatric patients and volunteers in clinical research (see Non-Patent Document 1 and Non-Patent Document 2). ). In addition, a nucleic acid sequence analysis method for detecting each genotype following identification of the causative mutant gene is desired.

  As a conventional nucleic acid sequence analysis technique, for example, there is a nucleic acid sequencing method (sequencing method). Nucleic acid sequencing can detect and identify nucleotide polymorphisms contained in nucleic acid sequences, but requires a great deal of effort to prepare template nucleic acids, DNA polymerase reaction, polyacrylamide gel electrophoresis, analysis of nucleic acid sequences, etc. And time is needed. Moreover, although labor saving is possible by using a recent automatic sequencer, there is a problem that an expensive apparatus is required.

  On the other hand, there are various known genetic diseases caused by point mutations in genes, and some of them know which part of the gene and how point mutations cause genetic diseases. Not a few.

As a method for detecting such a predicted point mutation, detection of a point mutation of a gene using a gene amplification method such as a PCR (polymerase chain reaction) method (see Patent Document 1 and Patent Document 2) has been conventionally performed. The method is known. A method (PCR-RFLP) is used in which amplified gene fragments are treated with a restriction enzyme that cleaves a specific nucleic acid sequence, and the size of the resulting fragments is judged. Similarly, as a detection method using the PCR method, the specificity of the primer used was used.
Alelle specific amplification method is used. In this method, as one of the pair of oligonucleotides used in the gene amplification method, a wild-type oligonucleotide that is completely complementary to the end region of the wild-type gene amplification region and the mutant-type gene amplification A mutant oligonucleotide that is completely complementary to the end region of the region is used. Mutant oligonucleotides have nucleotides complementary to the predicted point mutation at the 3 'end. Using such wild-type and mutant-type oligonucleotides simultaneously or separately, the sample gene is subjected to a gene amplification method (Non-patent Document 1 Cynthia)
DKBottema and Steves S. Sommer, Mutation Research, 288, 93-102, (1993)).

  If the sample gene is wild-type, nucleic acid amplification occurs when a wild-type oligonucleotide is used, but if a mutant-type oligonucleotide is used, the 3 ′ end of the oligonucleotide corresponds to the sample gene. Since it is not complementary to the nucleotide (mismatch), no extension reaction occurs, and no nucleic acid amplification occurs. On the other hand, if the sample gene is a mutant type, conversely, amplification does not occur when the wild-type oligonucleotide is used, and amplification occurs when the mutant-type oligonucleotide is used. Therefore, by examining whether amplification occurs when using each oligonucleotide, it is possible to determine whether the sample gene is a wild type or a mutant type, thereby identifying a point mutation in the sample gene. Can do. As a method to check whether amplification occurred at this time, after amplifying the amplified product with agarose gel, staining with a nucleic acid-specific binding fluorescent reagent such as ethidium bromide, and then irradiating with UV, the presence of amplified nucleic acid is detected. it can. Other methods include Southern blotting, in which amplified nucleic acid is immobilized on a nylon membrane and detected using a labeled probe, sandwich hybridization in which a detection probe is used after capturing with a supplementary probe immobilized on an individual carrier. Laws have been developed. However, in practice, the operation is complicated, and in order to analyze a large amount of samples quickly, it is necessary to construct a large-scale laboratory automation system which requires a lot of labor.

  For example, in the PCR-RFLP method, it is necessary to perform electrophoresis after performing restriction enzyme treatment after PCR. Although the operation after PCR can be simplified in the Alelle specific amplification method, when detecting by electrophoresis, it is necessary to separately perform wild type detection and mutation type detection. Further, Southern blotting and sandwich hybridization require a hybridization reaction with a probe, and the conditions must be strictly adjusted. Furthermore, a process for removing excess probes is required, and the operation is very complicated.

In addition, when performing amplification separately because wild type detection and mutation type detection are performed, the amplification cost increases in accordance with the number of mutations. There is a method in which a plurality of amplifications are amplified with one reaction system and detected with a DNA chip, but the DNA chip is still costly and difficult to use at the forefront of medicine.
Japanese Examined Patent Publication No. 4-67960 Japanese Patent Publication No. 4-67957 Gram and Brsen, European Consensus Conference on Pharmacogenetics.Commission of the European Communities, Luxembourg, 1990, pp. 87-96 Balant et al., Eur. J. Clin. Pharmacol. 36, 551-554, (1989)

Positional relationship between at least two types of oligonucleotide primer for base determination and oligonucleotide primer (B, C) Basic primer setting method for "mutation" or "substitution" Basic primer setting method for single nucleotide polymorphism Basic primer setting method for "Insert" Basic primer setting method for "deletion" Results of PCR analysis of the (E255K) mutant gene in the abl gene ATP binding region of the bcr-abl chimeric gene Results of PCR analysis of (T315I) mutant genes in the abl gene ATP binding region of the bcr-abl chimeric gene Results of PCR analysis of 681 (G → A) single nucleotide polymorphism of CYP2C19 gene Results of PCR analysis of 636 (G → A) single nucleotide polymorphism of CYP2C19 gene Results of PCR analysis of 1075 (A → C) single nucleotide polymorphism of CYP2C9 gene Detection results of exon 19 Del E746-A750 deletion mutation in human EGFR gene Detection results of exon 19 Del L747-S752 deletion mutation in human EGFR gene Detection results of exon 19 Del E746-T751 ins I deletion mutation of human EGFR gene Detection result of exon 19 Del L747-T751del P753 deletion mutation in human EGFR gene Detection results of exon 19 Del L747-A750 substitution P deletion mutation in human EGFR gene Detection results of human EGFR gene exon 19 Del L747-S752, Del E746-T751 ins I, Del L747-T751delP753, Del L747-A750 substitution P deletion mutation Results of analysis using the CYP2C9 gene 1075 (A → C) single nucleotide polymorphism probe Analysis of H.pylori CagA gene type by PCR

  Although it seems that the amplified nucleic acid can be detected by the method as described above and the mutation, substitution, single nucleotide polymorphism, insertion or deletion in the nucleic acid sequence can be easily identified, the operation is actually complicated. In order to analyze a large amount of samples quickly, it is necessary to construct a large-scale laboratory automation system that requires a lot of labor. In addition, there is a problem that the operation is very complicated.

  The object of the present invention is to solve the above-mentioned problems and to detect a mutation, substitution, single nucleotide polymorphism, insertion or deletion in a nucleic acid sequence clearly and reproducibly and a reagent therefor Is to provide.

The present invention relates to a method for determining at least one base site on a nucleic acid sequence contained in a sample solution, and at least one base determination oligonucleotide primer (A) containing the base site to be determined, and an oligonucleotide. Primers (B) and (C) are the following (1) to (3):
(1) Primer (A) is a sense primer or an antisense primer, and a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region combination;
(2) Primer (A) is a sense primer, and a combination of a sense primer (B) that is homologous to the downstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region (provided that Primer (C) is downstream of primer (B));
(3) Primer (A) is an antisense primer, and a combination of a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the upstream region (provided that , Primer (C) is downstream of primer (B));
A gene amplification reaction is performed using a combination selected from any of the above, and the amplification product (for example, electrophoresis, electrophoresis, detection method by hybridization, fluorescence detection method, melting curve analysis method, thermal elution chromatogram method, etc.) It has been found that the presence of a base to be determined can be easily determined by detection) (by an existing method), and the present invention has been completed. That is, the present invention has the following configuration.
1. In a method for determining at least one type of base site on a nucleic acid sequence contained in a sample solution, at least one type of oligonucleotide primer for base determination (A) containing the base site to be determined, and oligonucleotide primer (B) And (C), a base determination method comprising a step of performing a gene amplification reaction using:
The primers (A), (B) and (C) are selected from any combination of the following (1) to (3):
(1) Primer (A) is a sense primer or an antisense primer, and a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region combination;
(2) Primer (A) is a sense primer, and a combination of a sense primer (B) that is homologous to the downstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region (provided that Primer (C) is downstream of primer (B));
(3) Primer (A) is an antisense primer, and a combination of a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the upstream region (provided that Primer (C) is downstream of primer (B)).
2. Item 2. The base determination method according to Item 1, wherein the base site to be determined includes mutation, substitution, single nucleotide polymorphism, insertion or deletion.
3. Item 3. The method according to Item 1 or 2, wherein the first base from the 3 ′ end of the oligonucleotide primer for base determination (A) is the same or complementary base as the predicted base of the base site to be determined. Base determination method.
4). Item 3. The method according to Item 1 or 2, wherein the second base from the 3 ′ end of the oligonucleotide primer for base determination (A) is the same or complementary base as the expected base of the base site to be determined. Base determination method.
5. Item 3 or 3, wherein at least one of the 3 to 5 bases from the 3 ′ end of the oligonucleotide primer for base determination (A) is substituted with a base that is not complementary or homologous to the nucleic acid sequence used as a template. 4. The base determination method according to 4.
6). The Tm value of the oligonucleotide primer for base determination (A) has Tm values of at least two types of oligonucleotide primers (B, C) having sequences complementary or homologous to the upstream and / or downstream region of the base site to be determined. Item 6. The base determination method according to any one of Items 1 to 5, which is higher than the value.
7). At least two types of oligonucleotide primers (B, C) having concentrations complementary or homologous to the upstream and / or downstream region of the base site to be determined, when the concentration of the oligonucleotide primer for base determination (A) is used. Item 7. The base determination method according to any one of Items 1 to 6, wherein the concentration is higher than the concentration at the time of use.
8). Obtained by using any one of base primer oligonucleotide primer (A) and oligonucleotide primer (B, C) having a sequence complementary or homologous to the upstream and / or downstream region of the base site to be judged Item 8. The base determination method according to any one of Items 1 to 7, wherein the length of the amplified nucleic acid fragment varies depending on the base to be determined.
9. The 5 ′ end of at least two kinds of oligonucleotide primers (B, C) having a sequence complementary or homologous to the upstream and / or downstream region of the base site to be determined as a base determination oligonucleotide primer (A) is labeled Item 9. The base determination method according to any one of Items 1 to 8, wherein
10. Item 10. The base determination method according to any one of Items 1 to 9, wherein the label is any one selected from the group consisting of a fluorescent chemical substance, a luminophore, an enzyme, a fluorescent protein, a photoprotein, a magnetic substance, and a conductive substance. 11. Item 11. The base determination method according to any one of Items 1 to 10, wherein the gene amplification method is selected from the group consisting of PCR, NASBA, LCR, SDA, RCR and TMA.
12 Item 12. The base determination method according to any one of Items 1 to 11, wherein the gene amplification method is PCR, and the DNA polymerase used in the PCR does not have 5 ′ exonuclease activity.
13. In a method for determining at least one kind of base site on a nucleic acid sequence contained in a sample solution,
At least one base determination oligonucleotide primer (A) containing the base site to be determined, oligonucleotide primers (B) and (C), and at least one homologous or complementary to the nucleic acid sequence containing the base site A base determination method comprising a step of performing a gene amplification reaction using a kind of oligonucleotide probe (D),
The primers (A), (B) and (C) are selected from any combination of the following (1) to (3):
(1) Primer (A) is a sense primer or an antisense primer, and a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region combination;
(2) Primer (A) is a sense primer, and a combination of a sense primer (B) that is homologous to the downstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region (provided that Primer (C) is downstream of primer (B));
(3) Primer (A) is an antisense primer, and a combination of a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the upstream region (provided that Primer (C) is downstream of primer (B)).
14 In a method for determining at least one type of base site on a nucleic acid sequence contained in a sample solution, at least one type of oligonucleotide primer for base determination (A) containing the base site to be determined, and oligonucleotide primer (B) And (C), and a step of performing a gene amplification reaction using at least one kind of homologous or complementary oligonucleotide probe (E) having a sequence different from the nucleic acid sequence containing the base site by at least one base A base determination method characterized by
The primers (A), (B) and (C) are selected from any combination of the following (1) to (3):
(1) Primer (A) is a sense primer or an antisense primer, and a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region combination;
(2) Primer (A) is a sense primer, and a combination of a sense primer (B) that is homologous to the downstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region (provided that Primer (C) is downstream of primer (B));
(3) Primer (A) is an antisense primer, and a combination of a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the upstream region (provided that Primer (C) is downstream of primer (B)).
15. Item 15. The base determination method according to Item 13 or 14, wherein the oligonucleotide probe (D) or (E) is labeled.
16. Item 16. The base determination method according to Item 15, wherein the label is any one selected from the group consisting of a fluorescent chemical substance, a luminophore, an enzyme, a fluorescent protein, a photoprotein, a magnetic substance, and a conductive substance.
17. A base determination kit comprising at least one base determination oligonucleotide primer (A) containing the base site to be determined, and oligonucleotide primers (B) and (C),
The primers (A), (B) and (C) are selected from any combination of the following (1) to (3):
(1) Primer (A) is a sense primer or an antisense primer, and a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region combination;
(2) Primer (A) is a sense primer, and a combination of a sense primer (B) that is homologous to the downstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region (provided that Primer (C) is downstream of primer (B));
(3) Primer (A) is an antisense primer, and a combination of a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the upstream region (provided that Primer (C) is downstream of primer (B)).
18. Item 18. The kit for determining a base according to Item 17, further comprising at least one type of oligonucleotide probe (D) that is homologous or complementary to a nucleic acid sequence containing the base site.
19. Item 18. The kit for base determination according to Item 17, further comprising at least one kind of homologous or complementary oligonucleotide probe (E) having a sequence different from the nucleic acid sequence containing the base site by at least one base.
20. Item 20. The kit for base determination according to any one of Items 17 to 19, further comprising a DNA polymerase.

  As described above, the present invention provides a method that can clearly and easily determine the presence of a specific base in a sample nucleic acid. Since the method of the present invention does not require a complicated operation as in the conventional methods, results with good reproducibility can be obtained quickly and easily.

  If the chromosome or fragment thereof containing a specific base site related to mutation, substitution, single nucleotide polymorphism, insertion or deletion contained in a sample is a target nucleic acid containing a base site responsible for information of the target gene, There is no particular limitation. Examples of the target nucleic acid include Alu sequences, exons of genes encoding proteins, introns, promoters, and the like. More specifically, genes related to various diseases including genetic diseases, drug metabolism, lifestyle-related diseases (hypertension, diabetes, etc.) can be mentioned. For example, as a causative gene of chronic myelogenous leukemia, a bcr-abl chimeric gene point mutation, cytochrome P450 genetic polymorphism, cytokine genetic polymorphism and the like can be mentioned.

  In the present invention, the nucleic acid sequence may be simply referred to as a nucleic acid. In addition, a sequence in which a specific base exists may be referred to as a mutant type, and a sequence that does not have a specific type as a wild type. A mutant type is one in which at least one, preferably one nucleotide in a wild-type base sequence is point-mutated and replaced with another nucleotide, or inserted or deleted into a part of a wild-type nucleic acid. A nucleic acid containing It has been elucidated that the constitution is different due to such a difference in base sequence, and the method of the present invention examines whether or not the nucleic acid in the sample has such an expected sequence change. It is a method to do.

  In the present invention, the base site to be determined may be in another chromosome, may be in a remote position within the same gene, or may be adjacent.

  In the method base determination method of the present invention, the primers (A) to (C) may be used simultaneously to perform a gene amplification reaction, or any two of them may be used in combination to perform a gene amplification reaction. Preferably, the gene amplification reaction is performed in the presence of primers (A) to (C). In addition, the probe (D) and the probe (E) can be used in combination with the primers (A) to (C) simultaneously or separately.

  The primers used in the present invention can be amplified by PCR, NASBA, LCR, SDA, RCA, TMA, LAMP, and ICAN methods, which are known amplification methods, so that an amplification product is obtained so as to contain at least one base to be determined. design. The primer chain length may be 9 to 35 bases, preferably 11 to 30 bases, more preferably 15 to 28 bases, and further preferably 18 to 25 bases.

“Base site to be determined” includes “mutation”, “substitution”, “single nucleotide polymorphism”, “insertion” or “deletion”. What is necessary is just to set the oligonucleotide primer (A) for base determination containing the base site | part which wants to determine these. In addition, “base site to be determined” means “mutation”.
, “Substitution”, “single nucleotide polymorphism”, “insertion” or “deletion”. It is not always necessary for the primer (A) to contain an insertion sequence or a deletion sequence, and bases before and after the occurrence of “mutation”, “substitution”, “single nucleotide polymorphism”, “insertion” or “deletion”. The part may be included.

  In the description of the present invention, when explaining the orientation of the primer, the forward primer commonly used by the trader may be referred to as a sense primer, and the reverse primer may be referred to as an antisense primer.

In order to determine “mutation” or “substitution”, the base primer oligonucleotide primer (A) may be set so as to include a base after mutation, or a base that is the same as or complementary to the base after substitution. . Preferably, the first or second base from the 3 ′ end of the base determination oligonucleotide primer (A) may be the same as or complementary to the base expected to be “mutated” or “substituted”. More preferably, the second base from the 3 ′ end of the oligonucleotide primer for base determination (A) is the same as or complementary to the base expected to be “mutated” or “substituted”. Furthermore, it is preferable that at least one base from the 3rd to the 5th from the 3 ′ end of the oligonucleotide primer for base determination (A) is substituted with a base that is not complementary or homologous to the nucleic acid sequence used as a template. When an extension from this primer (A) is confirmed, it is judged that there is “mutation” or “substitution”.

  In order to determine “single nucleotide polymorphism”, the base determination oligonucleotide primer (A) may be set so as to include the same or complementary base as the mutant base. Preferably, if the first or second base from the 3 ′ end of the oligonucleotide primer for base determination (A) is the same as or complementary to the expected base variant of “single nucleotide polymorphism” Good. More preferably, the second base from the 3 ′ end of the oligonucleotide primer for base determination (A) should be the same as or complementary to the expected base variant of “single nucleotide polymorphism”. . Furthermore, it is preferable that at least one base from 3 to 5 from the 3 'end of the oligonucleotide primer (A) for base determination is substituted with a base that is not complementary or homologous to the nucleic acid sequence as a template. When an extension product from the primer (A) for determining this mutant type is confirmed, it is determined that there is at least a mutant type in the polymorphism analysis.

  Further, in order to determine “single nucleotide polymorphism”, the oligonucleotide primer for base determination (A) may be set so as to include the same or complementary base as the wild type base. Preferably, if the first or second base from the 3 ′ end of the oligonucleotide primer for base determination (A) is the same as or complementary to the wild type of the predicted base of the “single nucleotide polymorphism” Good. More preferably, the second base from the 3 ′ end of the oligonucleotide primer for base determination (A) should be the same as or complementary to the wild type of the predicted base of the “single nucleotide polymorphism”. . Furthermore, it is preferable that at least one base from 3 to 5 from the 3 'end of the oligonucleotide primer (A) for base determination is substituted with a base that is not complementary or homologous to the nucleic acid sequence as a template. When an extension from the primer (A) is confirmed to determine this wild type, it is determined that there is at least a wild type in the polymorphism analysis.

  Furthermore, when judging single nucleotide polymorphisms, the oligonucleotide primer for base judgment (A) set to include the same or complementary base as the mutant base and the base complementary to the wild type base or the same You may use simultaneously the oligonucleotide primer for base determination (A) set so that a base may be included. However, when the oligonucleotide primer (A) for judging the mutant type is used as the sense primer, the oligonucleotide primer (A) for judging the wild type is used as the antisense primer. Alternatively, when the oligonucleotide primer (A) for judging the mutant type is used as an antisense primer, the oligonucleotide primer (A) for judging the wild type is used as a sense primer. In this case, when an extension product only from the oligonucleotide primer (A) for judging the mutant type is confirmed, an extension product only from the oligonucleotide primer (A) for judging the mutant type homozygous and wild type When confirmed, when extension products from both the oligonucleotide homozygous for judging the wild type homozygous, mutant type and wild type (A) are confirmed, it can be judged to be heterozygous.

  When two types of mutants exist, the corresponding oligonucleotide primer (A) can be used. In this case, one is a sense primer and the other is an antisense primer as in the combination of the mutant type and the wild type.

  In addition, in the detection of single nucleotide polymorphisms or mutations, when using three or more primers in combination with the wild type and the mutant type, two or more types of sense primers and / or antisense primers) (one primer is two types) One type (total 3 types) of the other primer, or 2 types of both primers (total 4 types)), but in this case, by changing the length of the extension obtained, Simultaneous detection of all extensions is possible.

  In order to determine “insertion”, the base or base sequence including the base or base sequence that is the same as or complementary to the base or base sequence upstream and downstream of the expected site of “insertion”, and The oligonucleotide primer for base determination (A) may be set so as not to include the same or complementary base or base sequence. When an extension from this primer (A) is confirmed, it is determined that there is no “insertion”.

  In order to determine “insertion”, the base determination oligonucleotide primer (A) may be set so as to include the same or complementary base or base sequence as the “insert” base or base sequence. In this case, it is preferable that the 1 'to 5 bases at the 3' end of the primer (A) are designed so as not to coincide with the base or base sequence downstream of the base when it is not "inserted". When an extension from this primer (A) is confirmed, it is determined that there is “insertion”.

  In order to determine “deletion”, a base or base that contains the same or complementary base or base sequence upstream or downstream of the predicted site of “deletion” and that is complementary. The base determination oligonucleotide primer (A) may be set so as not to include the same or complementary base or base sequence as the sequence. When an extension from this primer (A) is confirmed, it is judged that there is a “deletion”.

  Further, in order to determine “deletion”, an oligonucleotide primer (A) for base determination may be set so as to include the same or complementary base or base sequence as the base or base sequence to be “deleted”. Good. In this case, it is preferable that the 1 ′ to 5 bases at the 3 ′ end of the primer (A) are designed so as not to coincide with the base or base sequence downstream of the “deleted” base or base sequence. When an extension from this primer (A) is confirmed, it is determined that there is no “deletion”.

  The Tm value of the oligonucleotide primer (A) for base determination is the Tm value of at least two types of oligonucleotide primers (B, C) having sequences complementary or homologous to the upstream and / or downstream region of the base site to be determined. Higher than that. The Tm value of the oligonucleotide primer is a temperature at which 50% of the double-stranded DNA molecules that have formed a hybrid dissociate. The calculation method is not particularly limited as long as it is a known method, but the Neighbor Neighbor method, It is obtained by either the Wallace method or the GC% method, and desirably satisfies the characteristics of the present invention. The sequences of the oligonucleotide primers (A), (B), and (C) to be used are determined by comparing the Tm values calculated by any of these methods. Note that a particularly preferable Tm value is calculated by the Nearest neighbor method. The difference in Tm value is not particularly limited as long as it is 0.5 ° C. or higher, but it is preferably 1 ° C. or higher.

Furthermore, since the amount of the nucleic acid molecule having the base sequence to be determined is very small, when it is desired to improve the detection sensitivity, the upstream and / or downstream regions of the base site whose concentration is to be determined when the determination oligonucleotide primer (A) is used. The concentration is preferably higher than that when using at least two kinds of oligonucleotide primers (B, C) having complementary or homologous sequences. The concentration of the oligonucleotide primer at the time of use is not particularly limited, but it is preferably used in the range of 0.5 to 50 μM, particularly preferably 1 to 20 μM. Within this range, the concentration of the oligonucleotide primer for base determination is determined based on the concentration of at least two types of oligonucleotide primers (B, C) having a sequence complementary or homologous to the upstream and / or downstream region of the base site to be determined. High is preferred. The difference in concentration is not particularly limited, and can be appropriately selected within a range higher than 1 time and lower than 100 times depending on the sequence of the oligonucleotide primer.
If there is only one base site to be determined,
At least one kind of base determination oligonucleotide primer (A) containing the base site to be determined, and oligonucleotide primers (B) and (C),
(1) Primer (A) is a sense primer or an antisense primer, and a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region combination;
(2) Primer (A) is a sense primer, and a combination of a sense primer (B) that is homologous to the downstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region (provided that Primer (C) is downstream of primer (B));
(3) Primer (A) is an antisense primer, and a combination of a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the upstream region (provided that , Primer (C) is downstream of primer (B));
The set formed from the primer (A, B, C) by the combination selected from either is required. For example, if there are two base sites to be determined, two sets of the primers (A, B, C) are usually required.

  However, when the base site to be determined is adjacent or in the vicinity, oligonucleotide primers (B, C) that are common to two or more base sites to be determined can be used. In this case, the oligonucleotide primer for base determination (A) containing the base site to be determined may be prepared for two or more base sites to be determined.

  For example, when the base site to be determined is “mutation”, “substitution”, or “single nucleotide polymorphism” and the base may be a wild type or a mutant type, an oligonucleotide primer for base determination Two types of (A) may be used, and the base can be determined even if only one type of wild type or mutant type is used.

  The two types of primers may be in the same direction (both sense primers or both antisense primers) or in different directions (one is a sense primer and the other is an antisense primer).

  When there are a plurality of bases to be judged (for example, 2 to 100 places, preferably 2 to 50 places, more preferably 2 to 20 places, particularly 2 to 10 places), each judgment place is specifically designated as desired. At least two types of oligonucleotide primers (B, C) having a sequence complementary or homologous to the upstream and downstream regions of the base site to be judged that can be amplified contain or lack the bases to be judged as multiple pairs A plurality of lost base determination oligonucleotide primers (A) may be designed. Multiple primers can be amplified in a single reaction system. In this case, considering the distance from the position of the base to be determined to the upstream or downstream primer (B or C), and designing the amplification product to have different chain lengths, it is desired to make a determination based on the amplification product chain length. The presence of a plurality of bases (for example, 2 to 100 sites, preferably 2 to 50 sites, more preferably 2 to 20 sites, particularly 2 to 10 sites) can be simultaneously determined. In addition, if a homologous or complementary oligonucleotide probe is used for each determination position, the presence of a specific base can be determined regardless of the length of the amplification product chain.

  In addition, when the base site to be determined is “insertion” or “deletion”, the number of “insertion” or “deletion” bases that can be detected by the present invention does not have to be one, and any number of two or more, for example, It may be 2 to 1000, especially 2 to 500. Of course, if the “inserted” or “deleted” bases are discontinuous (discontinuous) and constitute a series of “insertions” or “deletions”, then one set of primers (A, B, C), but if there is no correlation between the “insertion” or “deletion” sites, two or more primers (A, B , C) set can be used.

The at least two types of oligonucleotide primers (B, C) having a sequence complementary or homologous to the upstream and / or downstream region of the base site to be determined for use in the present invention are related to the primer (A),
(1) Primer (A) is a sense primer or an antisense primer, and a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region combination;
(2) Primer (A) is a sense primer, and a combination of a sense primer (B) that is homologous to the downstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region (provided that Primer (C) is downstream of primer (B));
(3) Primer (A) is an antisense primer, and a combination of a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the upstream region (provided that , Primer (C) is downstream of primer (B));
Any combination of the above can be applied.

  When using at least two kinds of oligonucleotide primers (A) for base determination, when the primer (A) is in the same direction, the primer (A) is between the oligonucleotide primers (B, C) (the The primer (A) may be located between the primer (B) and the primer (C) (see 1 in FIG. 1), or may be outside the primer (B) and the primer (C) ( (Refer to 2 and 3 in FIG. 1). In FIG. 1, X and X ′ indicate sites where bases are to be determined. The right primer indicates the forward primer, and the left primer indicates the reverse primer. (A) and (A ′) simply illustrate at least two types of oligonucleotide primers (A) for base determination, and are not limited to two types. In addition, primer (A) gave an example of the same direction (both sense primer or both antisense primer), but in 1 of FIG. 1, the direction is different (one is a sense primer and the other is an antisense primer). There may be.

  On the other hand, when at least two kinds of oligonucleotide primers (A) for base determination are in different directions, the primer (A) is between the oligonucleotide primers (B, C) (the primer (A) is a primer ( B) and the primer (C).

The base site to be determined may be a total of four bases including wild type and mutant type. Therefore, the primer (A) has 1 to 4 types. When the base site to be determined may have 3 types of bases, primer (A) can use 2 or 3 types, and when the base site to be determined may have 4 types of bases, The primer (A) can use 3 types or 4 types.
In the case of “deletion”, an oligonucleotide primer for base determination (A) designed to include a base or base sequence that is the same as or complementary to the base or base sequence to be “deleted”, and prediction of “deletion” Designed to include the same or complementary bases or base sequences as the upstream or downstream bases or base sequences, but not the same or complementary bases or base sequences to the deleted bases or base sequences Two types of base determination oligonucleotide primers (A) may be used simultaneously. In addition, since it is only necessary to determine the presence of a base or base sequence that is predicted to be deleted, at least one primer (A) may be used.
In the case of “insertion”, an oligonucleotide primer for base determination (A) designed to contain the same or complementary base or base sequence as the base or base sequence to be “inserted”, and the expected site of “insertion” Bases designed to contain the same or complementary bases or base sequences as the upstream and downstream bases or base sequences, and do not contain the same or complementary bases or base sequences as the inserted bases or base sequences Two types of oligonucleotide primer (A) for determination may be used simultaneously. In addition, since it is only necessary to determine the presence of a base or base sequence that is expected to be inserted, at least one primer (A) may be used.

In the present invention, the primers (A), (B), (C) are selected from any combination of the following (1) to (3):
(1) Primer (A) is a sense primer or an antisense primer, and a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region combination;
(2) Primer (A) is a sense primer, and a combination of a sense primer (B) that is homologous to the downstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region (provided that Primer (C) is downstream of primer (B));
(3) Primer (A) is an antisense primer, and a combination of a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the upstream region (provided that Primer (C) is downstream of primer (B)).

  These combinations are effectively combined and may be combined. That is, for example, in the combination (1), if there is a base site to be determined in the upstream region of the sense primer (B), a primer (A) for the sense primer may be newly added separately. If there is a base site to be determined in the downstream region of the antisense primer (C), a primer (A) for the antisense primer may be newly added. In the combination (2), if there is a base site to be determined in the downstream region of the antisense primer (C), a primer (A) for the antisense primer may be newly added. In the combination (3), if there is a base site to be determined in the upstream region of the sense primer (B), a primer (A) for the sense primer may be newly added separately.

In the present invention, the most important invention disclosure is that at least one base determination oligonucleotide primer (A) containing the base site to be determined, and oligonucleotide primers (B) and (C),
(1) Primer (A) is a sense primer or an antisense primer, and a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region combination;
(2) Primer (A) is a sense primer, and a combination of a sense primer (B) that is homologous to the downstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region (provided that Primer (C) is downstream of primer (B));
(3) Primer (A) is an antisense primer, and a combination of a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the upstream region (provided that , Primer (C) is downstream of primer (B));
Even if the primer (A, B, C) with a combination selected from any of the above is subjected to a gene amplification reaction at the same time, a non-specific amplification reaction does not occur unexpectedly, and the presence or absence of a base to be determined by the length of the amplification product In addition, it has been found that the location can be easily identified.

The invention will now be described by way of example. That is, in the method using the nucleic acid amplification method using the oligonucleotide primers (B and C) described in (1) above, the oligonucleotide primers (B) and (C ) Is recognized as being specifically amplified. In this reaction, when at least one type of base determination oligonucleotide primer (A) containing the base site to be determined is added, and when the target base is present in the nucleic acid sequence in the sample, the base determination oligo The nucleotide primer (A) and oligonucleotide primer (B) or (C) also specifically amplify the nucleic acid sequence having the target base, and it is easy to obtain another type of amplification product in addition to the amplification product described above. Recognized. However, even when there is no target base in the nucleic acid sequence in the sample, the sequence sandwiched between the oligonucleotide primers (B) and (C) is specifically amplified, and the nucleic acid fragment used as a template is geometrically Therefore, it was expected that a non-specific amplification reaction was caused by the oligonucleotide primer (A) for base determination, and as a result, another type of amplification product was obtained in addition to the amplification product described above. In this case, since two or more kinds of amplified nucleic acid fragments are always obtained regardless of the presence of a specific base, two kinds of oligonucleotides having sequences complementary or homologous to the upstream and downstream regions of the base site. In the presence of primer (B and C) and oligonucleotide primer (A) for base determination, it was considered difficult to determine a specific base (PCR
CLAMPING "
Peptide Nucleic Acids: Protocols and Applications, Horizon Scientific Press, Wymondham,
UK. 1999, p. 193-200).

  However, as a result of actual experiments, when there is a specific base in the nucleic acid sequence in the sample, it is complementary or homologous to the oligonucleotide primer for base determination (A) and the upstream and downstream regions of the base site. Even when the gene fragment obtained by the oligonucleotide primer (B or C) having the correct sequence is selectively generated and there is no mutation in the nucleic acid sequence, the oligonucleotide primer for base determination (A) is nonspecific It was confirmed that no amplification reaction occurred. It was completely unexpected that such a result was obtained.

This means that in the detection of mutant nucleic acid sequences using allele-specific oligonucleotides, it is no longer necessary to separately perform wild-type nucleic acid sequence detection and mutant nucleic acid sequence detection, which have been considered indispensable. . Accordingly, it is possible to provide an effective means for reducing detection errors such as amplification failure caused by the sample as well as economic effects such as halving the labor and reagents required for detection.
Depending on the combination of the oligonucleotide primer for base determination (A) containing the base site to be determined and the oligonucleotide primer (B, C), “mutation”, “substitution”, “single base” that is the “base site to be determined” “Polymorphism”, “insertion” or “deletion” can be easily determined. Hereinafter, as an example, the detection method will be described with reference to the drawings, but the present invention is not limited to this.
The case where the “base site to be determined” is “mutation” or “substitution” will be described with reference to FIG.
In FIG. 2, X represents a “mutation” or “substitution” base. The right primer indicates the forward primer, and the left primer indicates the reverse primer.
Fig. 2-1
To determine “mutation” or “substitution”, set the oligonucleotide primer for base determination (A) so that it contains the base after the mutation or the base after the substitution, and (1) the base site to be determined The combination of a sense primer (B) that is homologous to the upstream region and an antisense primer (C) that is complementary to the downstream region is selected.
If an amplification product of the primer (A, C) is obtained, it is determined that there is “mutation” or “substitution”.
Fig. 2-2
In order to determine “mutation” or “substitution”, the oligonucleotide primer for base determination (A) is set so that the base after the mutation or the base complementary to the base after the substitution is included, and (1) I want to determine This is a case where a combination of a sense primer (B) homologous to the upstream region of the base site and an antisense primer (C) complementary to the downstream region is selected.
If an amplification product of the primer (A, B) is obtained, it is determined that there is “mutation” or “substitution”.
Fig. 2-3
To determine “mutation” or “substitution”, set the oligonucleotide primer for base determination (A) so that it contains the base after the mutation or the base after the substitution, and (2) the base site to be determined A combination of a sense primer (B) that is homologous to the downstream region and an antisense primer (C) that is complementary to the downstream region (however, primer (C) is downstream of primer (B)) was selected Is the case.
If an amplification product of the primer (A, C) is obtained, it is determined that there is “mutation” or “substitution”.
Fig.2-4
To determine “mutation” or “substitution”, set the oligonucleotide primer for base determination (A) to include the base after the mutation or the base complementary to the base after the substitution, and (3) I want to make a determination A combination of a sense primer (B) homologous to the upstream region of the base site and an antisense primer (C) complementary to the upstream region (provided that primer (C) is downstream of primer (B)) This is the case.
If an amplification product of the primer (A, B) is obtained, it is determined that there is “mutation” or “substitution”.
The case where the “base site to be determined” is “single nucleotide polymorphism” will be described with reference to FIG.
In FIG. 3, X and Y represent different bases. The right primer indicates the forward primer, and the left primer indicates the reverse primer. (A) and (A ′) show different oligonucleotide primers for base determination (A).
Fig. 3-1
In order to determine “single nucleotide polymorphism”, the oligonucleotide primer for base determination (A) is set so as to include the same base as the base of X, and the base is set so as to include a base complementary to the base of Y. Set the oligonucleotide primer (A ') for determination, and (1) a combination of a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region. This is the case.
When X is a mutant type and Y is a wild type, if only the amplification product of primer (A, C) is obtained, only the amplification product of X (mutation type) homo and primer (A ′, B) can be obtained. , Y (wild type) homo, primer (A, C) amplification product and primer (A ', B) amplification product are obtained, X (mutant) and Y (wild type) heterozygous . When X is a mutant type and Y is another mutant type, if an amplification product of primer (A, C) is obtained, it can be determined that it is a mutation type to X, and the amplification product of primer (A ′) If it is obtained, it can be determined that the mutant type is Y.
Fig. 3-2
In order to determine “single nucleotide polymorphism”, an oligonucleotide primer for base determination (A) is set so as to include a base complementary to the base of Y, and (1) homologous to the upstream region of the base site to be determined. This is a case where a combination of a sense primer (B) and an antisense primer (C) complementary to the downstream region is selected.
When X is a mutant type and Y is a wild type, if only the amplification product of the primer (B, C) is obtained, if only the amplification product of the X (mutation type) homozygous primer (A, B) is obtained, If a Y (wild type) homozygote, an amplified product of primer (B, C) and an amplified product of primer (A, B) are obtained, it is determined that X (mutant) and Y (wild type) are heterozygous.
Fig.3-3
In order to determine “single nucleotide polymorphism”, an oligonucleotide primer for base determination (A) is set so that it contains the same base as the base of X, and (1) sense homologous to the upstream region of the base site to be determined. This is a case where a combination of the primer (B) and the antisense primer (C) complementary to the downstream region is selected.
When X is a mutant type and Y is a wild type, if only the amplified product of primer (A, C) is obtained, if only the amplified product of X (mutant type), primer (B, C) is obtained, If a Y (wild type) homozygote, an amplification product of primer (A, C) and an amplification product of primer (B, C) are obtained, it is determined that X (mutant type) and Y (wild type) are heterogeneous.
Fig.3-4
In order to determine “single nucleotide polymorphism”, an oligonucleotide primer for base determination (A) is set so that it contains the same base as the base of X, and (2) sense homologous to the downstream region of the base site to be determined. This is a case where a combination of the primer (B) and the antisense primer (C) complementary to the downstream region (provided that the primer (C) is downstream of the primer (B)) is selected. When X is a mutant type and Y is a wild type, it is determined that there is at least a mutant type to X if an amplification product of primer (A, C) is obtained. If only the amplification products of the primers (B, C) are obtained, it is determined that there is at least no X mutant.
Fig.3-5
In order to determine “single nucleotide polymorphism”, an oligonucleotide primer for base determination (A) is set so as to include a base complementary to the base of Y, and (3) homologous to the upstream region of the base site to be determined. This is a case where a combination of a sense primer (B) and an antisense primer (C) complementary to the upstream region (where primer (C) is downstream of primer (B)) is selected.
When X is a mutant type and Y is a wild type, it is determined that at least a wild type exists if an amplification product of the primer (A, B) is obtained. If only the amplification products of the primers (B, C) are obtained, it is determined that there is at least no wild type.
The case where “the base site to be determined” is “insertion” will be described with reference to FIG.
In FIG. 4, “i” indicates a predicted site of “insertion”, not a base. “IIIIIIIIII” indicates the inserted base or base sequence. The right primer indicates the forward primer, and the left primer indicates the reverse primer.
Fig.4-1
In order to determine “insertion”, the base or base sequence that includes the same base or base sequence as the base or base sequence upstream and downstream of the expected site (i) of “insertion” and is inserted (IIIIIIIIII ) Set the oligonucleotide primer (A) for base determination so that it does not contain the same base or base sequence as (1). (1) Sense primer (B) homologous to the upstream region of the base site to be determined and complementary to the downstream region This is a case where a combination with a typical antisense primer (C) is selected.
If only amplification products of primers (A, C) are obtained, it is determined that there is no “insertion”.
Fig.4-2
In order to determine “insertion”, the base or base sequence to be inserted includes a base or base sequence complementary to the base or base sequence upstream and downstream of the expected site (i) of “insertion”. The base primer oligonucleotide primer (A) is set so as not to contain a base or base sequence complementary to (IIIIIIIIII), (1) a sense primer (B) homologous to the upstream region of the base site to be determined, This is a case where a combination with an antisense primer (C) complementary to the downstream region is selected.
If only amplification products of the primers (A, B) are obtained, it is determined that there is no “insertion”.
Fig. 4-3
In order to determine “insertion”, the base or base sequence that includes the same base or base sequence as the base or base sequence upstream and downstream of the expected site (i) of “insertion” and is inserted (IIIIIIIIII ) Set the oligonucleotide primer (A) for base determination so that it does not contain the same base or base sequence as (2), and (2) sense primer (B) that is homologous to the downstream region of the base site to be determined and complementary to the downstream region This is a case where a combination with a typical antisense primer (C) (however, primer (C) is downstream of primer (B)) is selected.
If only amplification products of primers (A, C) are obtained, it is determined that there is no “insertion”.
Fig. 4-4
In order to determine “insertion”, the base or base sequence to be inserted includes a base or base sequence complementary to the base or base sequence upstream and downstream of the expected site (i) of “insertion”. (IIIIIIIIII) and a base primer oligonucleotide primer (A) so as not to contain a complementary base or base sequence, (3) a sense primer (B) homologous to the upstream region of the base site to be determined; This is a case where a combination of an upstream region and a complementary antisense primer (C) (however, primer (C) is downstream of primer (B)) is selected.
If only amplification products of the primers (A, B) are obtained, it is determined that there is no “insertion”.
Fig.4-5
In order to determine “insertion”, the base or base sequence that includes the same base or base sequence as the base or base sequence upstream or downstream of the expected site (i) of “insertion” and is inserted (IIIIIIIIII ) Set the oligonucleotide primer (A) for base determination so that it contains the same base or base sequence as (1), (1) sense primer (B) that is homologous to the upstream region of the base site to be determined, and complementary to the downstream region This is a case where a combination with the antisense primer (C) is selected.
If an amplification product of the primers (A, C) is obtained, it is determined that there is “insertion”.
Fig.4-6
In order to determine “insertion”, the base or base sequence to be inserted contains a base or base sequence that is complementary to the base or base sequence upstream or downstream of the expected site (i) of “insertion”. Set the oligonucleotide primer (A) for base determination so as to include a base or base sequence complementary to (IIIIIIIIII), (1) a sense primer (B) homologous to the upstream region of the base site to be determined, and downstream This is a case where a combination with an antisense primer (C) complementary to the region is selected.
If an amplification product of the primers (A, B) is obtained, it is determined that there is “insertion”.
Fig.4-7
In order to determine “insertion”, the base or base sequence that includes the same base or base sequence as the base or base sequence upstream or downstream of the expected site (i) of “insertion” and is inserted (IIIIIIIIII ) Set the oligonucleotide primer for base determination (A) so that it contains the same base or base sequence as (2), and (2) sense primer (B) homologous to the downstream region of the base site to be determined and complementary to the downstream region This is a case where a combination with an antisense primer (C) (where primer (C) is downstream of primer (B)) is selected.
If an amplification product of the primers (A, C) is obtained, it is determined that there is “insertion”.
Fig.4-8
In order to determine “insertion”, the base or base sequence to be inserted includes a base or base sequence complementary to the base or base sequence upstream and downstream of the expected site (i) of “insertion”. Set the oligonucleotide primer (A) for base determination so as to include a base or base sequence complementary to (IIIIIIIIII), (3) a sense primer (B) homologous to the upstream region of the base site to be determined, and upstream This is a case where a combination of a region and a complementary antisense primer (C) (however, primer (C) is downstream of primer (B)) is selected. If an amplification product of the primers (A, B) is obtained, it is determined that there is “insertion”.
The case where the “base site to be determined” is “deletion” will be described with reference to FIG.
In FIG. 5, “DDDDDDDDDD” indicates a base or base sequence that is predicted to be deleted. “D” indicates not the base but the site after “deletion”. The right primer indicates the forward primer, and the left primer indicates the reverse primer.
Fig.5-1
In order to determine "deletion", the base or base sequence to be deleted includes the same base or base sequence as the base or base sequence upstream and downstream of the predicted site of "deletion", and "DDDDDDDDDD" Set the oligonucleotide primer (A) for base determination so that it does not contain the same base or base sequence as (1). (1) Sense primer (B) homologous to the upstream region of the base site to be determined and complementary to the downstream region This is a case where a combination with the antisense primer (C) is selected.
If an amplification product of the primers (A, C) is obtained, it is determined that there is “deletion”.
Fig.5-2
In order to determine “deletion”, a base or base sequence that includes a base or base sequence that is complementary to a base or base sequence upstream and downstream of the predicted site of “deletion” and that is to be deleted is “ Set the oligonucleotide primer (A) for base determination so that it does not contain a base or base sequence complementary to “DDDDDDDDDD”, (1) a sense primer (B) that is homologous to the upstream region of the base site to be determined, and downstream This is a case where a combination with an antisense primer (C) complementary to the region is selected.
If an amplification product of the primer (A, B) is obtained, it is judged that there is “deletion”.
Fig.5-3
In order to determine "deletion", the base or base sequence to be deleted includes the same base or base sequence as the base or base sequence upstream and downstream of the predicted site of "deletion", and "DDDDDDDDDD" Set the oligonucleotide primer for base determination (A) so that it does not contain the same base or base sequence as (1), (2) sense primer (B) that is homologous to the downstream region of the base site to be determined, and complementary to the downstream region This is a case where a combination with an antisense primer (C) (where primer (C) is downstream of primer (B)) is selected.
If an amplification product of the primers (A, C) is obtained, it is judged that there is “deletion”.
Fig.5-4
In order to determine “deletion”, a base or base sequence that includes a base or base sequence that is complementary to a base or base sequence upstream and downstream of the predicted site of “deletion” and that is to be deleted is “ Set the oligonucleotide primer (A) for base determination so that it does not contain a base or base sequence complementary to “DDDDDDDDDD”, (3) a sense primer (B) that is homologous to the upstream region of the base site to be determined, and upstream This is a case where a combination of a region and a complementary antisense primer (C) (however, primer (C) is downstream of primer (B)) is selected.
If an amplification product of the primer (A, B) is obtained, it is judged that there is “deletion”.
Fig.5-5
In order to determine “deletion”, a base or base sequence complementary to the base or base sequence to be deleted (DDDDDDDDDD) is included, and 1 to 5 bases at the 3 ′ end are bases to be “deleted”. Alternatively, a base determination oligonucleotide primer (A) that does not match the base or base sequence downstream of the base sequence is set, (1) a sense primer (B) that is homologous to the upstream region of the base site to be determined, This is a case where a combination with an antisense primer (C) complementary to the downstream region is selected.
If an amplification product of the primer (A, B) is obtained, it is determined that there is no “deletion”.
Fig.5-6
In order to determine “deletion”, the same base or base sequence as the “deletion” base or base sequence (DDDDDDDDDD) is included, and 1 to 5 bases at the 3 ′ end are the bases or bases to be “deletion” Set a base determination oligonucleotide primer (A) that does not match the base or base sequence downstream of the sequence, (1) a sense primer (B) that is homologous to the upstream region of the base site to be determined, and the downstream region When a combination with an antisense primer (C) complementary to is selected.
If an amplification product of the primer (A, C) is obtained, it is determined that there is no “deletion”.
Fig.5-7
In order to determine “deletion”, the same base or base sequence as the “deleted” base or base sequence (DDDDDDDDDD) is included, and 1 to 5 bases at the 3 ′ end are the bases or bases to be “deleted” Set a base determination oligonucleotide primer (A) that does not match the base or base sequence downstream of the sequence, (2) a sense primer (B) that is homologous to the downstream region of the base site to be determined, and the downstream region And a combination with a complementary antisense primer (C) (provided that primer (C) is downstream of primer (B)).
If an amplification product of the primer (A, C) is obtained, it is determined that there is no “deletion”.
Fig.5-8
In order to determine “deletion”, a base or base sequence complementary to the base or base sequence to be deleted (DDDDDDDDDD) is included, and 1 to 5 bases at the 3 ′ end are bases to be “deleted”. Alternatively, a base determination oligonucleotide primer (A) that does not match the base sequence or base sequence downstream of the base sequence is set, (3) a sense primer (B) that is homologous to the upstream region of the base site to be determined, This is a case where a combination of an upstream region and a complementary antisense primer (C) is selected (provided that primer (C) is downstream of primer (B)).
If an amplification product of the primer (A, B) is obtained, it is determined that there is no “deletion”.

  In the present invention, whether or not the amplification reaction is correct can be determined at a time. In the present invention, even when an amplification product from the extension reaction from the primer (A) cannot be obtained, an amplification product by at least the combination of the primer (B) and the primer (C) is obtained. If no amplification product was obtained when the base was determined according to the present invention, this indicates that the amplification reaction itself was unsuccessful. This means that if no amplification product is obtained in the conventional determination method, it cannot be determined whether there is no mutation, substitution, single nucleotide polymorphism, insertion or deletion, or whether the amplification reaction has been unsuccessful. It overcomes the problem.

  The at least one kind of oligonucleotide probe (D) that is homologous or complementary to the nucleic acid sequence containing the base site includes the base site (wild type or mutant) and is homologous or complementary to the nucleic acid sequence of the product. Oligonucleotides with base sequences are widely included.

  In addition, as the at least one kind of homologous or complementary oligonucleotide probe (E) having a sequence different from the nucleic acid sequence containing the base site by at least one base, the oligonucleotide probe (D) and at least one base (preferably One base) includes oligonucleotides having different sequences. The probe is used as a labeled probe. The labeled probe is designed to hybridize to the newly generated sequence, so that the newly generated extension product can be detected efficiently without being affected by the existing nucleic acid. can do. As the label of the probe, the same label as that of the primer is used. When both the primer and the probe are labeled, different labels can be preferably used.

In the so-called TaqMan (registered trademark) assay using an oligonucleotide probe pre-labeled with a fluorescent dye and a polymerase having 5 'nuclease activity, a sample capable of detecting a single nucleotide mutation (polymorphism) together with a PCR reaction is prepared. This is an extremely effective method for analysis. Alternatively, the sequence can also be specified by performing a melting curve analysis using an oligonucleotide probe. As another method, the obtained nucleic acid fragment can be detected by fluorescence using an intercalator such as SYBR (registered trademark) Green I or ethylene bromide, and the amplified fragment may be detected by electrophoresis.
An oligonucleotide probe substantially homologous or complementary to the point mutation site may be immobilized on a water-insoluble simple substance. Examples of the water-insoluble carrier include synthetic polymers, biopolymers, metals, and glass. That is, the PCR product is bound on the carrier by adding the PCR product that has been previously made single-stranded to the oligonucleotide probe immobilized on the water-insoluble carrier. If the bound PCR product is labeled in advance, the sequence of the PCR product can be specified by detecting the label.
Oligonucleotide probes may be arranged in an array on these water-insoluble carriers and used as a DNA array.

In the present invention, the oligonucleotide primer can be basically extended by using a conventional method. Usually, a target nucleic acid is used as a template by allowing four types of deoxynucleoside triphosphates (dNTPs) and DNA polymerase to act on a chromosome or fragment thereof containing a specific nucleotide polymorphism site denatured into a single strand. The oligonucleotide is extended. The extension reaction is molecular
It can be performed according to the method described in Cloning, A Laboratory Manual (Sambrook et al., 1989).

Examples of nucleic acid amplification methods include PCR, NASBA (Nucleic acid sequence-based amplification method; Nature, vol. 350, page 91 (1991)), LCR (International Publication No. 89/12696, JP-A-2-2934), SDA. (Strand
Displacement Amplification: Nucleic acid research Volume 20, 1691 (1992)), RCR (International Publication No. 90/1069), TMA (Transcription
mediated amplification method; J. Clin. Microbiol. Vol. 31, p. 3270 (1993)) LAMP (loop-mediated
isothermal amplification method: J Clin Microbiol. 2004 Volume 42: 1,956), ICAN (isothermal
and chimeric primer-initiated amplification of nucleic acids: Kekkaku. 2003, 78, 533).

In particular, the PCR method repeats a cycle consisting of three steps of denaturation, annealing, and extension in the presence of a sample nucleic acid, four types of deoxynucleoside triphosphates, a pair of oligonucleotides, and a heat-resistant DNA polymerase, and thereby In this method, the region of the sample nucleic acid sandwiched between the oligonucleotides is exponentially amplified. That is, the sample nucleic acid is denatured in the denaturation step, each oligonucleotide is hybridized with a region on the complementary single-stranded sample nucleic acid in the subsequent annealing step, and each oligonucleotide is the starting point in the subsequent extension step. As described above, a DNA strand complementary to each single-stranded sample nucleic acid serving as a template is extended by the action of DNA polymerase to form double-stranded DNA. By this one cycle, one double-stranded DNA is amplified into two double-stranded DNAs. Therefore, if this cycle is repeated n times, the region of the sample DNA sandwiched between the pair of oligonucleotides is theoretically amplified 2 ⁿ times. Since the amplified DNA region exists in a large amount, it can be easily detected by a method such as electrophoresis. Therefore, if a gene amplification method is used, it is possible to detect even a very small amount of sample nucleic acid that could not be detected in the past (single molecule is acceptable), which is a very widely used technique recently. .

When the gene amplification method is PCR, the presence of the base to be determined can be easily determined based on the length of the amplified nucleic acid fragment to be obtained. That is, the obtained amplified nucleic acid fragment contains or lacks a base to be determined as any one of oligonucleotide primers (B, C) having a sequence complementary or homologous to the upstream and downstream regions of the base site to be determined. Amplified nucleic acid fragment as described above, which is based on the lost nucleotide determination oligonucleotide primer (A) or the oligonucleotide primer (B, C) having a sequence complementary or homologous to the upstream and downstream regions of the base site to be determined The presence of the base can be determined by detecting whether the amplified nucleic acid fragment has a longer chain length by a known method such as electrophoresis. The DNA polymerase used for PCR amplification is not particularly limited, and any DNA polymerase can be used.
(1) In the case of a combination of a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region, it preferably has 5 ′ exonuclease activity. Not a DNA polymerase can be used. As a DNA polymerase having no 5 ′ exonuclease activity, Pyrococcus furiosus (Pyrococcus)
furiosus) -derived thermostable DNA polymerase (Pfu polymerase, WO92 / 09689, JP-A-5-328969), Thermusococcus
thermostable DNA polymerase derived from litoralis (Tli polymerase, JP-A-6-7160) Pyrococcus sp. KOD1-derived heat-resistant DNA polymerase
(KOD polymerase, JP-A-7-298879) and the like.
(2) A combination of a sense primer (B) that is homologous to the downstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region (provided that primer (C) is more than primer (B)) (3) A combination of a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the upstream region (provided that primer (C) is a primer) In the case of a combination with (B), a DNA polymerase having a 5 ′ exonuclease activity can be preferably used. Examples of DNA polymerase having 5 ′ exonuclease activity include Taq polymerase, Tfl polymerase, Tth polymerase, Bst
For example, DNA polymerase. These enzymes are merely examples, and are not limited at all. Polymerases having the same action can also be used in the present invention.

  The method for detecting the nucleic acid fragment (amplification product) obtained by the present invention is not particularly limited, but electrophoresis method, detection method by hybridization, fluorescence detection method, melting curve analysis method, thermal elution chromatogram method For example, existing methods can be used as appropriate.

  Electrophoresis can be used as a very simple detection method. That is, the obtained amplified nucleic acid fragment contains or lacks a base to be determined as any one of oligonucleotide primers (B, C) having a sequence complementary or homologous to the upstream and downstream regions of the base site to be determined. Amplified nucleic acid fragment by the lost nucleotide determination oligonucleotide primer (A) and the above-mentioned amplified nucleic acid by the oligonucleotide primer (B, C) having a sequence complementary or homologous to the upstream and downstream regions of the base site to be determined If the chain lengths of the fragments are different, they can be easily distinguished.

  When an amplified nucleic acid fragment obtained by the oligonucleotide primer for base determination (A) is obtained, at least one base is different from the wild type sequence. Therefore, a detection method by hybridization, a melting curve analysis method, and a thermal elution chromatogram method are used. It is possible. Further, preferably, when at least one non-complementary base is introduced 3 to 5 from the 3 ′ end of the base determination oligonucleotide, the sequence differs from the wild-type nucleic acid sequence by at least 2 bases. Therefore, the specificity in hybrid detection is extremely high.

  Moreover, the primer to be used can also use what was labeled depending on the case. In addition, a labeled probe is preferably used. The label used can be selected from the group consisting of a fluorescent chemical substance, a luminophore, an enzyme, a fluorescent protein, a photoprotein, a magnetic substance, and a conductive substance.

  The detection of the label is not particularly limited as long as it is a known method. However, when the label is a fluorescent chemical substance or a fluorescent protein, light of a specific wavelength is irradiated to excite the fluorescent chemical substance or the fluorescent protein to bring it to the ground state. It is possible to measure the amount of fluorescence of a specific wavelength generated when converted. Examples of the fluorescent chemical substance used in these include FITC, FAM, TAMRA, Texas Red, VIC, Cy3, Cy5, HEX and the like, and examples of the fluorescent protein include GFP, YFP, and RFP. When the label is an enzyme, it can be measured by detecting the reaction product produced by adding the enzyme substrate. Enzyme and substrate combinations used in these include alkaline phosphatase and paranitrophenyl phosphate, CDP-star, AMPPD, DDAOphospate, BCIP-NBT, etc., peroxidase and TMB, Lumi-Light (Roche Diagnostics) ), Combinations of SAT-1 (Dojindo), combinations of diaphorase and NTB, various oxidases and substrates, combinations of various dehydrogenases and substrates, etc. It can be used without any problems. When the label is a magnetic substance, measurement can be performed by detecting magnetism. When the label is a conductive substance, the measurement can be performed by detecting the current value.

  In the present invention, the kit includes a DNA polymerase and at least two types of oligonucleotide primers (B, C) having sequences complementary or homologous to the upstream and downstream regions of the base site to be determined. Or at least 1 type of oligonucleotide primer (A) for base determination which was deleted should just be included. Furthermore, four types of deoxynucleoside triphosphates may be included.

Samples used in the assay can be biomaterials (blood, plasma, mucous membranes, nails, hair, body fluids, mucus, tissues or tissue fragments), environmental samples (river water, sludge, drainage, soil or air), or food (agricultural products, Livestock products, processed foods or marine products), viruses, bacteria or culture solutions thereof, but are not limited thereto. These samples may be prepared by known methods. Typically, the phenol / chloroform extraction method (Biochimica et Biophysica acta).
72, 619-629, 196
3 years) and alkaline SDS method (Nucleic Acid Research Vol. 7, pp. 1513-1523, 1979). As a system using a nucleic acid binding carrier for nucleic acid isolation, a method using glass particles and a sodium iodide solution (Proc.
Natl. Acad) USA Vol. 76-2, 615-619, 197
9), and a method using hydroxyapatite (Japanese Patent Laid-Open No. 63-263093). As another method, a method using silica particles and chaotropic ions (J) Clinical Microbiology
28-3, 495-503, 1990, JP-A-2-289596).

  According to the present invention, it is possible to provide an effective means and kit for reducing detection errors such as amplification failure caused by a sample as well as economical effects such as halving the labor and reagents required for detection. .

Hereinafter, based on an Example, this invention is demonstrated more concretely. However, the present invention is not limited to the following examples.
(1) Synthesis of oligonucleotide for detecting mutation in abl gene ATP binding region of bcr-abl chimera gene Bases shown in SEQ ID NOs: 1 to 4 by the phosphoamidite method using DNA synthesizer type 392 manufactured by PerkinElmer. Oligonucleotides having sequences (hereinafter referred to as oligos 1 to 4) were synthesized. The synthesis was performed according to the manual, and the deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C. overnight. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, we commissioned DNA synthesis contractors (Japan Bioservices, Operon, Proligo, Sigma Genosys, etc.). Oligo 1 is a common sense primer (B), oligo 2 is a common antisense primer (C), and both common primers are combined and used as an oligonucleotide for an amplification reaction. Oligo 3 is a primer (A) for amplifying the (E255K) mutant gene in the abl gene ATP binding region of the bcr-abl chimeric gene. Oligo 4 is a primer (A) for amplifying the (T315I) gene mutation in the abl gene ATP binding region of the bcr-abl chimeric gene.

[Example 1]
(2) Analysis of (E255K) gene mutation in abl gene ATP binding region of bcr-abl chimeric gene by PCR method
(Preparation of total RNA)
Total RNA extracted from the leukemia cell line was added to the column (QIAampRNA Bloo
d Mini Kit; manufactured by QIAGEN), the column was washed, and then adsorbed RN
A was eluted with DEPC water (diethylpyrocarbonate treated dilated and deionized water). The concentration and purity of RNA were confirmed by the ratio of absorbance A260nm / A280nm.

(Preparation of cDNA)
2 μg of the total RNA obtained above was mixed with 500 ng of random primer, 200 units of reverse transcriptase (SuperScript II; manufactured by GIBCO), 40 units of RNase inhibitor (GIBCO) and 1 mM.
It was prepared by reverse transcription in a 45 μl reaction solution containing dNTPs.

(Perform PCR)
2.25 μl of cDNA prepared above, Oligo 1 (SEQ ID NO: 1), Oligo 2 (SEQ ID NO: 2), Oligo 3 (SEQ ID NO: 3) each 5 pmoles, and 10 × PCRmix, 0.2 mM
dNTPs, 1 mM MgSO ₄ , and KOD-plus DNA polymerase were added to milliQ water to make a total volume of 25 μl. The PCR reaction was initially performed at 94 ° C. for 2 minutes, followed by a denaturation step at 94 ° C. for 15 seconds, an annealing at 62.5 ° C. for 30 seconds, and a DNA extension reaction step at 68 ° C. for 30 seconds as one cycle. This was carried out by performing a total of 35 cycles.

(Detection using electrophoresis)
5 μl of the amplification reaction solution was electrophoresed on a 10% acrylamide gel. The results are shown in FIG.

[Example 2]
Analysis of (T315I) gene mutation in the abl gene ATP binding region of the bcr-abl chimeric gene by PCR (Preparation of total RNA)
Total RNA extracted from the leukemia cell line was adsorbed on a column (QIAampRNA Blood Mini Kit; manufactured by QIAGEN), the column was washed, and the adsorbed RNA was eluted with DEPC water (diethylpyrocarbonate hydrated and distilled water). The concentration and purity of RNA were confirmed by the ratio of absorbance A260nm / A280nm.

(Preparation of cDNA)
2 μg of the total RNA obtained above was mixed with 500 ng of random primer, 200 units of reverse transcriptase (SuperScript II; manufactured by GIBCO), 40 units of RNase inhibitor (GIBCO) and 1 mM.
It was prepared by reverse transcription in a 45 μl reaction solution containing dNTPs.

(Perform PCR)
2.25 μl of cDNA prepared above, Oligo 1 (SEQ ID NO: 1), Oligo 2 (SEQ ID NO: 2), Oligo 4 (SEQ ID NO: 4) each 5 pmoles, and 10 × PCRmix, 0.2 mM
dNTPs, 1 mM MgSO ₄ , and KOD-plus DNA polymerase were added to milliQ water to make a total volume of 25 μl. The PCR reaction was initially performed at 94 ° C. for 2 minutes, followed by a denaturation step at 94 ° C. for 15 seconds, an annealing at 62.5 ° C. for 30 seconds, and a DNA extension reaction step at 68 ° C. for 30 seconds as one cycle. This was carried out by performing a total of 35 cycles.

(Detection using electrophoresis)
5 μl of the amplification reaction solution was electrophoresed using 1% acrylamide gel. The results are shown in FIG.

  As described above, at least one pair of oligonucleotides that amplify a region containing a mutation, DNA polymerase, and at least one oligonucleotide that specifically amplifies a mutated nucleic acid sequence can be used simultaneously and quickly. The type could be clearly determined.

[Example 3]
(1) Synthesis of oligonucleotide for detecting 681 (G → A) single nucleotide mutation polymorphism of CYP2C19 gene Using DNA synthesizer type 392 manufactured by PerkinElmer, Inc., as shown in SEQ ID NOs: 5 to 8 by the phosphoramidite method Oligonucleotides having a base sequence (hereinafter referred to as oligos 5 to 8) were synthesized. The synthesis was performed according to the manual, and the deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C. overnight. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, we commissioned DNA synthesis contractors (Japan Bioservices, Operon, Proligo, Sigma Genosys, etc.). Oligo 5 (SEQ ID NO: 5) is a common sense primer (B), oligo 6 (SEQ ID NO: 6) is a common antisense primer (C), and both common primers are combined and used as an oligonucleotide for an amplification reaction. . Oligo 7 (SEQ ID NO: 7) is a primer (A) for amplifying the 681G gene (wild type) of the CYP2C19 gene. Oligo 8 (SEQ ID NO: 8) is a primer (A) for amplifying the 681A gene (mutant type) of the CYP2C19 gene.

Analysis of 681 (G → A) single nucleotide polymorphism of CYP2C19 gene by PCR method Using DNA solution extracted from human leukocytes by phenol-chloroform method as a sample, the following reagents were added, and human CYP2C19 Gene base polymorphism
(681G → A) was analyzed.

(Perform PCR)
1 μl of the sample prepared above, 5 pmoles of oligos 5 to 8, and 10 × PCRmix, 0.2 mM dNTPs, 1 mM MgSO ₄ , and KOD-plus DNA polymerase were added to milliQ water to make a total volume of 25 μl. The PCR reaction was initially performed at 94 ° C. for 2 minutes, followed by a denaturation step at 94 ° C. for 15 seconds, an annealing at 60 ° C. for 30 seconds, and a DNA extension reaction step at 68 ° C. for 30 seconds as one cycle. This was carried out by performing a total of 35 cycles.

(Detection using electrophoresis)
5 μl of the amplification reaction solution was electrophoresed using a 3% agarose gel. The results are shown in FIG.
As the electrophoresis results show, the amplified fragments obtained by the samples are different, so that the sequence can be easily determined.

[Example 4]
(1) Synthesis of oligonucleotide for detecting 636 (G → A) single nucleotide mutation polymorphism of CYP2C19 gene Using DNA synthesizer type 392 manufactured by PerkinElmer, Inc., as shown in SEQ ID NOs: 9 to 12 by the phosphoramidite method Oligonucleotides having a base sequence (hereinafter referred to as oligos 9 to 12) were synthesized. The synthesis was performed according to the manual, and the deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C. overnight. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, we commissioned DNA synthesis contractors (Japan Bioservices, Operon, Proligo, Sigma Genosys, etc.). Oligo 9 is a common sense primer (A), oligo 10 is a common antisense primer (B), and both common primers are combined and used as an oligonucleotide for an amplification reaction. Oligo 11 is a primer for amplifying the 636G gene (wild type) of the CYP2C19 gene. Oligo 12 is a primer for amplifying the 636A gene (mutant type) of the CYP2C19 gene.

Analysis of 636 (G → A) single nucleotide polymorphism of CYP2C19 gene by PCR method Using DNA solution extracted from human leukocytes by phenol-chloroform method as a sample, the following reagents were added, and human CYP2C19 under the following conditions: Gene base polymorphism
(636G → A) was analyzed.

(Perform PCR)
1 μl of the sample prepared above, 10 pmoles of oligo 9, 5 pmoles of oligo 10, 30 pmoles of oligo 11, 7.5 pmoles of oligo 12 and 10 × PCRmix, 0.2 mM
dNTPs, 1 mM MgSO ₄ , and KOD-plus DNA polymerase were added to milliQ water to make a total volume of 25 μl. The PCR reaction was initially performed at 94 ° C. for 2 minutes, followed by a denaturation step at 94 ° C. for 15 seconds, an annealing at 60 ° C. for 30 seconds, and a DNA extension reaction step at 68 ° C. for 30 seconds as one cycle. This was carried out by performing a total of 35 cycles.

(Detection using electrophoresis)
5 μl of the amplification reaction solution was electrophoresed using a 3% agarose gel. The results are shown in FIG.
GG or G / G represents wild type homo, A / A represents mutant homo, and G / A represents hetero. As the electrophoresis results show, the amplified fragments obtained by the samples are different, so that the sequence can be easily determined.

[Example 5]
(1) Synthesis of oligonucleotide detecting 1075 (A → C) single nucleotide polymorphism of CYP2C9 gene Using DNA synthesizer type 392 manufactured by PerkinElmer, Inc., as shown in SEQ ID NOs: 13 to 16 by the phosphoramidite method Oligonucleotides having a base sequence (hereinafter referred to as oligos 13 to 16) were synthesized. The synthesis was performed according to the manual, and the deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C. overnight. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, we commissioned DNA synthesis contractors (Japan Bioservices, Operon, Proligo, Sigma Genosys, etc.). Oligo 13 is a common sense primer (B), oligo 14 is a common antisense primer (C), and both common primers are combined and used as an oligonucleotide for an amplification reaction. Oligo 15 is a primer (A) for amplifying the CYP2C9 gene 1075A gene (wild type). Oligo 16 is a primer (A) for amplifying the 1075C gene (mutant type) of the CYP2C9 gene.

Analysis of 1075 (A → C) single nucleotide polymorphism of CYP2C9 gene by PCR method DNA solution (samples 1 and 2) extracted from human leukocytes by phenol-chloroform method and plasmid DNA solution (sample 3) where the mutant gene was cloned ) As a sample, add the following reagents, and base polymorphism of human CYP2C9 gene under the following conditions
(1075A → C) was analyzed.

(Perform PCR)
1 μl of the sample prepared above, Oligo 13 at 10 pmoles, Oligo 14 at 2.5 pmoles, Oligos 15 and 16 at 30 pmoles each, and 10 × PCRmix, 0.2 mM
dNTPs, 1 mM MgSO 4, and KOD-plus DNA polymerase were added to milliQ water to make a total volume of 25 μl. The PCR reaction was initially performed at 94 ° C. for 2 minutes, followed by a denaturation step at 94 ° C. for 15 seconds, an annealing at 60 ° C. for 30 seconds, and a DNA extension reaction step at 68 ° C. for 30 seconds as one cycle. This was carried out by performing a total of 35 cycles.

(Detection using electrophoresis)
5 μl of the amplification reaction solution was electrophoresed using a 3% agarose gel. The results are shown in FIG. AA represents wild type homo, CC represents mutant homo, and AC represents hetero. As the electrophoresis results show, the amplified fragments obtained by the samples are different, so that the sequence can be easily determined.

[Example 6]
(1) Synthesis of oligonucleotide for detecting exon 19 Del E746-A750 deletion mutation of human EGFR gene The nucleotide sequence shown in SEQ ID NOs: 17 to 20 by the phosphoramidite method using DNA synthesizer type 392 manufactured by PerkinElmer Were synthesized (hereinafter referred to as oligos 17 to 20). The synthesis was performed according to the manual, and the deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C. overnight. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, we commissioned DNA synthesis contractors (Japan Bioservices, Operon, Proligo, Sigma Genosys, etc.). Oligo 17 is the exon 21 common sense primer (B), and oligo 18 is the exon 21 common antisense primer (C). The two primers are combined and used as an oligonucleotide for the amplification reaction. Oligo 19 is an exon 19 common sense primer, and oligo 20 is an exon 19 of the EGFR gene.
It is a primer (A) for amplifying a Del E746-A750 deletion mutant gene (mutant type).

(2) Analysis of deletion mutation of human EGFR gene by PCR method DNA solution (sample 1) extracted from human leukocytes by phenol-chloroform method, and human EGFR exon 19
Plasmid DNA solution containing Del E746-A750 deletion mutant gene (Sample 2), hereinafter referred to as Del L747-S752 (Sample 3), Del E746-T751 ins
A plasmid DNA solution containing each deletion mutant gene of I (sample 4), Del L747-T751del P753 (sample 5), Del L747-A750 substitution P (sample 6) and a plasmid DNA solution containing the human EGFR exon 21 wild type gene Using the mixed DNA solution and the plasmid DNA solution containing human EGFR exon 19 wild type gene and the DNA solution mixed with plasmid DNA solution containing human EGFR exon 21 wild type gene (sample 7) as samples, the following reagents were used: In addition, the deletion mutation of the human EGFR gene was analyzed under the following conditions.

(Perform PCR)
1 μl of the sample prepared above, Oligo 17 at 20 pmoles, Oligo 18 at 1 pmoles, Oligo 19 at 30 pmoles, Oligo 20 at 20 pmoles, and 10 × PCRmix, 0.2 mM
dNTPs, 1 mM MgSO 4, and KOD-plus DNA polymerase were added to milliQ water to make a total volume of 25 μl. The PCR reaction was initially performed at 94 ° C. for 2 minutes, followed by a denaturation step at 94 ° C. for 15 seconds, an annealing at 60 ° C. for 30 seconds, and a DNA extension reaction step at 68 ° C. for 30 seconds as one cycle. This was carried out by performing a total of 35 cycles.

(Detection using electrophoresis)
5 μl of the amplification reaction solution was electrophoresed using a 3% agarose gel. The results are shown in FIG.
As shown by the electrophoresis results, only the sample 2 was amplified.

[Example 7]
(1) Synthesis of oligonucleotide for detecting exon 19 Del L747-S752 deletion mutation of human EGFR gene It is shown in SEQ ID NOs: 17 to 19 and 21 by the phosphoramidite method using DNA synthesizer type 392 manufactured by PerkinElmer. Oligonucleotides having a base sequence (hereinafter referred to as oligos 17 to 19 and 21) were synthesized. The synthesis was performed according to the manual, and the deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C. overnight. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, we commissioned DNA synthesis contractors (Japan Bioservices, Operon, Proligo, Sigma Genosys, etc.). Oligo 17 is the exon 21 common sense primer (B), and oligo 18 is the exon 21 common antisense primer (C). The two primers are combined and used as an oligonucleotide for the amplification reaction. Oligo 19 is an exon 19 common sense primer, and oligo 21 is an EGFR gene exon 19
It is a primer (A) for amplifying a Del L747-S752-deficient mutant gene (mutant type).

(2) Analysis of deletion mutation of human EGFR gene by PCR method DNA solution (sample 1) extracted from human leukocytes by phenol-chloroform method, and human EGFR exon 19
Plasmid DNA solution containing Del E746-A750 deletion mutant gene (Sample 2), hereinafter referred to as Del L747-S752 (Sample 3), Del E746-T751 ins
A plasmid DNA solution containing each deletion mutant gene of I (sample 4), Del L747-T751del P753 (sample 5), Del L747-A750 substitution P (sample 6) and a plasmid DNA solution containing the human EGFR exon 21 wild type gene Using the mixed DNA solution and the plasmid DNA solution containing human EGFR exon 19 wild type gene and the DNA solution mixed with plasmid DNA solution containing human EGFR exon 21 wild type gene (sample 7) as samples, the following reagents were used: In addition, the deletion mutation of the human EGFR gene was analyzed under the following conditions.

(Perform PCR)
1 μl of the sample prepared above, Oligo 17 at 20 pmoles, Oligo 18 at 1 pmoles, Oligo 19 at 30 pmoles, Oligo 21 at 30 pmoles, and 10 × PCRmix, 0.2 mM
dNTPs, 1 mM MgSO 4, and KOD-plus DNA polymerase were added to milliQ water to make a total volume of 25 μl. The PCR reaction was initially performed at 94 ° C. for 2 minutes, followed by a denaturation step at 94 ° C. for 15 seconds, an annealing at 60 ° C. for 30 seconds, and a DNA extension reaction step at 68 ° C. for 30 seconds as one cycle. This was carried out by performing a total of 35 cycles.

(Detection using electrophoresis)
5 μl of the amplification reaction solution was electrophoresed using a 3% agarose gel. The results are shown in FIG.
As shown by the electrophoresis results, the mutant type was amplified only in sample 3.

[Example 8]
(1) Synthesis of oligonucleotide for detecting exon 19 Del E746-T751 ins I deletion mutation of human EGFR gene Using DNA synthesizer type 392 manufactured by PerkinElmer, using phosphoamidite method, SEQ ID NOs: 17 to 19 and 22 Oligonucleotides having the indicated base sequences (hereinafter referred to as oligos 17 to 19 and 22) were synthesized. The synthesis was performed according to the manual, and the deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C. overnight. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, we commissioned DNA synthesis contractors (Japan Bioservices, Operon, Proligo, Sigma Genosys, etc.). Oligo 17 is the exon 21 common sense primer (B), and oligo 18 is the exon 21 common antisense primer (C). The two primers are combined and used as an oligonucleotide for the amplification reaction. Oligo 19 is the exon 19 common sense primer, and oligo 22 is the EGFR gene exon 19
It is a primer (A) for amplifying Del E746-T751 ins I deletion mutant gene (mutant type).

(2) Analysis of deletion mutation of human EGFR gene by PCR method DNA solution (sample 1) extracted from human leukocytes by phenol-chloroform method, and human EGFR exon 19
Plasmid DNA solution containing Del E746-A750 deletion mutant gene (Sample 2), hereinafter referred to as Del L747-S752 (Sample 3), Del E746-T751 ins
A plasmid DNA solution containing each deletion mutant gene of I (sample 4), Del L747-T751del P753 (sample 5), Del L747-A750 substitution P (sample 6) and a plasmid DNA solution containing the human EGFR exon 21 wild type gene Using the mixed DNA solution and the plasmid DNA solution containing human EGFR exon 19 wild type gene and the DNA solution mixed with plasmid DNA solution containing human EGFR exon 21 wild type gene (sample 7) as samples, the following reagents were used: In addition, the deletion mutation of the human EGFR gene was analyzed under the following conditions.

(Perform PCR)
1 μl of the sample prepared above, Oligo 17 at 20 pmoles, Oligo 18 at 1 pmoles, Oligo 19 at 30 pmoles, Oligo 22 at 40 pmoles, and 10 × PCRmix, 0.2 mM
dNTPs, 1 mM MgSO 4, and KOD-plus DNA polymerase were added to milliQ water to make a total volume of 25 μl. The PCR reaction was initially performed at 94 ° C. for 2 minutes, followed by a denaturation step at 94 ° C. for 15 seconds, an annealing at 60 ° C. for 30 seconds, and a DNA extension reaction step at 68 ° C. for 30 seconds as one cycle. This was carried out by performing a total of 35 cycles.

(Detection using electrophoresis)
5 μl of the amplification reaction solution was electrophoresed using a 3% agarose gel. The results are shown in FIG.
As shown by the electrophoresis results, only the sample 4 was amplified.

[Example 9]
(1) Synthesis of oligonucleotide for detecting exon 19 Del L747-T751del P753 deletion mutation of human EGFR gene Using DNA synthesizer type 392 manufactured by PerkinElmer, Inc., shown in SEQ ID NOs: 17 to 19 and 23 by the phosphoramidite method Oligonucleotides having the following nucleotide sequences (hereinafter referred to as oligos 17 to 19 and 23) were synthesized. The synthesis was performed according to the manual, and the deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C. overnight. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, we commissioned DNA synthesis contractors (Japan Bioservices, Operon, Proligo, Sigma Genosys, etc.). Oligo 17 is the exon 21 common sense primer (B), and oligo 18 is the exon 21 common antisense primer (C). The two primers are combined and used as an oligonucleotide for the amplification reaction. Oligo 19 is the exon 19 common sense primer, and oligo 23 is the EGFR gene exon 19
This is a primer (A) for amplifying the Del L747-T751del P753-deficient mutant gene (mutant type).

(2) Analysis of deletion mutation of human EGFR gene by PCR method DNA solution (sample 1) extracted from human leukocytes by phenol-chloroform method, and human EGFR exon 19
Plasmid DNA solution containing Del E746-A750 deletion mutant gene (Sample 2), hereinafter referred to as Del L747-S752 (Sample 3), Del E746-T751 ins
A plasmid DNA solution containing each deletion mutant gene of I (sample 4), Del L747-T751del P753 (sample 5), Del L747-A750 substitution P (sample 6) and a plasmid DNA solution containing the human EGFR exon 21 wild type gene Using the mixed DNA solution and the plasmid DNA solution containing human EGFR exon 19 wild type gene and the DNA solution mixed with plasmid DNA solution containing human EGFR exon 21 wild type gene (sample 7) as samples, the following reagents were used: In addition, the deletion mutation of the human EGFR gene was analyzed under the following conditions.

(Perform PCR)
1 μl of the sample prepared above, Oligo 17 at 20 pmoles, Oligo 18 at 1 pmoles, Oligo 19 at 30 pmoles, Oligo 23 at 30 pmoles, and 10 × PCRmix, 0.2 mM
dNTPs, 1 mM MgSO 4, and KOD-plus DNA polymerase were added to milliQ water to make a total volume of 25 μl. The PCR reaction was initially performed at 94 ° C. for 2 minutes, followed by a denaturation step at 94 ° C. for 15 seconds, an annealing at 60 ° C. for 30 seconds, and a DNA extension reaction step at 68 ° C. for 30 seconds as one cycle. This was carried out by performing a total of 35 cycles.

(Detection using electrophoresis)
5 μl of the amplification reaction solution was electrophoresed using a 3% agarose gel. The results are shown in FIG.
As the electrophoresis result shows, only the sample 5 was amplified.

[Example 10]
(1) Exon 19 of human EGFR gene Synthesis of oligonucleotide detecting Del P747-A750 substitution P-deficient mutation Using DNA synthesizer type 392 manufactured by PerkinElmer, using phosphoramidite method, SEQ ID NOs: 17 to 19 and 24 Oligonucleotides having the indicated base sequences (hereinafter referred to as oligos 17 to 19 and 24) were synthesized. The synthesis was performed according to the manual, and the deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C. overnight. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, we commissioned DNA synthesis contractors (Japan Bioservices, Operon, Proligo, Sigma Genosys, etc.). Oligo 17 is the exon 21 common sense primer (B), and oligo 18 is the exon 21 common antisense primer (C). The two primers are combined and used as an oligonucleotide for the amplification reaction. Oligo 19 is the exon 19 common sense primer, and oligo 24 is the EGFR gene exon 19
A primer (A) for amplifying a Del L747-A750 substitution P-deficient mutant gene (mutant type).

(2) Analysis of deletion mutation of human EGFR gene by PCR method DNA solution (sample 1) extracted from human leukocytes by phenol-chloroform method, and human EGFR exon 19
Plasmid DNA solution containing Del E746-A750 deletion mutant gene (Sample 2), hereinafter referred to as Del L747-S752 (Sample 3), Del E746-T751 ins
A plasmid DNA solution containing each deletion mutant gene of I (sample 4), Del L747-T751del P753 (sample 5), Del L747-A750 substitution P (sample 6), and a plasmid DNA solution containing human EGFR exon 21 wild type gene Using the mixed DNA solution and the plasmid DNA solution containing the human EGFR exon 19 wild type gene and the plasmid DNA solution containing the human EGFR exon 21 wild type gene (sample 7) as samples, use the following reagents: In addition, the deletion mutation of the human EGFR gene was analyzed under the following conditions.

(Perform PCR)
1 μl of the sample prepared above, Oligo 17 at 20 pmoles, Oligo 18 at 1 pmoles, Oligo 19 at 30 pmoles, Oligo 24 at 30 pmoles, and 10 × PCRmix, 0.2 mM
dNTPs, 1 mM MgSO 4, and KOD-plus DNA polymerase were added to milliQ water to make a total volume of 25 μl. The PCR reaction was initially performed at 94 ° C. for 2 minutes, followed by a denaturation step at 94 ° C. for 15 seconds, an annealing at 60 ° C. for 30 seconds, and a DNA extension reaction step at 68 ° C. for 30 seconds as one cycle. This was carried out by performing a total of 35 cycles.

(Detection using electrophoresis)
5 μl of the amplification reaction solution was electrophoresed using a 3% agarose gel. The results are shown in FIG.
As the result of electrophoresis shows, only the sample 6 was amplified.

[Example 11]
(1) Exon 19 Del L747-S752 of human EGFR gene, Del E746-T751 ins
I, Del L747-T751del P753, Del L747-A750 substitution Synthesis of oligonucleotide detecting P-deficient mutation Using a DNA synthesizer type 392 manufactured by PerkinElmer, SEQ ID NOs: 17-19 and 21-24 by the phosphoramidite method Were synthesized (hereinafter referred to as oligos 17 to 19 and 21 to 24). The synthesis was performed according to the manual, and the deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C. overnight. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, we commissioned DNA synthesis contractors (Japan Bioservices, Operon, Proligo, Sigma Genosys, etc.). Oligo 17 is the exon 21 common sense primer (B), and oligo 18 is the exon 21 common antisense primer (C). The two primers are combined and used as an oligonucleotide for the amplification reaction. Oligo 19 is an exon 19 common sense primer, and oligos 21 to 24 are primers (A) for amplifying an exon 19-deficient mutant gene (mutant type) of the EGFR gene.

(2) Analysis of human EGFR gene deletion mutation by PCR method Plasmid DNA solution containing human EGFR exon 19 Del E746-A750 deletion mutation gene (Sample 1), hereinafter referred to as Del
L747-S752 (Sample 2), Del E746-T751 ins I (Sample 3), Del L747-T751del P753 (Sample 4), Del
L747-A750 substitution P (sample 5) plasmid DNA solution containing each deletion mutant gene and plasmid DNA solution containing human EGFR exon 21 wild type gene, and plasmid DNA containing human EGFR exon 19 wild type gene Using the DNA solution (sample 6) mixed with the solution and plasmid DNA solution containing human EGFR exon 21 wild type gene, and the DNA solution (sample 7) extracted from human leukocytes by the phenol-chloroform method as samples, the following reagents Was added, and the deletion mutation of the human EGFR gene was analyzed under the following conditions.

(Perform PCR)
1 μl of the sample prepared above, Oligo 17 20 pmoles, Oligo 18 1 pmole, Oligo 19 30 pmoles, Oligo 21 30 pmoles, Oligo 23 30 pmoles, Oligo 24 30 pmoles, and 10 × PCRmix, 0.2 mM
dNTPs, 1 mM MgSO 4, and KOD-plus DNA polymerase were added to milliQ water to make a total volume of 25 μl. The PCR reaction was initially performed at 94 ° C. for 2 minutes, followed by a denaturation step at 94 ° C. for 15 seconds, an annealing at 60 ° C. for 30 seconds, and a DNA extension reaction step at 68 ° C. for 30 seconds as one cycle. This was carried out by performing a total of 35 cycles.

(Detection using electrophoresis)
5 μl of the amplification reaction solution was electrophoresed using a 3% agarose gel. The results are shown in FIG.
As the electrophoresis results show, the mutant types of samples 2 to 5 were amplified.

[Example 12]
(1) Synthesis of oligonucleotide for detecting 1075 (A → C) single nucleotide polymorphism of CYP2C9 gene Using DNA synthesizer type 392 manufactured by PerkinElmer, using the phosphoamidite method, SEQ ID NOs: 13, 14, 16, Oligonucleotides having the base sequence shown in 25 (hereinafter referred to as oligos 13, 14, 16, 25) were synthesized. The synthesis was performed according to the manual, and the deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C. overnight. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, we commissioned DNA synthesis contractors (Japan Bioservices, Operon, Proligo, Sigma Genosys, etc.). Oligo 13 is a common sense primer (B), oligo 14 is a common antisense primer (C), and both common primers are combined and used as an oligonucleotide for amplification reaction. Oligo 16 is a primer (A) for amplifying the 1075C gene (mutant type) of the CYP2C9 gene. Oligo 25 is a probe containing a detection site, and the 3 ′ end is labeled with Texas Red.

(2) Analysis of 1075 (A → C) single nucleotide polymorphism of CYP2C9 gene by PCR method DNA solution extracted from human leukocytes by phenol-chloroform method (samples 1 and 2) and plasmid DNA solution obtained by cloning mutant gene Using (Sample 3) as a sample, add the following reagents, and base polymorphism of human CYP2C9 gene under the following conditions
(1075A → C) was analyzed.

(Perform PCR)
1 μl of the sample prepared above, Oligo 13 at 10 pmoles, Oligo 14 at 2.5 pmoles, Oligo 16 at 30 pmoles, and 10 × PCRmix, 0.2 mM
dNTPs, 1 mM MgSO ₄ , KOD-plus DNA polymerase, SYBR GreenI (1/10000) was added to milliQ water to make a total volume of 25 μl. The PCR reaction was initially performed at 94 ° C. for 2 minutes, followed by a denaturation step at 94 ° C. for 15 seconds, an annealing at 60 ° C. for 30 seconds, and a DNA extension reaction step at 68 ° C. for 30 seconds as one cycle. This was carried out by performing a total of 35 cycles.

(Detection using probe: melting curve analysis)
Add 10 pmoles of Oligo 25 to 25 μl of the amplification reaction solution, incubate at 95 ° C. for 5 minutes, increase to 40 ° C., increase the temperature to 80 ° C. at a rate of 0.5 ° C./sec. The change in fluorescence intensity per unit time was measured. The results are shown in FIG. AA represents wild type homo, CC represents mutant homo, and AC represents hetero.
As the melting curve analysis results show, the peak positions obtained by the samples are different, so that the sequence can be easily determined.

[Example 13]
(1) Synthesis of oligonucleotide for detecting H. pylori CagA gene type Oligonucleotide having the nucleotide sequence shown in SEQ ID NOs: 26 to 28 by the phosphoramidite method using DNA synthesizer type 392 manufactured by PerkinElmer ( Hereinafter, oligos 26, 27, and 28) were synthesized. The synthesis was performed according to the manual, and the deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C. overnight. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, we commissioned DNA synthesis contractors (Japan Bioservices, Operon, Proligo, Sigma Genosys, etc.). Oligo 26 is a common sense primer (B), oligo 27 is a common antisense primer (C), and both common primers are combined and used as an oligonucleotide for an amplification reaction. Oligo 28 is a primer (A) for amplifying the western type of the H. pylori CagA gene.

(2) Analysis of H.pylori CagA gene type by PCR method DNA solution (sample 16) extracted from human gastric biopsy tissue with Dneasy Tissue Kit (Quiagen), H.pylori East Asian type gene, H.pylori Using the plasmid DNA solution (samples 2 to 15) that cloned the Western European gene as a sample, add the following reagents, and H. pylori under the following conditions:
The type of CagA gene was analyzed.

(Perform PCR)
1 μl of the sample prepared above, Oligo 26 at 10 pmoles, Oligo 27 at 10 pmoles, Oligo 28 at 20 pmoles, and 10 × PCRmix, 0.2 mM dNTPs, 1 mM
MgSO ₄ and KOD-plus DNA polymerase were added to milli-Q water to make a total volume of 25 μl. The PCR reaction was initially performed at 94 ° C. for 2 minutes, followed by a denaturation step at 94 ° C. for 15 seconds, an annealing at 60 ° C. for 30 seconds, and a DNA extension reaction step at 68 ° C. for 30 seconds as one cycle. This was carried out by performing a total of 35 cycles.

(Detection using electrophoresis)
5 μl of the amplification reaction solution was electrophoresed using a 3% agarose gel. The results are shown in FIG. As shown by the electrophoresis results, it was confirmed that CagA East Asian type was amplified by 10 ⁴ Copy and CagA Western type was amplified by 10 ³ Copy, and detection was possible from samples extracted from gastric biopsy tissue. .

  According to the present invention, wild-type nucleic acid sequence detection and mutant-type nucleic acid sequence detection, which have been indispensable in the detection by electrophoresis, are separately performed in the detection of mutation, substitution, single nucleotide polymorphism, insertion or deletion until now. Because it is no longer necessary, it is possible to provide an effective means for reducing detection errors such as poor amplification due to samples as well as economic effects such as halving the labor and reagents required for detection. However, it is expected to greatly contribute to the industry.

Claims (14)

In a method for determining at least one type of base site on a nucleic acid sequence contained in a sample solution, one type of oligonucleotide primer for base determination (A) containing the base site to be determined and including the base site to be determined; In the simultaneous presence of oligonucleotide primers (B) and (C), a base determination method comprising a step of performing a gene amplification reaction by PCR using KOD-plus (trademark) DNA polymerase,
The primers (A), (B) and (C) are selected from any combination of the following (1) to (3):
(1) Primer (A) is a sense primer or an antisense primer, comprising a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region combination;
(2) Primer (A) is a sense primer, a combination of a sense primer (B) homologous to the downstream region of the base site to be determined and an antisense primer (C) complementary to the downstream region (provided that Primer (C) is downstream of primer (B));
(3) A combination of a primer (A) which is an antisense primer and a sense primer (B) homologous to the upstream region of the base site to be determined and an antisense primer (C) complementary to the upstream region (provided that , Primer (C) is downstream of primer (B)). The base determination method according to claim 1, wherein the base site to be determined includes mutation, substitution, single nucleotide polymorphism, insertion or deletion. 3. The base determination oligonucleotide primer (A), wherein the first base from the 3 ′ end is the same as or complementary to an expected base of a base site to be determined. Base determination method. 3. The base determination oligonucleotide primer (A), wherein the second base from the 3 ′ end is the same as or complementary to the predicted base of the base site to be determined. Base determination method. The base primer oligonucleotide primer (A) is characterized in that at least one base from 3 to 5 from the 3 'end is substituted with a base that is not complementary or homologous to a nucleic acid sequence as a template. Or the base determination method of 4. The Tm value of at least two types of oligonucleotide primers (B, C) having a sequence complementary or homologous to the upstream and / or downstream region of the base site to be determined is determined by the Tm value of the oligonucleotide primer (A) for base determination It is higher than a value, The base determination method in any one of Claims 1-5 characterized by the above-mentioned. At least two types of oligonucleotide primers (B, C) having concentrations complementary or homologous to the upstream and / or downstream region of the base site to be determined are determined when the oligonucleotide primer for base determination (A) is used. The base determination method according to claim 1, wherein the concentration is higher than the concentration at the time of use. Obtained by using any one of base primer oligonucleotide primer (A) and oligonucleotide primer (B, C) having a sequence complementary or homologous to the upstream and / or downstream region of the base site to be judged The base determination method according to claim 1, wherein the length of the amplified nucleic acid fragment varies depending on the base to be determined. The 5 ′ end of at least two kinds of oligonucleotide primers (B, C) having a sequence complementary or homologous to the upstream and / or downstream region of the base site to be determined as the base determination oligonucleotide primer (A) is labeled The base determination method according to claim 1, wherein the base determination method is performed. The base determination method according to any one of claims 1 to 9, wherein the label is any one selected from the group consisting of a fluorescent chemical substance, a luminophore, an enzyme, a fluorescent protein, a photoprotein, a magnetic substance, and a conductive substance. In a method for determining at least one type of base site on a nucleic acid sequence contained in a sample solution, one type of base determination oligonucleotide primer (A) per base site to be determined, including the base site to be determined; In the presence of oligonucleotide primers (B) and (C), gene amplification reaction by PCR using at least one kind of oligonucleotide probe (D) that is homologous or complementary to the nucleic acid sequence containing the base site Is a base determination method characterized by comprising a step of performing melting curve analysis by using KOD-plus (trademark) DNA polymerase,
The primers (A), (B) and (C) are selected from any combination of the following (1) to (3):
(1) Primer (A) is a sense primer or an antisense primer, comprising a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region combination;
(2) Primer (A) is a sense primer, a combination of a sense primer (B) homologous to the downstream region of the base site to be determined and an antisense primer (C) complementary to the downstream region (provided that Primer (C) is downstream of primer (B));
(3) A combination of a primer (A) which is an antisense primer and a sense primer (B) homologous to the upstream region of the base site to be determined and an antisense primer (C) complementary to the upstream region (provided that , Primer (C) is downstream of primer (B)). In a method for determining at least one type of base site on a nucleic acid sequence contained in a sample solution, one type of oligonucleotide primer for base determination (A) containing the base site to be determined and including the base site to be determined; In the presence of oligonucleotide primers (B) and (C), at least one type of oligonucleotide probe (E) that is homologous or complementary and has a sequence that differs from the nucleic acid sequence containing the base site by at least one base is used. A base determination method comprising a step of performing a gene amplification reaction by PCR using KOD-plus (trademark) DNA polymerase and performing a melting curve analysis,
The primers (A), (B) and (C) are selected from any combination of the following (1) to (3):
(1) Primer (A) is a sense primer or an antisense primer, comprising a sense primer (B) that is homologous to the upstream region of the base site to be determined and an antisense primer (C) that is complementary to the downstream region combination;
(2) Primer (A) is a sense primer, a combination of a sense primer (B) homologous to the downstream region of the base site to be determined and an antisense primer (C) complementary to the downstream region (provided that Primer (C) is downstream of primer (B));
(3) A combination of a primer (A) which is an antisense primer and a sense primer (B) homologous to the upstream region of the base site to be determined and an antisense primer (C) complementary to the upstream region (provided that , Primer (C) is downstream of primer (B)). The base determination method according to claim 11 or 12, wherein the oligonucleotide probe (D) or (E) is labeled. The base determination method according to claim 13, wherein the label is any one selected from the group consisting of a fluorescent chemical substance, a luminophore, an enzyme, a fluorescent protein, a photoprotein, a magnetic substance, and a conductive substance.