WO2006051990A1 - Diagnostic method using nucleic acid amplification - Google Patents

Diagnostic method using nucleic acid amplification Download PDF

Info

Publication number
WO2006051990A1
WO2006051990A1 PCT/JP2005/020963 JP2005020963W WO2006051990A1 WO 2006051990 A1 WO2006051990 A1 WO 2006051990A1 JP 2005020963 W JP2005020963 W JP 2005020963W WO 2006051990 A1 WO2006051990 A1 WO 2006051990A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
nucleic acid
primer
specimen
target nucleic
Prior art date
Application number
PCT/JP2005/020963
Other languages
French (fr)
Japanese (ja)
Inventor
Yoshihide Hayashizaki
Toshizo Hayashi
Yasumasa Mitani
Original Assignee
Riken
Kabushiki Kaisha Dnaform
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from JP2005129830A external-priority patent/JP2008029205A/en
Application filed by Riken, Kabushiki Kaisha Dnaform filed Critical Riken
Publication of WO2006051990A1 publication Critical patent/WO2006051990A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • the present invention relates to a rapid genetic diagnosis method for cells or tissues associated with a disease.
  • cancer cells having properties specific to the disease are known.
  • cancer cells having various properties are known even in cancer of the same tissue.
  • the effects of therapeutic drugs, cancer progression, prognosis, etc. differ depending on the nature of such cancer cells. Therefore, investigating the nature of cancer cells and classifying them by molecular biology techniques provides important information related to drug selection, treatment selection, and surgical procedure selection during surgery. .
  • a nucleic acid amplification method As a nucleic acid amplification method, an isothermal amplification method that does not require complicated temperature control as in the PCR method is known.
  • a strand displacement amplification method SDA method; Japanese Patent Publication No. 7-114718. Publication
  • 3SR method self-sustained sequence amplification method
  • Q ⁇ replicase method Japanese Patent No. 2710159 publication
  • NASBA method Japanese Patent No. 2650159 publication
  • LAMP method International Publication No. ⁇ 28082 pamphlet
  • ICAN method WO02Z16639 pamphlet
  • the rolling circle method and the WO2004Z040019 pamphlet are known.
  • the present inventors detect a genetic mutation associated with a disease using the first specimen and the second specimen obtained from the subject as samples, and the first specimen and the second specimen. By comparing the detection results between the cells, it was found that cells related to the disease contained in the first sample or cells derived therefrom can be detected quickly and accurately in the second sample.
  • the present invention is based on these findings.
  • an object of the present invention is to provide a method for rapidly and accurately detecting a disease-related cell or a cell derived therefrom contained in a first specimen in a second specimen.
  • the method for detecting a disease-related cell is a method for detecting a cell related to a disease contained in a first sample from a subject or a cell derived therefrom in a second sample.
  • a disease-related cell contained in the first specimen or a cell derived therefrom is detected by the second specimen force when the detected gene mutation is identical to each other. It is.
  • a plurality of specimens collected at different times, or a plurality of specimens collected from different parts of a living body are used for disease-related cells or cells contained in one specimen. It is possible to quickly and accurately examine whether or not cells derived from this are contained in other specimens. In particular, since the detection method according to the present invention can be carried out in a short time, it is possible to obtain detection results during surgery.
  • Fig. 1 is a diagram schematically showing the action mechanism of a nucleic acid amplification reaction using a first primer.
  • FIG. 2 is a diagram illustrating the structure of a second primer.
  • FIG. 3a is a diagram schematically showing the action mechanism of a nucleic acid amplification reaction using a first primer and a second primer.
  • FIG. 3b is a diagram schematically showing the action mechanism of a nucleic acid amplification reaction using the first primer and the second primer.
  • FIG. 4 is a diagram showing mutation sites in the human P 53 gene nucleotide sequence (SEQ ID NO: 1).
  • FIG. 5 is a graph showing the time course of the amplification reaction in the tissue at the center of the primary lesion in case 1.
  • a genetic mutation associated with a disease is detected from the genome contained in each of the first specimen and the second specimen of subject power.
  • the first specimen may be any disease as long as it contains cells associated with the target disease.
  • a person skilled in the art can select as appropriate.
  • a tissue obtained by collecting the force at the center of a lesion before treatment of the target disease can be used.
  • the second sample is a sample to be examined for the presence or amount of the disease-related cells or cells derived from the first sample, and is appropriately selected by those skilled in the art according to the target disease. Is done.
  • the second specimen can be tissue around the primary lesion of the cancer or tissue at a site suspected of metastasis.
  • the first sample may be collected by biopsy or the like before the operation, and the second sample may be collected during the operation.
  • One sample may be collected during the operation, and the second sample may be collected by biopsy etc. after the operation.
  • the first sample is collected before the treatment of the target disease
  • the second sample is collected during or after the treatment of the disease. It is supposed to be.
  • Such treatment is appropriately selected by those skilled in the art depending on the target disease, and examples thereof include drug therapy, radiation therapy, hyperthermia, hormone therapy, immunotherapy, and surgical therapy.
  • the first specimen is taken from the lesion of the target disease
  • the second specimen is taken from another site. It is said.
  • a cell associated with a disease has a gene mutation associated with the disease, preferably a gene mutation specific for the disease, and is appropriately selected by those skilled in the art depending on the target disease. Gene mutations related to this are known for various diseases, and those skilled in the art can appropriately use combinations of these diseases and gene mutations. Examples of such gene mutations include mutations in the p53 gene specific for cancer. There are differences.
  • a cell derived from a disease-related cell is not a disease-related cell itself, it is a cell that has proliferated and proliferated in a disease-related cell at a site other than the primary lesion of the disease. Accordingly, cells derived from disease-related cells have the same genotype as disease-related cells.
  • the disease-related cell is a cancer cell or a carcinoma cell.
  • cancer-specific gene mutations are detected in the genomes contained in the first specimen and the second specimen. Cancer invasion or metastasis can be evaluated by using the first specimen as the primary tumor tissue and the second specimen as the tissue from the periphery of the primary lesion. In addition, cancer metastasis to the site can be evaluated by using the first specimen as a primary cancer tissue and the second specimen as a tissue with other site strength. Furthermore, whether the first specimen is a cancer tissue of the primary lesion and the second specimen is a cancer tissue present in another site, so that the cancer tissue present in that site is metastatic cancer from the primary lesion. Can be evaluated.
  • the p53 protein which also expresses this gene power, is a transcription factor that controls the cell cycle, apoptosis, and DNA repair, and plays a role in preventing the accumulation of mutations (such as canceration) at the individual level.
  • mutations in the p53 gene are found in more than 50% of cases, of which 80% are point mutations with amino acid substitutions. This mutation rate is much higher than other known oncogenes.
  • the p53 protein is a DNA binding protein, and many of the mutations found in cancer are concentrated in the DNA binding domain.
  • mutations in the p53 gene that are common in colorectal cancer are mutations at 175, 248, and 273, followed by CG, followed by 196, 213, 245, There is a tendency for mutations to concentrate at the 282nd codon.
  • the types of point mutations also have a clear tendency, and there are overwhelmingly many transversions from GC to AT.
  • any one or a combination of two or more of such gene mutations can be used.
  • the detection method of the present invention it is possible to obtain such information during surgery.
  • the primary cancer tissue is the first specimen
  • the second specimen is the washing liquid obtained by washing the thoracic cavity, resulting in the primary lesion present in the washing liquid.
  • the presence or absence of cancer cells can be examined, which makes it possible to accurately grasp cancer transition. Accurate information about cancer metastasis is very useful in determining the procedure.
  • the presence or absence of cancer invasion and metastasis can be examined by using the cancer tissue of the primary lesion as the first specimen and the yarn and tissue from the periphery of the primary lesion as the second specimen.
  • Such information is useful for determining surgical procedures, selecting postoperative treatments, selecting drugs to be administered, and determining therapeutic effects.
  • Detection of gene mutation can be performed by standard methods well known in the art, such as a method using PCR, a method using a microarray, an invader method, a TaqMan PCR method, a primer extension method, and the like.
  • detection of a gene mutation is performed by a nucleic acid amplification method.
  • the nucleic acid amplification method it is possible to detect the mutation based on the presence or absence of the amplification product by using at least one kind of primer containing a nucleotide residue that is resistant to the mutation.
  • a ply containing a mutant type as a nucleotide residue that is resistant to mutation When a mer is used, the presence of the amplification product indicates the presence of the mutation.
  • nucleic acid amplification method any method known to those skilled in the art may be used, but preferably the target nucleic acid can be amplified by a reaction under isothermal conditions.
  • isothermal nucleic acid amplification methods include the LAMP method (International Publication No. 00Z28082 pamphlet), the I CAN method (International Publication No. 02Z16639 pamphlet), the SDA method (Japanese Patent Publication No. 7-114718), and the autonomous replication method. , NASBA method (Japanese Patent No. 2650159), TMA method, Q ⁇ replicase method (Japanese Patent No. 2710159), International Publication No. 2004Z040019, etc.
  • the “isothermal” refers to maintaining an approximately constant temperature condition such that the enzyme and the primer can substantially function.
  • the “substantially constant temperature condition” is not only to maintain the set temperature accurately, but is allowed as long as the temperature changes to the extent that the substantial function of the enzyme and the primer is not impaired. Means that.
  • the nucleic acid amplification reaction under a certain temperature condition can be carried out by keeping the temperature at which the activity of the enzyme used can be maintained.
  • the reaction temperature is preferably set to a temperature near the melting temperature (Tm) of the primer or lower.
  • Tm melting temperature
  • this temperature is preferably about 20 ° C to about 75 ° C, and more preferably about 35 ° C to about 65 ° C.
  • the isothermal nucleic acid amplification method comprises a primer set capable of amplifying the target nucleic acid sequence, wherein the first primer force contained in the primer set comprises the target nucleic acid sequence.
  • a sequence (Ac ′) that hybridizes to the sequence (A) at the 3 ′ end portion is included at the 3 ′ end portion, and is a sequence that is present 5 ′ from the sequence (A) in the target nucleic acid sequence (
  • the sequence ( ⁇ ′) that hybridizes to the complementary sequence (Be) of B) is the 5 of the sequence (Ac).
  • the primer set comprising the 'side is used.
  • the nucleic acid amplification method developed by the present inventors is used as the isothermal nucleic acid amplification method.
  • a primer set comprising at least two kinds of primers capable of amplifying a target nucleic acid sequence, wherein the first primer included in the primer set is a sequence of the 3 ′ end portion of the target nucleic acid sequence.
  • sequence (B) comprising 3 'terminal portion of hybridizing sequence (Ac) in (A) and existing in the target nucleic acid sequence V and 5' to sequence (A).
  • a sequence ( ⁇ ′) that hybridizes to the sequence (Be) is included on the 5 ′ side of the sequence (Ac ′), and the second primer included in the primer set is complementary to the target nucleic acid sequence.
  • “noblybize” means that the primer hybridizes to the target nucleic acid under stringent conditions and does not hybridize to nucleic acid molecules other than the target nucleic acid.
  • Stringent conditions can be determined depending on the melting temperature Tm (° C) of the duplex of the primer according to the present invention and its complementary strand, the salt concentration of the hybridization solution, etc. Reference can be made to J. Sambrook, EF Frisch, T. Maniatis; Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory (1989). For example, when hybridization is performed at a temperature slightly lower than the melting temperature of the primer to be used, the primer can be specifically hybridized to the target nucleic acid.
  • a primer that hybridizes to a target nucleic acid comprises a sequence of all or part of a nucleic acid molecule complementary to the target nucleic acid.
  • Fig. 1 schematically shows the mechanism of nucleic acid synthesis by the first primer.
  • the first primer comprises the sequence (Ac ') and further comprises the sequence ( ⁇ ') on the 5 'side.
  • Array (Ac ') Is hybridized to the sequence (A), and the sequence ( ⁇ ′) is hybridized to the complementary sequence (Be) of the sequence ( ⁇ ).
  • the first primer may contain an intervening sequence that does not affect the reaction between the sequence (Ac ′) and the sequence ( ⁇ ′).
  • the sequence (Ac) in the primer is nobbreviated to the sequence (A) of the target nucleic acid sequence (FIG. 1 (a)).
  • a primer extension reaction occurs in this state, a nucleic acid containing a complementary sequence of the target nucleic acid sequence is synthesized.
  • the sequence ( ⁇ ′) present on the 5 ′ end side of the synthesized nucleic acid hybridizes to the sequence (Be) present in the nucleic acid, and thereby the stem in the 5 ′ end portion of the synthesized nucleic acid.
  • a loop structure is formed.
  • the sequence (A) on the vertical nucleic acid becomes a single strand, and another primer having the same sequence as the first primer hybridizes to this part (FIG. 1 (b)).
  • the strand displacement reaction causes an extension reaction from the newly hybridized first primer, and at the same time, the previously synthesized nucleic acid is separated from the vertical nucleic acid (FIG. 1 (c)).
  • the phenomenon that the sequence ( ⁇ ') hybridizes to the sequence (Be) is typically caused by the presence of complementary regions on the same strand.
  • the dissociation of a partial force or a part of the other end that is relatively unstable begins.
  • the base pair of the terminal portion is in an equilibrium state of dissociation and binding at a relatively high temperature, and the double strand is maintained as a whole.
  • a stem-loop structure can be formed as a metastable state.
  • the same other primer binds to the complementary strand portion (sequence (A) on the vertical nucleic acid) that was exposed due to the formation of the structure, and the polymerase immediately
  • sequence (A) on the vertical nucleic acid
  • the design criteria for the first primer in a preferred embodiment of the present invention are as follows. First, in order for a new primer to efficiently anneal to the homologous nucleic acid after the complementary strand of the truncated nucleic acid is synthesized by extension of the primer, a stem loop structure is formed at the 5 'end of the synthesized complementary strand. The part of the sequence (A) on the vertical nucleic acid is Must be single stranded.
  • Ratio to X (X—Y) ZX is important.
  • the efficiency V the formation of the stem loop structure, that is, the efficiency!
  • the distance between (X + Y) is important.
  • the optimum temperature for the primer extension reaction is at most around 72 ° C, and at such a low temperature, it is difficult for the extended strand to dissociate over a long region. Therefore, in order for the sequence ( ⁇ ′) to hybridize efficiently to the sequence (Be), it is considered preferable that the number of bases between both sequences is small.
  • sequence ( ⁇ ′) in order for the sequence ( ⁇ ′) to hybridize to the sequence (Be) and the portion of the sequence (A) on the cage nucleic acid to be a single strand, the sequence ( ⁇ ′) and the sequence (Be) It is considered preferable that the number of bases between and is larger.
  • the first primer according to a preferred embodiment of the present invention is the case where there is no intervening sequence between the sequence (Ac) and the sequence ( ⁇ ') constituting the primer.
  • (X— ⁇ ) ⁇ is 1.00 or more, preferably 0.00 or more, more preferably 0.05 or more, more preferably 0.10 or more, and 1.00 or less, preferably 0.75. In the following, it is designed to be more preferably 0.50 or less, and still more preferably 0.25 or less.
  • ( ⁇ + ⁇ ) is preferably 15 or more, more preferably 20 or more, more preferably 30 or more, and is preferably 50 or less, more preferably 48 or less, and even more preferably 42 or less.
  • the first primer according to a preferred embodiment of the present invention ⁇ X— ( ⁇ — ⁇ ′ ⁇ is 1 1.00 or more, preferably 0.00 or more, more preferably 0.05 or more, more preferably 0.10 or more, and 1.00 or less, It is preferably designed to be 0.75 or less, more preferably 0.50 or less, and even more preferably 0.25 or less, and ( ⁇ + ⁇ + ⁇ ′) is preferably 15 or more, more preferably Is 20 or more, more preferably 30 Also, it is preferably 100 or less, more preferably 75 or less, and still more preferably 50 or less.
  • the first primer has a chain length such that base pairing with a target nucleic acid can be carried out while maintaining necessary specificity under given conditions.
  • the chain length of this primer is preferably 15 to 100 nucleotides, more preferably 20 to 60 nucleotides.
  • the lengths of the sequence (Ac ′) and the sequence ( ⁇ ′) constituting the first primer are each preferably 5 to 50 nucleotides, more preferably 7 to 30 nucleotides. If necessary, insert an intervening sequence that does not affect the reaction between sequence (Ac ') and sequence ( ⁇ ').
  • the second primer is added to the sequence (C) of the 3 'end portion of the complementary sequence of the target nucleic acid sequence (the strand opposite to the strand to which the first primer hybridizes).
  • a folded sequence (D-Dc) comprising two nucleic acid sequences hybridizing to each other on the same strand, comprising a sequence (Cc) to be hybridized (Cc) at the 3 ′ end.
  • the structure of such a second primer is, for example, as shown in FIG. 2, but is not limited to the sequence and the number of nucleotides shown in FIG.
  • the length of the sequence (Cc ′) constituting the second primer is preferably 5 to 50 nucleotides, more preferably 10 to 30 nucleotides.
  • the length of the folded sequence (D-Dc ′) is preferably 2 to 1000 nucleotides, more preferably 2 to 100 nucleotides, further preferably 4 to 60 nucleotides, and further preferably 6 to 40 nucleotides.
  • the number of nucleotides in the base pair formed by hybridization within the folded sequence is preferably 2 to 500 bp, more preferably 2 to 50 bp, more preferably 2 to 30 bp, more preferably 2 to 30 bp. Furthermore, it is preferably 3 to 20 bp.
  • the nucleotide sequence of the folded sequence (D-Dc) may be! Or any sequence, and is not particularly limited, but is preferably a sequence that does not hybridize to the target nucleic acid sequence. If necessary, an intervening sequence that does not affect the reaction may be inserted between the sequence (Cc ′) and the folded sequence (D-Dc ′).
  • FIG. 3 A possible mechanism of action of the nucleic acid amplification reaction by the first primer and the second primer will be described with reference to Fig. 3 (Figs. 3a and 3b).
  • two sequences to be hybridized are referred to as complementary sequences. iS This does not limit the invention.
  • the first primer hybridizes to the sense strand of the target nucleic acid, and the primer extension reaction occurs (FIG. 3 (a)).
  • a stem-loop structure is formed on the extended strand (one), and the new first primer hybridizes to the sequence (A) on the target nucleic acid sense strand that has become a single strand (Fig.
  • a hairpin-type double-stranded nucleic acid in which the extension strand () is bound to the 3 ′ side of the extension strand (+) via the sequence (A) and the sequence (Be) is generated.
  • the first primer hybridizes to the sequence (A) and sequence (Be) (Fig. 3 (g)), and the extension chain (-) is generated by the extension reaction (Figs. 3 (h) and (0)).
  • the folded sequence present at the 3 ′ end of the hairpin double-stranded nucleic acid provides a free 3 ′ end (FIG. 3 (h)), and an extension reaction therefrom (FIG.
  • a single-stranded nucleic acid having a folded sequence at both ends and containing an extended strand (+) and an extended strand (one) alternately through the sequences derived from the first and second primers (FIG. 3).
  • the folded sequence present at the 3 'end provides a free 3' end (starting point of complementary strand synthesis) (Fig. 3 (k)).
  • Anti The chain length is doubled per extension reaction (Fig. 3 (1) and (m)), and the extended strand from the first primer released in Fig. 3 (- ) Provides a free 3 'end (the complementary strand synthesis origin) due to the folding sequence present at the 3' end (Fig. 3 (n)).
  • a loop structure is formed, producing a single-stranded nucleic acid containing an extended strand (+) and an extended strand (one) alternately via a sequence derived from the primer ( Figure 3 (o)).
  • extension reactions occur one after another. Sequences derived from the first and second primers are included between the extended strand (+) and the extended strand (-). Because, each primer extension reaction was Haiburidizu This can significantly amplify the sense and antisense strands of the target nucleic acid.
  • the primer set is a third primer that hybridizes to the target nucleic acid sequence or its complementary sequence, and a third primer that does not compete with other primers for hybridization to the target nucleic acid sequence or its complementary sequence.
  • a third primer that does not compete with other primers for hybridization to the target nucleic acid sequence or its complementary sequence.
  • “do not compete!” Means that the primer does not interfere with the provision of the complementary strand synthesis starting point by hybridizing to the target nucleic acid.
  • the amplification product has the target nucleic acid sequence and its complementary sequence alternately. There is a folded sequence or loop structure at the 3 ′ end of the amplified product, and extension reactions occur one after another from the complementary strand synthesis starting point provided.
  • the third primer can anneal to the target sequence present in the single-stranded part when such an amplification product is partially in a single-stranded state. As a result, a new complementary strand synthesis origin is provided in the target nucleic acid sequence in the amplification product, and an extension reaction takes place therefrom, so that the nucleic acid amplification reaction is performed more rapidly.
  • the third primer is not necessarily limited to one type, but two or more types of the third primer may be used simultaneously in order to improve the speed and specificity of the nucleic acid amplification reaction. These third primers typically have different sequence powers than the first primer and the second primer, and may hybridize to a partially overlapping region as long as they do not compete with these primers.
  • the chain length of the third primer is preferably 2 to: LOO nucleotides, more preferably 5 to 50 nucleotides, and even more preferably 7 to 30 nucleotides.
  • the third primer mainly has an auxiliary function for proceeding more rapidly with the nucleic acid amplification reaction by the first primer and the second primer. Accordingly, it is preferable that the third primer has a Tm lower than the Tm of the third and third ends of the first primer and the second primer, respectively.
  • the amplification reaction of the third primer The amount added to the solution is preferably smaller than the amount added to each of the first primer and the second primer.
  • the primer set is designed so that the mutation site is included in the sequence (A), the sequence (B), or the sequence (C). can do. Accordingly, it is possible to determine the presence or absence of the mutation by confirming the presence or absence of the amplification product, and it is possible to determine the amount of the gene having the mutation by quantifying the amplification product.
  • the primer set is designed such that a nucleotide residue related to mutation is included in the sequence (A).
  • the first primer when the target nucleic acid sequence is contained in the nucleic acid sample, the first primer is annealed to the sequence (A) in the nucleic acid amplification reaction, so that an amplification product is obtained. If the nucleic acid sample contains a nucleic acid sequence that is different from the target nucleic acid sequence at the mutation site, it is difficult for the first primer to anneal to the sequence (A) in the nucleic acid amplification reaction. Therefore, no amplification product is obtained or the amount of amplification product obtained is significantly reduced.
  • the nucleotide residue involved in the mutation is preferably included in the 5 ′ end of the sequence (A) (corresponding to the 3 ′ end in the first primer).
  • the sequence (Ac ′) contained in the first primer is preferably a sequence complementary to the sequence (A).
  • the primer set is designed such that a nucleotide residue related to mutation is included in the sequence (C).
  • the amplification product is obtained because the second primer anneals to the sequence (C) in the nucleic acid amplification reaction. If the nucleic acid sample contains a nucleic acid sequence that is different from the target nucleic acid sequence at the mutation site, it is difficult for the second primer to anneal to the sequence (C) in the nucleic acid amplification reaction. Therefore, no amplification product is obtained or the amount of amplification product obtained is significantly reduced.
  • the nucleotide residue involved in the mutation is preferably contained in the 5 ′ end of the sequence (C) (corresponding to the 3 ′ end in the second primer).
  • the sequence (Cc ′) contained in the second primer is preferably a sequence complementary to the sequence (C).
  • the primer set may comprise a mutation-related nucleotide. It is designed such that a residue is included in the sequence (B).
  • the first primer is annealed to the sequence (A) and the extension reaction is performed. Since the contained sequence ( ⁇ ') hybridizes to the sequence (Be) on the extended strand, a stem-loop structure is efficiently formed. The formation of this efficient stem loop structure allows the other first primer to anneal in a saddle shape, and the mechanism of action shown in FIG. 1 proceeds efficiently, resulting in an amplification product.
  • the nucleic acid sample contains a nucleic acid sequence that is different from the target nucleic acid sequence at the mutation site, it is difficult to form the stem-loop structure in the nucleic acid amplification reaction.
  • the mechanism of action shown in 1 is hindered and no amplification product is obtained, or the amount of amplification product obtained is significantly reduced.
  • the sequence ( ⁇ ') contained in the first primer is preferably the same sequence as the sequence ( ⁇ ).
  • a stem-and-loop structure can be formed as a metastable state.
  • this stem loop structure does not exist stably, especially when there are non-complementary nucleotides between the sequence ( ⁇ ') and the sequence (Be) part that form the stem. Or the stem is not formed at all.
  • the hybridization between the vertical sequence (A) and the sequence (Ac ') in the primer is more dominant, and the sequence (A) is not a single strand.
  • the next first primer cannot be annealed. Therefore, it is extremely difficult to cause the continuous reaction shown in Fig. 1.
  • the site force sequence (A) or sequence (B) related to the deletion or insertion can be designed to be placed between the force contained in sequence (C) or between sequence (A) and sequence (B). This makes it possible to determine the presence or absence of a sequence deletion or insertion by confirming the presence or absence of an amplification product, and the amount of a gene having the deletion or insertion by quantifying the amplification product. Can be determined.
  • the primer set is designed such that a site related to deletion or insertion is included in the sequence (A).
  • the first primer is annealed to the sequence (A) in the nucleic acid amplification reaction, so that an amplification product is obtained.
  • the nucleic acid sample contains a nucleic acid sequence that is different from the target nucleic acid sequence due to deletion Z insertion, it will be difficult for the first primer to anneal to the sequence (A) in the nucleic acid amplification reaction. No amplification product is obtained or the amount of amplification product obtained is significantly reduced.
  • the sequence (Ac) contained in the first primer is preferably a sequence complementary to the sequence (A).
  • the primer set is designed such that a site for deletion or insertion is included in the sequence (C).
  • the amplification product is obtained because the second primer anneals to the sequence (c) in the nucleic acid amplification reaction.
  • the nucleic acid sample contains a nucleic acid sequence that is different from the target nucleic acid sequence due to deletion Z insertion, it will be difficult for the second primer to anneal to the sequence (C) in the nucleic acid amplification reaction. No amplification product is obtained or the amount of amplification product obtained is significantly reduced.
  • the sequence (Cc) contained in the second primer is preferably a sequence complementary to the sequence (C).
  • the primer set is designed such that a site related to deletion or insertion is included in the sequence (B).
  • the nucleic acid amplification reaction includes the first primer after annealing to the sequence (A) and the extension reaction, and then the primer is contained in the primer. Since the sequence ( ⁇ ') hybridizes to the sequence (Be) on the extended strand, the stem loop structure It is formed efficiently. The formation of this efficient stem-loop structure allows the other first primer to anneal in a saddle shape, and the mechanism of action shown in Fig. 1 proceeds efficiently, resulting in an amplified product. .
  • the nucleic acid sample contains a nucleic acid sequence that is different from the target nucleic acid sequence due to deletion Z insertion, formation of the stem-loop structure in the nucleic acid amplification reaction becomes difficult.
  • the mechanism of action shown in FIG. 1 is hindered, and no amplification product is obtained, or the amount of amplification product obtained is significantly reduced.
  • the details are as described above for the mutation detection according to the present invention.
  • the sequence ( ⁇ ⁇ ′) contained in the first primer is preferably the same sequence as the sequence ( ⁇ ).
  • the primer set is designed such that a site related to deletion or insertion is located between the sequence ( ⁇ ) and the sequence ( ⁇ ).
  • the first primer is annealed to the sequence ( ⁇ ) and the extension reaction is performed, and then the primer is added to the primer. Since the contained sequence ( ⁇ ') hybridizes to the sequence (Be) on the extended strand, a stem loop structure is efficiently formed. The formation of this efficient stem-loop structure allows the other first primer to anneal in a saddle shape, and the mechanism of action shown in Figure 1 proceeds efficiently, resulting in an amplified product. .
  • the nucleic acid sample contains a nucleic acid sequence that is different from the target nucleic acid sequence due to deletion Z insertion, the sequence ( ⁇ ') contained in the first primer and the sequence on the extended strand ( Therefore, it is difficult to form the stem-loop structure in the nucleic acid amplification reaction. Therefore, in this case, the mechanism of action shown in FIG. 1 is hindered and no amplification product is obtained, or the amount of amplification product obtained is significantly reduced.
  • the primer set is designed such that a site for deletion or insertion is located between the sequence (A) and the sequence (C). .
  • the primer when the target nucleic acid sequence is contained in the nucleic acid sample, the primer is annealed to the sequence (A) in the nucleic acid amplification reaction and the extension reaction is performed. Since the sequence ( ⁇ ′) contained in is hybridized with the sequence (Be) on the extended strand, a stem-loop structure is efficiently formed. The formation of this efficient stem-loop structure allows the other first primer to anneal in a saddle shape. Since the mechanism of action shown in (1) proceeds efficiently, an amplification product is obtained.
  • the nucleic acid sample contains a nucleic acid sequence that differs from the target nucleic acid sequence due to deletion z insertion, no amplification product is obtained or the amount of amplification product obtained is significantly reduced.
  • a nucleic acid sequence that is different from the target nucleic acid sequence is included in the nucleic acid sample! Since (efficiency) is significantly reduced, no amplification product is obtained or the amount of amplification product obtained is significantly reduced.
  • a nucleic acid sequence that differs from the target nucleic acid sequence is contained in the nucleic acid sample due to the deletion of the sequence between sequence (A) and sequence (C).
  • the sequence ( ⁇ ') contained in the first primer cannot be hybridized on the extended strand, making it impossible or difficult to form a stem-loop structure. Therefore, the mechanism of action shown in FIG. 1 is hindered, and no amplification product is obtained, or the amount of amplification product obtained is significantly reduced. Furthermore, a nucleic acid sequence that differs from the target nucleic acid sequence due to the deletion of the sequence between sequence ( ⁇ ) and sequence (C) is contained in the nucleic acid sample. Even if no deletion occurs, the speed (efficiency) of nucleic acid amplification is reduced, so that no amplification product is obtained or the amount of amplification product obtained is significantly reduced.
  • any one having room temperature, medium temperature, or heat resistance can be suitably used as long as it has strand displacement activity (strand displacement ability).
  • this polymerase may be either a natural body or a mutant having an artificial mutation.
  • An example of such a polymerase is DNA polymerase.
  • this DNA polymerase has substantially no 5 ⁇ 3 exonuclease activity.
  • DNA polymerases include thermophilic Bacillus such as Bacillus stearothermophilus (hereinafter referred to as “B. st”), Bacillus caldotenax (hereinafter referred to as “B. ca”), and the like.
  • DNA polymerase 5 ′ ⁇ 3 of DNA polymerase derived from genus bacteria, mutants lacking exonuclease activity, and Tarenow fragment of DNA polymerase I derived from E. coli.
  • DNA polymerases used in nucleic acid amplification reactions include Vent DNA polymerase, Vent (Exo-) DNA polymerase, DeepVent DNA polymerase, DeepVent (Ex o-) DNA polymerase, ⁇ 29 phage DNA polymerase, MS-2 phage DNA polymerase, Z-Taq DNA polymerase, Pfo DNA polymerase, Pfo turbo DNA polymerase, KOD DNA polymerase, 9 ° Nm DNA polymerase, Therminater DNA polymerase, etc. Can be mentioned.
  • Examples of other reagents used in the nucleic acid amplification reaction include catalysts such as magnesium chloride, magnesium acetate, and magnesium sulfate, substrates such as dNTP mix, tris hydrochloride buffer, tricine buffer, sodium phosphate buffer, A buffer such as potassium phosphate buffer can be used. Furthermore, additives such as dimethyl sulfoxide and betaine ( ⁇ , ⁇ , ⁇ -trimethylglycine), acidic substances and cation complexes described in WO 99/54455 pamphlets may be used. .
  • a melting temperature adjusting agent can be added to the reaction solution in order to increase the nucleic acid amplification efficiency.
  • the melting temperature (Tm) of a nucleic acid is generally determined by the specific nucleotide sequence of the double stranded portion in the nucleic acid. By adding a melting temperature adjusting agent to the reaction solution, this melting temperature can be changed. Therefore, under a certain temperature, it is possible to adjust the intensity of double strand formation in the nucleic acid.
  • a general melting temperature adjusting agent has an effect of lowering the melting temperature. By adding such a melting temperature adjusting agent, it is possible to lower the melting temperature of the duplex forming part between two nucleic acids, in other words, to reduce the strength of the duplex formation.
  • the melting temperature adjusting agent is not particularly limited, but is preferably dimethyl sulfoxide (DMSO), betaine, formamide or glycerol, or any combination thereof, more preferably dimethyl sulfoxide (DMSO). ).
  • an enzyme stabilizer can be added to the reaction solution.
  • the enzyme in the reaction solution is stabilized, so that the nucleic acid amplification efficiency can be increased.
  • the enzyme stabilizer used in the present invention is not particularly limited and may be any one known in the art such as glycerol, urine serum albumin, saccharides and the like.
  • a reagent for enhancing the heat resistance of an enzyme such as DNA polymerase or reverse transcriptase can be added to the reaction solution as an enzyme stabilizer.
  • an enzyme stabilizer As a result, the enzyme in the reaction solution is stabilized, so that the nucleic acid synthesis efficiency and amplification efficiency can be increased.
  • Such reagents are well known in the art and may be rugged, but are not particularly limited, but are preferably saccharides, more preferably monosaccharides or oligosaccharides, more preferably It can be trehalose, sorbitol or mannitol, or a mixture of two or more of these.
  • the nucleic acid amplification reaction is carried out in the presence of a mismatch recognition protein, which makes it possible to detect mutations more accurately.
  • Misuma Tutsi gene diagnostic method to detect mismatches utilizes a binding protein
  • a binding protein have been developed (M. Gotoh et al., Genet . Anal, 14, 47-50, 1997) specific in 0 in a nucleic acid
  • a mismatched control nucleic acid is hybridized with a test nucleic acid suspected of having a mutation, and a mismatch recognition protein is introduced into the mismatched nucleic acid.
  • detecting There are known methods for detecting.
  • mismatch means adenine (A), gyonin (G), cytosine (C), and Means that one base pair selected from thymine (T) (uracil (U) in the case of RNA) is not a normal base pair (A and T combination or G and C combination) .
  • Mismatches include not only one mismatch but also multiple consecutive mismatches, mismatches caused by insertion and Z or deletion of one or more bases, and combinations thereof.
  • heteroduplex structure means a double-stranded structure containing a non-complementary region by having one or more mismatches, which is a substantially complementary double-stranded structure. . Such a heteroduplex structure results in a false amplification product that should not be produced by nature.
  • mismatch binding protein binds to the heteroduplex structure as described above, and the subsequent amplification reaction is prevented. Therefore, by using a mismatch binding protein, it is possible to prevent the generation of an erroneous amplification product.
  • the mismatch binding protein used in the present invention may be any protein known to those skilled in the art as long as it is a protein capable of recognizing a mismatch in a double-stranded nucleic acid and binding to the mismatch site. It may be a thing.
  • the mismatch-binding protein used in the present invention has one or more amino acid substitutions, deletions, additions, and / or insertions in the amino acid sequence of the wild-type protein as long as the mismatch in the double-stranded nucleic acid can be recognized. It may be a protein (mutant) that also has an amino acid sequence ability! /. Such mutants can also be created artificially by forces that may occur in nature. Many methods are known for introducing amino acid mutations into proteins.
  • site-directed mutagenesis methods include WP Deng and JA Nickoloff's method (Anal. Biochem., 200, 81, 1992), ⁇ ⁇ . Makamaye and F. Eckstein's method (Nucleic Adids Res., 14, 9679). -9698, 1986), etc.
  • the random mutagenesis method uses a method using Escherichia coli XLl-Red strain (Stratagene) lacking the basic repair system, sodium nitrite, etc.
  • methods for chemically modifying bases J.-J. Diaz et al., BioTechnique, 11, 204-211, 1 991) are known.
  • mismatch binding proteins are known, such as MutM, Mut S and their analogs (Radman, M. et al. Annu. Rev. Genet. 20: 523-538 (1986)). Radaman, M. etal., Sci. Amer., August 1988, pp40-46; Modric h, P., J. Biol. Chem. 264: 6597-6600 (1989); Lahue.RS et al, Science 245: 160-164 (198 8); Jiricny, J. et al '. Nucl. Acids Res. 16: 7843-7853 (1988); Su, SSet al., J. Biol. Chem.
  • the mismatch binding protein used in the present invention is preferably derived from MutS, MutH, MutL, or yeast, and more preferably MutS, MutH, or MutL.
  • a mismatch binding protein may also bind to a single-stranded nucleic acid, and it is known that binding of such a mismatch binding protein to a single-stranded nucleic acid is inhibited by the single-stranded binding protein. ing. Therefore, when a mismatch binding protein is used in the nucleic acid amplification reaction, it is preferable to use a single-stranded binding protein in combination. Mismatch-binding proteins can also bind to double-stranded nucleic acids that do not contain mismatches. Such misbinding of mismatch-binding proteins can be activated by using a activating agent to activate the mismatch-binding proteins. It is known that it is inhibited by keeping it. Therefore, when using a mismatch binding protein in a nucleic acid amplification reaction, it is preferable to use a protein that has been activated in advance by an activator.
  • the single-stranded binding protein (SSB) used to inhibit the binding of the mismatch binding protein to the single-stranded nucleic acid can be any SSB known in the art.
  • Preferred SSBs include single-stranded binding proteins from Escherichia coli, Drosophila, and Xenopus, and gene 32 protein from T4 butteriophage, and their equivalents from other species. Can be mentioned.
  • mismatch binding proteins used in this case are MutS, MutH, MutL, HexA, MSH1-6, Rep3, RNaseA, uracil-DNA glycosidase, T4 endonuclease VII, resolvase, etc., preferably MutS, MSH2 or MSH6, or a mixture of two or more of these, more preferably MutS Is done.
  • the active agent for activating the mismatch binding protein can be appropriately selected by those skilled in the art, and is not particularly limited, but preferably ATP (adenosine 5'-triphosphate). , ADP (adenosine 5'-diphosphate), ATP- ⁇ 3 (adenosine 5 '0 (3-thio triphosphate)), AMP-PNP (adenosine 5' [ J 8, ⁇ imido] triphosphate), etc. Or one of the nucleotides that can bind to the mismatch binding protein.
  • the activity of the mismatch binding protein can be performed by incubating the mismatch binding protein and the active agent at room temperature for several seconds to several minutes.
  • the presence of the amplification product obtained by the nucleic acid amplification method can be detected by many various methods.
  • One method is the detection of amplification products of a specific size by general gel electrophoresis. In this method, for example, it can be detected by a fluorescent substance such as ethidium bromide or cyber green.
  • detection can be performed by using a labeled probe having a label such as piotin and hybridizing it to the amplification product.
  • Piotin can be detected by binding to fluorescently labeled avidin, avidin bound to an enzyme such as peroxidase, and the like.
  • Yet another method is to use immunochromatography.
  • the amplification efficiency in the nucleic acid amplification reaction is very high, so that the amplification product can also be detected indirectly by using the fact that pyrophosphate is generated as an amplification byproduct.
  • a method of visually observing the white turbidity of the reaction solution by utilizing the fact that pyrophosphoric acid binds to magnesium in the reaction solution to cause white precipitation of magnesium pyrophosphate.
  • the Another method is that pyrophosphate binds strongly to metal ions such as magnesium.
  • a metal indicator whose color tone changes according to the magnesium ion concentration (for example, Eriochrome Black T, Hydroxy Naphthol Blue, etc.) is added to the reaction solution, and the color change of the reaction solution is visually observed. This makes it possible to detect the presence or absence of amplification.
  • Calcein, etc. the increase in fluorescence accompanying the amplification reaction can be observed visually, so that amplification products can be detected in real time.
  • the gene mutation detected from the first specimen is then compared with the gene mutation detected from the second specimen. In this comparison, if these detected gene mutations are identical to each other, it indicates that a disease-related cell contained in the first sample or a cell derived therefrom was detected in the second sample. It is.
  • the sample collected before the treatment of the target disease is the first sample
  • the sample collected during or after the treatment of the disease is the second sample.
  • a method for evaluating the effect of treatment of a disease in a subject comprising: (a) a genome contained in a first sample collected from a subject before the treatment of the disease. And (b) detecting a gene mutation in a genome contained in a second specimen collected from the subject during or after the treatment of the disease. And (c) comparing a first gene force detected gene mutation and a second sample force detected gene mutation.
  • the detected gene mutation when the detected gene mutation is different from each other or when the gene mutation is not detected in the second specimen, it is evaluated that the therapeutic effect is high.
  • the treatment is appropriately selected by those skilled in the art depending on the target disease, and examples thereof include drug therapy, radiation therapy, hyperthermia, hormone therapy, immunotherapy, and surgical treatment.
  • the sample collected from the lesion of the target disease is used as the first sample, and the sample collected from other sites is used as the second sample. It becomes possible to evaluate the primary focus of the disease and metastasis to other sites (for example, cancer metastasis). Therefore, according to the present invention, the primary lesion force of the disease in the subject is transferred to other sites.
  • a method of evaluating comprising: (a) detecting a genetic mutation associated with the disease in a genome contained in a first specimen collected from the primary lesion of the disease of the subject, (b) the subject A step of detecting the genetic mutation in the genome contained in the second specimen from which the other site forces were also collected, and (c) the genetic mutation detected from the first specimen and the second specimen force. Comparing the mutation.
  • the detected gene mutations are identical to each other, it is evaluated that there is metastasis from the primary lesion site to another site.
  • the disease is cancer or tumor, and metastasis to these other sites is evaluated.
  • cancer or tumor infiltration can be evaluated by using a cancer tissue or tumor tissue in the primary lesion as the first specimen and a tissue in the periphery of the primary lesion as the second specimen. Further, by setting the cancer tissue of the primary lesion as the first specimen and the cancer tissue present in the other part as the second specimen, the cancer tissue present in that part is metastatic cancer from the primary lesion. It can be evaluated whether or not there is.
  • Example ⁇ ⁇ Primary lesion of colorectal cancer indexed by 53 gene differences
  • mutations in the human p53 gene (SEQ ID NO: 1) were examined by nucleic acid amplification.
  • the mutations to be examined were nucleotide substitutions at the 175th, 213rd, 245th, 248th, and 273rd codons (boxed in Fig. 4).
  • R1 5'—GGATATATATATATCCAGATTCTCTTCCTCTGTGCG—3 ′ (SEQ ID NO: 15);
  • R2 5′—GGATATATATATATCCCTGGAGTCTTCCAGTGTGAT—3 ′ (SEQ ID NO: 16);
  • R3 5 GGATATATATATATCCCTCAGGCGGCTCATAGGGCA-3 ′ (SEQ ID NO: 17)
  • R4 5'— GGATATATATATATCCCATCGCTATCTGAGCAGCGC— 3 '(SEQ ID NO: 18)
  • the number attached to the beginning of the name of the forward primer indicates the codon number in the p53 gene to be examined.
  • the underlined portion of the forward primer sequence is a nucleotide residue that can be used for the mutation to be examined.
  • F indicates a forward primer
  • R indicates a reverse primer.
  • W means that the primer contains a wild type sequence
  • M means that the primer contains a mutant type sequence.
  • the sequence at the 3, terminal side (about 20 mer) is ringed in a cage shape, and the sequence at the 5 'end (lOmer) is on the extension strand of the primer. It is designed to hybridize to the region starting 16 bases downstream of the 3 'terminal residue.
  • the reverse primer is designed to have a structure in which the sequence at the 3rd end (20mer) anneals in a cage shape and the sequence at the 5 'end (16mer) folds within that region. .
  • the forward primer contains nucleotide residues that are resistant to mutation Therefore, it is possible to determine whether or not a residue that can be mutated is included in the forward primer depending on the presence or absence of an amplification product by the nucleic acid amplification reaction.
  • tissue suspension (1 ⁇ 1) was added to a reaction solution (24 L) having the following composition: Tris—HCl (20 mM, pH 8.8), KCl (lOmM) ⁇ (NH) SO ( lOmM) ⁇ MgSO (8mM), DMSO
  • the graph shown in FIG. 5 shows the time course of the amplification reaction in the tissue at the center of the primary lesion in case 1.
  • Tables 1 and 2 below summarize the results of amplification reactions for Case 1 and Case 2, respectively.
  • the amplification reaction results are +++, ++, +, and king, starting from the one with the highest increase rate of the amplification product (the one with the fastest rise of the curve in the graph). What did not exist was taken as one.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

A method of quickly and accurately detecting a disease-associated cell or a cell originating therefrom, which is contained in a first specimen, from a second specimen. Namely, a method of detecting a disease-associated cell or a cell originating therefrom, which is contained in a first specimen obtained from a subject, from a second specimen comprising: (a) the step of detecting a gene mutation associating the disease as described above in a genome contained in the first specimen; (b) the step of detecting the above-described gene mutation in a genome contained in the second specimen; and (c) the step of comparing the gene mutation detected in the first specimen with the gene mutation detected in the second specimen. In this method, it is pointed out that a disease-associated cell or a cell originating therefrom, which is contained in the first specimen, is detected from the second specimen in the case where the gene mutations thus detected are the same as each other.

Description

明 細 書  Specification
核酸増幅を用いた診断方法  Diagnostic method using nucleic acid amplification
関連出願の参照  Reference to related applications
[0001] 本特許出願は、先に出願された米国仮出願第 60Z627, 181号（出願日：2004 年 11月 15日）および日本国特許出願である特願 2005— 129830号（出願日： 200 5年 4月 27日）に基づく優先権の主張を伴うものである。これら先の特許出願におけ る全開示内容は、引用することにより本明細書の一部とされる。  [0001] This patent application consists of previously filed US Provisional Application No. 60Z627,181 (filing date: November 15, 2004) and Japanese Patent Application No. 2005-129830 (filing date: 200). This is accompanied by a priority claim based on April 27, 5). The entire disclosure of these earlier patent applications is hereby incorporated by reference.
発明の背景  Background of the Invention
[0002] 発明の分野 [0002] Field of the Invention
本発明は、疾患に関連する細胞または組織の迅速な遺伝子診断法に関する。  The present invention relates to a rapid genetic diagnosis method for cells or tissues associated with a disease.
[0003] 普晋 術 [0003] Art of Putuo
様々な疾患において、その疾患に特異的な性質を有する細胞が知られている。例 えば、癌細胞としては、同じ組織の癌においても様々な性質のものが知られている。 さらに、このような癌細胞の性質によって、治療用薬剤の効果、癌の進行、予後など が異なることが知られている。よって、癌細胞の性質を調べて癌を分子生物学的手法 により分類することにより、薬剤の選択、治療法の選択、手術の際の術式の選択など に関連する重要な情報が提供される。  In various diseases, cells having properties specific to the disease are known. For example, cancer cells having various properties are known even in cancer of the same tissue. Furthermore, it is known that the effects of therapeutic drugs, cancer progression, prognosis, etc. differ depending on the nature of such cancer cells. Therefore, investigating the nature of cancer cells and classifying them by molecular biology techniques provides important information related to drug selection, treatment selection, and surgical procedure selection during surgery. .
[0004] 疾患に関連する細胞の性質を診断するためには、その疾患の病巣部から組織を採 取し、これに含まれる細胞の特性を検査することが考えられる。例えば、癌の診断を 行なうためには、癌組織を生検によって採取し、該組織に含まれる細胞の特性を調 ベることが考えられる。しかし、従来の検査技術では、検査結果が得られるまでに長 時間を要する。従って、疾患に関連する細胞または組織の迅速な検査を可能とする 技術が必要とされている。  [0004] In order to diagnose the nature of a cell associated with a disease, it is conceivable to take a tissue from the lesion of the disease and examine the characteristics of the cells contained therein. For example, in order to diagnose cancer, it is conceivable to collect cancer tissue by biopsy and examine the characteristics of cells contained in the tissue. However, with conventional inspection techniques, it takes a long time to obtain inspection results. Therefore, there is a need for techniques that allow rapid examination of cells or tissues associated with disease.
[0005] 近年、癌などの疾病の分子生物学的な研究が進んでおり、癌細胞などの疾患関連 細胞に特異的な遺伝子変異などが数多く見出されている。  [0005] In recent years, molecular biological studies of diseases such as cancer have progressed, and many gene mutations specific to disease-related cells such as cancer cells have been found.
[0006] 一方で、遺伝子中の標的領域を増幅しうる様々な核酸増幅法が開発されており、こ のような方法としては、最も良く知られている PCR法 (米国特許第 4683195号明細 書;米国特許第 4683202号明細書;および米国特許第 4800159号明細書)をはじ めとして、 RNAを铸型とする RT—PCR法（Trends in Biotechnology 10, ppl46- 152, 1992)などが知られている。 [0006] On the other hand, various nucleic acid amplification methods capable of amplifying a target region in a gene have been developed. As such a method, the most well-known PCR method (US Pat. No. 4,683,195) As well as RT-PCR (Trends in Biotechnology 10, ppl46-152, 1992), which uses RNA as a cage, as well as U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,800,159. ing.
[0007] 核酸増幅方法としてはまた、 PCR法のような複雑な温度調節を必要としな 、等温増 幅法が知られており、例えば、鎖置換増幅法 (SDA法;特公平 7— 114718号公報） 、自己維持配列増幅法（3SR法）、 Q βレプリカーゼ法（日本国特許第 2710159号 公報）、 NASBA法（日本国特許第 2650159号公報）、 LAMP法（国際公開第 ΟθΖ 28082号パンフレット）、 ICAN法（国際公開第 02Z16639号パンフレット）、ローリ ングサークル法、国際公開第 2004Z040019号パンフレットに記載の方法などが知 られている。 [0007] As a nucleic acid amplification method, an isothermal amplification method that does not require complicated temperature control as in the PCR method is known. For example, a strand displacement amplification method (SDA method; Japanese Patent Publication No. 7-114718). Publication), self-sustained sequence amplification method (3SR method), Q β replicase method (Japanese Patent No. 2710159 publication), NASBA method (Japanese Patent No. 2650159 publication), LAMP method (International Publication No. ΟθΖ28082 pamphlet) The methods described in the ICAN method (WO02Z16639 pamphlet), the rolling circle method, and the WO2004Z040019 pamphlet are known.
[0008] さらに、核酸増幅法を利用した遺伝子変異の検出法として、上記の LAMP法を用 いた変異検出法 (特開 2003— 159100号公報）、国際公開第 01/34838号パンフ レットに記載の方法などが知られている。  [0008] Further, as a method for detecting a gene mutation using a nucleic acid amplification method, a mutation detection method using the above-mentioned LAMP method (Japanese Patent Laid-Open No. 2003-159100), described in WO 01/34838 pamphlet Methods are known.
発明の概要  Summary of the Invention
[0009] 本発明者らは、被験者から得られた第一の検体および第二の検体をサンプルとし て、疾患に関連する遺伝子変異を検出し、前記第一の検体と第二の検体との間で検 出結果を比較することにより、第一の検体に含まれる疾患に関連する細胞またはこれ に由来する細胞を第二の検体において迅速かつ正確に検出することができることを 見出した。本発明はこれら知見に基づくものである。  [0009] The present inventors detect a genetic mutation associated with a disease using the first specimen and the second specimen obtained from the subject as samples, and the first specimen and the second specimen. By comparing the detection results between the cells, it was found that cells related to the disease contained in the first sample or cells derived therefrom can be detected quickly and accurately in the second sample. The present invention is based on these findings.
[0010] 従って、本発明の目的は、第一の検体に含まれる疾患関連細胞またはこれに由来 する細胞を第二の検体において迅速かつ正確に検出する方法を提供することにある  [0010] Therefore, an object of the present invention is to provide a method for rapidly and accurately detecting a disease-related cell or a cell derived therefrom contained in a first specimen in a second specimen.
[0011] そして、本発明による疾患関連細胞の検出法は、被験者からの第一の検体に含ま れる疾患に関連する細胞またはこれに由来する細胞を、第二の検体において検出 する方法であって、 The method for detecting a disease-related cell according to the present invention is a method for detecting a cell related to a disease contained in a first sample from a subject or a cell derived therefrom in a second sample. ,
(a)第一の検体に含まれるゲノムにおいて、前記疾患に関連する遺伝子変異を検出 する工程、  (a) detecting a genetic mutation associated with the disease in the genome contained in the first specimen;
(b)第二の検体に含まれるゲノムにおいて、前記遺伝子変異を検出する工程、なら びに (b) a step of detecting the gene mutation in the genome contained in the second specimen; Bini
(c)第一の検体力 検出された遺伝子変異と第二の検体力 検出された遺伝子変異 とを比較する工程  (c) Step of comparing the first specimen force detected gene mutation and the second specimen force detected gene mutation
を含んでなり、検出された遺伝子変異が相互に同一である場合に、第一の検体に含 まれる疾患関連細胞またはこれに由来する細胞が第二の検体力 検出されたことが 示される方法である。  A disease-related cell contained in the first specimen or a cell derived therefrom is detected by the second specimen force when the detected gene mutation is identical to each other. It is.
[0012] 本発明によれば、異なる時期に採取された複数の検体、または生体中の異なる部 位から採取された複数の検体にお!、て、一つの検体に含まれる疾患関連細胞また はこれに由来する細胞が他の検体に含まれているか否かを迅速かつ正確に調べるこ とが可能となる。特に、本発明による検出法は短時間で実施できるため、手術中に検 出結果を得ることも可能である。  [0012] According to the present invention, a plurality of specimens collected at different times, or a plurality of specimens collected from different parts of a living body are used for disease-related cells or cells contained in one specimen. It is possible to quickly and accurately examine whether or not cells derived from this are contained in other specimens. In particular, since the detection method according to the present invention can be carried out in a short time, it is possible to obtain detection results during surgery.
図面の簡単な説明  Brief Description of Drawings
[0013] [図 1]図 1は、第一のプライマーを用いた核酸増幅反応の作用機序を模式的に示した 図である。  [0013] [Fig. 1] Fig. 1 is a diagram schematically showing the action mechanism of a nucleic acid amplification reaction using a first primer.
[図 2]図 2は、第二のプライマーの構造を例示した図である。  FIG. 2 is a diagram illustrating the structure of a second primer.
[図 3a]図 3aは、第一のプライマーおよび第二のプライマーを用いた核酸増幅反応の 作用機序を模式的に示した図である。  FIG. 3a is a diagram schematically showing the action mechanism of a nucleic acid amplification reaction using a first primer and a second primer.
[図 3b]図 3bは、第一のプライマーおよび第二のプライマーを用いた核酸増幅反応の 作用機序を模式的に示した図である。  FIG. 3b is a diagram schematically showing the action mechanism of a nucleic acid amplification reaction using the first primer and the second primer.
[図 4]図 4は、ヒト P53遺伝子のヌクレオチド配列（配列番号 1)中の変異部位を示す図 である。 [4] FIG. 4 is a diagram showing mutation sites in the human P 53 gene nucleotide sequence (SEQ ID NO: 1).
[図 5]図 5は、症例 1の原発巣中心部の組織における増幅反応の経時変化を示すグ ラフである。  FIG. 5 is a graph showing the time course of the amplification reaction in the tissue at the center of the primary lesion in case 1.
発明の具体的説明  Detailed description of the invention
[0014] 本発明による疾患関連細胞の検出法では、被験者力 の第一の検体および第二 の検体について、それぞれに含まれるゲノムから、疾患に関連する遺伝子変異が検 出される。  [0014] In the method for detecting a disease-related cell according to the present invention, a genetic mutation associated with a disease is detected from the genome contained in each of the first specimen and the second specimen of subject power.
[0015] 第一の検体は、目的とする疾患に関連する細胞を含むものであればよぐその疾患 に応じて当業者であれば適宜選択することができる。このような第一の検体としては、 例えば、目的とする疾患の治療前における病巣の中心部力 採取した組織を用いる ことができる。第二の検体は、第一の検体に含まれる疾患関連細胞またはこれに由 来する細胞の有無またはその量を調べる対象となる検体であり、目的とする疾患に応 じて当業者により適宜選択される。例えば、目的とする疾患が癌である場合には、第 二の検体は、その癌の原発巣の周辺組織または転移の疑われる部位の組織などと することができる。また、疾患の病巣部を外科的手術により摘出する場合には、第一 の検体を手術の前にバイオプシー等によって採取し、第二の検体を手術中に採取し てもよく、あるいは、第一の検体を手術中に採取し、第二の検体を手術後にバイオプ シ一等によって採取してもよ 、。 [0015] The first specimen may be any disease as long as it contains cells associated with the target disease. A person skilled in the art can select as appropriate. As such a first specimen, for example, a tissue obtained by collecting the force at the center of a lesion before treatment of the target disease can be used. The second sample is a sample to be examined for the presence or amount of the disease-related cells or cells derived from the first sample, and is appropriately selected by those skilled in the art according to the target disease. Is done. For example, when the target disease is cancer, the second specimen can be tissue around the primary lesion of the cancer or tissue at a site suspected of metastasis. Also, when the lesion of the disease is removed by surgical operation, the first sample may be collected by biopsy or the like before the operation, and the second sample may be collected during the operation. One sample may be collected during the operation, and the second sample may be collected by biopsy etc. after the operation.
[0016] 本発明の一つの実施態様によれば、第一の検体は目的とする疾患の治療前に採 取されたものとされ、第二の検体は前記疾患の治療中または治療後に採取されたも のとされる。このような治療は目的とする疾患に応じて当業者により適宜選択されるが 、例えば、薬物療法、放射線療法、温熱療法、ホルモン療法、免疫療法、外科的治 療等が挙げられる。この実施態様では、治療前に存在していた疾患関連細胞がその 疾患の治療により消滅した力否力、または治療による該細胞の数の変化を調べること ができ、これにより治療効果を評価することが可能となる。  [0016] According to one embodiment of the present invention, the first sample is collected before the treatment of the target disease, and the second sample is collected during or after the treatment of the disease. It is supposed to be. Such treatment is appropriately selected by those skilled in the art depending on the target disease, and examples thereof include drug therapy, radiation therapy, hyperthermia, hormone therapy, immunotherapy, and surgical therapy. In this embodiment, it is possible to examine the force majeure of the disease-related cells that existed before the treatment disappeared by the treatment of the disease, or the change in the number of the cells by the treatment, thereby evaluating the therapeutic effect. Is possible.
[0017] 本発明の他の実施態様によれば、第一の検体は目的とする疾患の病巣部から採 取されたものとされ、第二の検体は他の部位カゝら採取されたものとされる。この実施 態様では、目的とする疾患の病巣部に見られる疾患関連細胞が、病巣部の周辺をは じめとする他の部位に存在するか否か、またはその存在量を調べることができ、これ により該疾患の原発巣力 他の部位への転移 (例えば、癌転移)を評価することが可 能となる。  [0017] According to another embodiment of the present invention, the first specimen is taken from the lesion of the target disease, and the second specimen is taken from another site. It is said. In this embodiment, it is possible to examine whether or not the disease-related cells found in the lesion of the target disease are present in other sites including the periphery of the lesion, or the abundance thereof. This makes it possible to evaluate the primary focus of the disease and metastasis to other sites (for example, cancer metastasis).
[0018] 疾患に関連する細胞は、その疾患に関連する遺伝子変異、好ましくはその疾患に 特異的な遺伝子変異を有するものであり、目的とする疾患に応じて当業者により適宜 選択される。様々な疾患についてこれに関連する遺伝子変異が知られており、当業 者であればこれらの疾患と遺伝子変異との組み合わせを適宜利用することが可能で ある。このような遺伝子変異としては、例えば、癌に特異的な p53遺伝子における変 異が挙げられる。また、疾患関連細胞に由来する細胞は、疾患関連細胞そのもので はないものの、該疾患の原発巣以外の部位において疾患関連細胞力 ***し、増 殖した細胞である。従って、疾患関連細胞に由来する細胞は、疾患関連細胞と同じ 遺伝子型を有する。 [0018] A cell associated with a disease has a gene mutation associated with the disease, preferably a gene mutation specific for the disease, and is appropriately selected by those skilled in the art depending on the target disease. Gene mutations related to this are known for various diseases, and those skilled in the art can appropriately use combinations of these diseases and gene mutations. Examples of such gene mutations include mutations in the p53 gene specific for cancer. There are differences. In addition, although a cell derived from a disease-related cell is not a disease-related cell itself, it is a cell that has proliferated and proliferated in a disease-related cell at a site other than the primary lesion of the disease. Accordingly, cells derived from disease-related cells have the same genotype as disease-related cells.
[0019] 本発明の好ま ヽ実施態様によれば、疾患関連細胞は癌細胞または類癌腫細胞と される。この実施態様では、第一の検体および第二の検体に含まれるゲノムにおい て、癌に特異的な遺伝子変異が検出される。第一の検体を原発巣の癌組織とし、第 二の検体を原発巣の周辺部からの組織とすることにより、癌の浸潤または転移を評価 することができる。また、第一の検体を原発巣の癌組織とし、第二の検体を他の部位 力もの組織とすることにより、その部位への癌の転移を評価することができる。さらに、 第一の検体を原発巣の癌組織とし、第二の検体を他の部位に存在する癌組織とする ことにより、その部位に存在する癌組織が前記原発巣からの転移癌であるか否かを 評価することができる。  [0019] According to a preferred embodiment of the present invention, the disease-related cell is a cancer cell or a carcinoma cell. In this embodiment, cancer-specific gene mutations are detected in the genomes contained in the first specimen and the second specimen. Cancer invasion or metastasis can be evaluated by using the first specimen as the primary tumor tissue and the second specimen as the tissue from the periphery of the primary lesion. In addition, cancer metastasis to the site can be evaluated by using the first specimen as a primary cancer tissue and the second specimen as a tissue with other site strength. Furthermore, whether the first specimen is a cancer tissue of the primary lesion and the second specimen is a cancer tissue present in another site, so that the cancer tissue present in that site is metastatic cancer from the primary lesion. Can be evaluated.
[0020] 癌細胞に特異的な遺伝子変異は数多く知られているが、中でも最も多いのが p53 遺伝子の変異である。この遺伝子力も発現する p53タンパク質は転写因子の一つで あり、細胞周期、アポトーシス、および DNA修復の制御を行ない、個体レベルでの変 異の蓄積 (癌化など)を防ぐ役割を果たしている。ヒトの癌では、その 50%以上の症 例において p53遺伝子の変異が見られ、その中の 80%がアミノ酸置換を伴う点突然 変異である。この変異率は他に知られている癌遺伝子と比較してかなり高くなつてい る。 p53タンパク質は DNA結合タンパク質であり、癌で見られる変異の多くは DNA 結合ドメインに集中している。さらに、 p53遺伝子の中でも際だって変異の集中する ホットスポット »報告 れて ヽる (Lopez M et al., Use of cytological specimens for p53 gene alteration detection in oral squamous cell carcinoma risk patients. Clin. Oncol . (R Coll Radiol). 2004 Aug; 16(5): 366— 70 ; Soussi T et al., Reassessment of the TP5 3 mutation database in human disease by data mining with a library of TP53 missens e mutations, Hum. Mutat. 25(1): 6-17, 2004； Sudhir bnvastava, et al., Biomarkers fo r Early Detection of Colon Cancer, Clinical Cancer Research vol. 7, 1118, 2001； Tni erry Soussi, et al., Significance of TP53 Mutation in Human Cancer: A Critical Anal ysis of Mutations at CpG Dinucleotides, Human. Mutation Vol.21, 192, 2003)。また 、臓器によって変異の頻度や変異のおこりやすい位置は異なる。ヒトの癌の中で、 p5 3遺伝子の変異の頻度が高いものは、肺癌、大腸癌、脾臓癌などであり、これらの癌 の 70%近くに変異が見られる。例えば、大腸癌に多く見られる p53遺伝子中の変異 は、第 175コドン、第 248コドン、および第 273コドンに見られる CGでの変異であり、 次いで第 196コドン、第 213コドン、第 245コドン、第 282コドンなどに変異が集中す る傾向がある。点突然変異の種類にも明確な傾向があり、圧倒的に GCから ATへの トランスバージョンが多い。本発明による検出法においては、このような遺伝子変異の いずれか 1つまたは 2以上の組み合わせを用いることができる。 [0020] Many gene mutations specific to cancer cells are known, but the most common is the mutation of the p53 gene. The p53 protein, which also expresses this gene power, is a transcription factor that controls the cell cycle, apoptosis, and DNA repair, and plays a role in preventing the accumulation of mutations (such as canceration) at the individual level. In human cancer, mutations in the p53 gene are found in more than 50% of cases, of which 80% are point mutations with amino acid substitutions. This mutation rate is much higher than other known oncogenes. The p53 protein is a DNA binding protein, and many of the mutations found in cancer are concentrated in the DNA binding domain. In addition, hot spots where mutations are conspicuously concentrated in the p53 gene have been reported (Lopez M et al., Use of cytological specimens for p53 gene alteration detection in oral squamous cell carcinoma risk patients. Clin. Oncol. 2004 Aug; 16 (5): 366—70; Soussi T et al., Reassessment of the TP5 3 mutation database in human disease by data mining with a library of TP53 missens e mutations, Hum. Mutat. 25 ( 1): 6-17, 2004; Sudhir bnvastava, et al., Biomarkers for Early Detection of Colon Cancer, Clinical Cancer Research vol. 7, 1118, 2001; Tni erry Soussi, et al., Significance of TP53 Mutation in Human Cancer: A Critical Anal ysis of Mutations at CpG Dinucleotides, Human. Mutation Vol. 21, 192, 2003). Moreover, the frequency of mutation and the position where the mutation is likely to occur vary depending on the organ. Among human cancers, those with a high frequency of mutations in the p53 gene are lung cancer, colon cancer, spleen cancer, and mutations are found in nearly 70% of these cancers. For example, mutations in the p53 gene that are common in colorectal cancer are mutations at 175, 248, and 273, followed by CG, followed by 196, 213, 245, There is a tendency for mutations to concentrate at the 282nd codon. The types of point mutations also have a clear tendency, and there are overwhelmingly many transversions from GC to AT. In the detection method according to the present invention, any one or a combination of two or more of such gene mutations can be used.
[0021] 癌組織を摘出するための外科的手術では、実際の病巣部を直接観察することがで き、より正確で細力な組織像の観察が可能となる。その際に医師が知りたい情報は、 例えば、浸潤の程度、複数個の病巣が観察される場合におけるそれらの起源の同一 性、転移の可能性などである。本発明による検出法によれば、これらの情報を手術中 に得ることが可能となる。例えば、肺癌の摘出手術においては、原発巣の癌組織を第 一の検体とし、胸腔内を洗浄して得られる洗浄液を第二の検体とすることにより、洗 浄液中に存在する原発巣由来の癌細胞の有無を調べることができ、これにより、癌転 移について正確に把握することが可能となる。癌転移についての正確な情報は、術 式の決定に非常に有用である。また、原発巣の癌組織を第一の検体とし、原発巣の 周辺部からの糸且織を第二の検体とすることにより、癌の浸潤および転移の有無を調 ベることができる。このような情報は、術式の決定、術後の治療法の選択、投与薬剤 の選択、治療効果の判定などに有用である。  [0021] In a surgical operation for removing a cancer tissue, an actual lesion can be directly observed, and a more accurate and detailed tissue image can be observed. The information that the doctor wants to know is, for example, the degree of infiltration, the identity of their origin when multiple lesions are observed, and the possibility of metastasis. According to the detection method of the present invention, it is possible to obtain such information during surgery. For example, in lung cancer excision surgery, the primary cancer tissue is the first specimen, and the second specimen is the washing liquid obtained by washing the thoracic cavity, resulting in the primary lesion present in the washing liquid. The presence or absence of cancer cells can be examined, which makes it possible to accurately grasp cancer transition. Accurate information about cancer metastasis is very useful in determining the procedure. In addition, the presence or absence of cancer invasion and metastasis can be examined by using the cancer tissue of the primary lesion as the first specimen and the yarn and tissue from the periphery of the primary lesion as the second specimen. Such information is useful for determining surgical procedures, selecting postoperative treatments, selecting drugs to be administered, and determining therapeutic effects.
[0022] 遺伝子変異の検出は、当技術分野において周知の標準的な方法、例えば、 PCR を用いる方法、マイクロアレイを用いる方法、インベーダー法、 TaqMan PCR法、プ ライマーエクステンション法などによって行なうことができる。  [0022] Detection of gene mutation can be performed by standard methods well known in the art, such as a method using PCR, a method using a microarray, an invader method, a TaqMan PCR method, a primer extension method, and the like.
[0023] 本発明の好ましい実施態様によれば、遺伝子変異の検出は、核酸増幅法によって 行なわれる。核酸増幅法では、変異に力かるヌクレオチド残基を含む少なくとも一種 のプライマーを用いることにより、増幅産物の有無によってその変異を検出することが 可能となる。例えば、変異に力かるヌクレオチド残基として変異型のものを含むプライ マーを用いる場合には、増幅産物の存在により該変異の存在が示される。このような 方法では、目的とする遺伝子変異の有無が検出されるだけでなぐ存在する変異型 遺伝子を定量することも可能である。また、変異に力かるヌクレオチド残基として野生 型のものを含むプライマーを用いる核酸増幅反応を同時に行なうことにより、野生型 遺伝子と変異型遺伝子の存在比率を知ることも可能である。 [0023] According to a preferred embodiment of the present invention, detection of a gene mutation is performed by a nucleic acid amplification method. In the nucleic acid amplification method, it is possible to detect the mutation based on the presence or absence of the amplification product by using at least one kind of primer containing a nucleotide residue that is resistant to the mutation. For example, a ply containing a mutant type as a nucleotide residue that is resistant to mutation When a mer is used, the presence of the amplification product indicates the presence of the mutation. In such a method, it is also possible to quantify the existing mutant gene simply by detecting the presence or absence of the target gene mutation. It is also possible to know the abundance ratio of the wild type gene and the mutant gene by simultaneously carrying out a nucleic acid amplification reaction using a primer containing a wild type nucleotide residue that is effective for mutation.
[0024] 核酸増幅法としては、当業者に公知のいかなる方法を用いてもよいが、好ましくは 等温下での反応によって標的核酸の増幅を可能とするものとされる。このような等温 核酸増幅法としては、例えば、 LAMP法（国際公開第 00Z28082号パンフレット）、 I CAN法（国際公開第 02Z16639号パンフレット）、 SDA法（特公平 7— 114718号 公報）、自立複製法、 NASBA法（日本国特許第 2650159号公報）、 TMA法、 Q β レプリカーゼ法（日本国特許第 2710159号公報）、国際公開第 2004Z040019号 パンフレットに記載の方法等が挙げられる。  [0024] As a nucleic acid amplification method, any method known to those skilled in the art may be used, but preferably the target nucleic acid can be amplified by a reaction under isothermal conditions. Examples of such isothermal nucleic acid amplification methods include the LAMP method (International Publication No. 00Z28082 pamphlet), the I CAN method (International Publication No. 02Z16639 pamphlet), the SDA method (Japanese Patent Publication No. 7-114718), and the autonomous replication method. , NASBA method (Japanese Patent No. 2650159), TMA method, Qβ replicase method (Japanese Patent No. 2710159), International Publication No. 2004Z040019, etc.
[0025] ここで、「等温」とは、酵素およびプライマーが実質的に機能しうるような、ほぼ一定 の温度条件下に保つことをいう。さらに、「ほぼ一定の温度条件」とは、設定された温 度を正確に保持することのみならず、酵素およびプライマーの実質的な機能を損な わな 、程度の温度変化であれば許容されることを意味する。一定の温度条件下での 核酸増幅反応は、使用する酵素の活性を維持できる温度に保つことにより実施する ことができる。また、この核酸増幅反応において、プライマーが標的核酸にァユーリン グするためには、例えば、反応温度を、そのプライマーの融解温度 (Tm)付近の温 度、もしくはそれ以下に設定することが好ましぐさらには、プライマーの融解温度 (T m)を考慮し、ストリンジエンシーのレベルを設定することが好ましい。従って、この温 度は、好ましくは、約 20°C〜約 75°Cであり、さらに好ましくは、約 35°C〜約 65°Cとす る。  [0025] Here, "isothermal" refers to maintaining an approximately constant temperature condition such that the enzyme and the primer can substantially function. Furthermore, the “substantially constant temperature condition” is not only to maintain the set temperature accurately, but is allowed as long as the temperature changes to the extent that the substantial function of the enzyme and the primer is not impaired. Means that. The nucleic acid amplification reaction under a certain temperature condition can be carried out by keeping the temperature at which the activity of the enzyme used can be maintained. In this nucleic acid amplification reaction, in order for the primer to ring the target nucleic acid, for example, the reaction temperature is preferably set to a temperature near the melting temperature (Tm) of the primer or lower. Furthermore, it is preferable to set the stringency level in consideration of the melting temperature (Tm) of the primer. Therefore, this temperature is preferably about 20 ° C to about 75 ° C, and more preferably about 35 ° C to about 65 ° C.
[0026] 本発明の好ま 、実施態様によれば、等温核酸増幅法には、標的核酸配列を増 幅しうるプライマーセットであって、前記プライマーセットに含まれる第一のプライマー 力 標的核酸配列の 3'末端部分の配列 (A)にハイブリダィズする配列 (Ac')を 3 '末 端部分に含んでなり、かつ前記標的核酸配列において前記配列 (A)よりも 5 '側に存 在する配列（B)の相補配列（Be)にハイブリダィズする配列（Β')を前記配列 (Ac )の 5 '側に含んでなるものである前記プライマーセットが用いられる。 [0026] According to a preferred embodiment of the present invention, the isothermal nucleic acid amplification method comprises a primer set capable of amplifying the target nucleic acid sequence, wherein the first primer force contained in the primer set comprises the target nucleic acid sequence. A sequence (Ac ′) that hybridizes to the sequence (A) at the 3 ′ end portion is included at the 3 ′ end portion, and is a sequence that is present 5 ′ from the sequence (A) in the target nucleic acid sequence ( The sequence (Β ′) that hybridizes to the complementary sequence (Be) of B) is the 5 of the sequence (Ac). The primer set comprising the 'side is used.
[0027] 本発明の特に好ましい実施態様によれば、等温核酸増幅法として、本発明者ら〖こ よって開発された核酸増幅法が用いられる。該方法では、標的核酸配列を増幅しうる 少なくとも 2種のプライマーを含んでなるプライマーセットであって、前記プライマーセ ットに含まれる第一のプライマーが、標的核酸配列の 3 '末端部分の配列 (A)にハイ ブリダィズする配列 (Ac )を 3 '末端部分に含んでなり、かつ前記標的核酸配列にお V、て前記配列 (A)よりも 5 '側に存在する配列（B)の相補配列（Be)にハイブリダィズ する配列（Β')を前記配列 (Ac')の 5 '側に含んでなるものであり、前記プライマーセッ トに含まれる第二のプライマーが、前記標的核酸配列の相補配列の 3 '末端部分の 配列（C)にハイブリダィズする配列（Cc')を 3 '末端部分に含んでなり、かつ相互にハ イブリダィズする 2つの核酸配列を同一鎖上に含む折返し配列（D-Dc')を前記配列（ Cc')の 5 '側に含んでなるものである前記プライマーセットが用いられる。  [0027] According to a particularly preferred embodiment of the present invention, the nucleic acid amplification method developed by the present inventors is used as the isothermal nucleic acid amplification method. In the method, a primer set comprising at least two kinds of primers capable of amplifying a target nucleic acid sequence, wherein the first primer included in the primer set is a sequence of the 3 ′ end portion of the target nucleic acid sequence. Complementary of sequence (B) comprising 3 'terminal portion of hybridizing sequence (Ac) in (A) and existing in the target nucleic acid sequence V and 5' to sequence (A). A sequence (Β ′) that hybridizes to the sequence (Be) is included on the 5 ′ side of the sequence (Ac ′), and the second primer included in the primer set is complementary to the target nucleic acid sequence. A folded sequence (D-) containing two nucleic acid sequences hybridizing to the sequence (Cc ') at the 3' end portion and hybridizing to each other on the same strand. Dc ') on the 5' side of the sequence (Cc ') Wherein the primer set is used.
[0028] 本発明にお 、て「ノヽイブリダィズする」とは、プライマーがストリンジェントな条件下で 標的核酸にハイブリダィズし、標的核酸以外の核酸分子にはハイブリダィズしな 、こ とを意味する。ストリンジェントな条件は、本発明によるプライマーとその相補鎖との二 重鎖の融解温度 Tm (°C)およびハイブリダィゼーシヨン溶液の塩濃度などに依存して 決定することができ、例えば、 J. Sambrook, E. F. Frisch, T. Maniatis; Molecular Clon ing 2nd edition, Cold Spring Harbor Laboratory (1989)等を参照することができる。例 えば、使用するプライマーの融解温度よりわずかに低!、温度下でハイブリダィゼーシ ヨンを行なうと、プライマーを標的核酸に特異的にハイブリダィズさせることができる。 このようなプライマーは、市販のプライマー構築ソフト、例えば、 Primer3 (Whitehead Institute for Biomedical Research社製）などを用いて設計することができる。本発明 の好ましい実施態様によれば、ある標的核酸にハイブリダィズするプライマーは、そ の標的核酸に相補的な核酸分子の全部または一部の配列を含んでなるものである。  In the present invention, “noblybize” means that the primer hybridizes to the target nucleic acid under stringent conditions and does not hybridize to nucleic acid molecules other than the target nucleic acid. Stringent conditions can be determined depending on the melting temperature Tm (° C) of the duplex of the primer according to the present invention and its complementary strand, the salt concentration of the hybridization solution, etc. Reference can be made to J. Sambrook, EF Frisch, T. Maniatis; Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory (1989). For example, when hybridization is performed at a temperature slightly lower than the melting temperature of the primer to be used, the primer can be specifically hybridized to the target nucleic acid. Such a primer can be designed using commercially available primer construction software such as Primer3 (manufactured by Whitehead Institute for Biomedical Research). According to a preferred embodiment of the present invention, a primer that hybridizes to a target nucleic acid comprises a sequence of all or part of a nucleic acid molecule complementary to the target nucleic acid.
[0029] 前記第一のプライマーによる核酸合成の作用機序を図 1に模式的に示す。まず、 铸型となる核酸中の標的核酸配列を決定し、その標的核酸配列の 3 '末端部分の配 列 (A)、および配列 (A)よりも 5 '側に存在する配列 (B)を決定する。第一のプライマ 一は、配列 (Ac')を含んでなり、さらにその 5 '側に配列（Β')を含んでなる。配列 (Ac') は、配列 (A)にハイブリダィズするものであり、配列（Β')は、配列（Β)の相補配列（Be )にハイブリダィズするものである。ここで、第一のプライマーは、前記配列 (Ac')と前 記配列（Β')の間に、反応に影響を与えない介在配列を含んでいてもよい。このような プライマーを铸型核酸にアニーリングさせると、プライマー中の配列 (Ac )が標的核 酸配列の配列 (A)にノ、イブリダィズした状態となる（図 1(a))。この状態でプライマー伸 長反応が起こると、標的核酸配列の相補配列を含む核酸が合成される。そして、合 成された核酸の 5'末端側に存在する配列 (Β')が、同核酸中に存在する配列 (Be)に ハイブリダィズし、これにより、合成された核酸の 5'末端部分においてステム—ルー プ構造が形成される。その結果、铸型核酸上の配列 (A)がー本鎖となり、この部分に 先の第一のプライマーと同一の配列を有する他のプライマーがハイブリダィズする（ 図 l(b))。その後、鎖置換反応により、新たにハイブリダィズした第一のプライマーか らの伸長反応が起こると同時に、先に合成された核酸が铸型核酸から分離される（図 1(c)) o [0029] Fig. 1 schematically shows the mechanism of nucleic acid synthesis by the first primer. First, determine the target nucleic acid sequence in the nucleic acid to be a cage, and determine the sequence (A) at the 3 'end of the target nucleic acid sequence (A) and the sequence (B) that is 5' to the sequence (A). decide. The first primer comprises the sequence (Ac ') and further comprises the sequence (Β') on the 5 'side. Array (Ac ') Is hybridized to the sequence (A), and the sequence (Β ′) is hybridized to the complementary sequence (Be) of the sequence (Β). Here, the first primer may contain an intervening sequence that does not affect the reaction between the sequence (Ac ′) and the sequence (Β ′). When such a primer is annealed to a vertical nucleic acid, the sequence (Ac) in the primer is nobbreviated to the sequence (A) of the target nucleic acid sequence (FIG. 1 (a)). When a primer extension reaction occurs in this state, a nucleic acid containing a complementary sequence of the target nucleic acid sequence is synthesized. Then, the sequence (Β ′) present on the 5 ′ end side of the synthesized nucleic acid hybridizes to the sequence (Be) present in the nucleic acid, and thereby the stem in the 5 ′ end portion of the synthesized nucleic acid. —A loop structure is formed. As a result, the sequence (A) on the vertical nucleic acid becomes a single strand, and another primer having the same sequence as the first primer hybridizes to this part (FIG. 1 (b)). After that, the strand displacement reaction causes an extension reaction from the newly hybridized first primer, and at the same time, the previously synthesized nucleic acid is separated from the vertical nucleic acid (FIG. 1 (c)).
[0030] 上記の作用機序にお!、て、配列（Β')が配列（Be)にハイブリダィズする現象は、典 型的には、同一鎖上に相補領域が存在することにより起こる。一般に、二本鎖核酸が 一本鎖に解離するときは、その末端あるいはそれ以外の比較的不安定な部分力 部 分的な解離が始まる。上記第一のプライマーによる伸長反応で生成した二本鎖核酸 は、比較的高温では末端部分の塩基対は解離と結合の平衡状態にあり、全体として は二本鎖を保っている。そのような状態で末端の解離した部分に相補的な配列が同 一鎖上に存在すると、準安定な状態としてステム ループ構造を形成することができ る。このステムループ構造は安定的には存在しないが、その構造の形成により剥き出 しとなつた相補鎖部分 (铸型核酸上の配列 (A) )に同一の他のプライマーが結合し、 すぐさまポリメラーゼが伸長反応を行うことにより、先に合成された鎖が置換されて遊 離すると同時に、新たな二本鎖核酸を生成することができる。  [0030] In the above mechanism of action, the phenomenon that the sequence (Β ') hybridizes to the sequence (Be) is typically caused by the presence of complementary regions on the same strand. In general, when a double-stranded nucleic acid is dissociated into a single strand, the dissociation of a partial force or a part of the other end that is relatively unstable begins. In the double-stranded nucleic acid generated by the extension reaction using the first primer, the base pair of the terminal portion is in an equilibrium state of dissociation and binding at a relatively high temperature, and the double strand is maintained as a whole. In such a state, if a sequence complementary to the dissociated portion of the terminal is present on the same strand, a stem-loop structure can be formed as a metastable state. Although this stem-loop structure does not exist stably, the same other primer binds to the complementary strand portion (sequence (A) on the vertical nucleic acid) that was exposed due to the formation of the structure, and the polymerase immediately By performing an extension reaction, the previously synthesized strand is displaced and released, and at the same time, a new double-stranded nucleic acid can be generated.
[0031] 本発明の好ましい実施態様における第一のプライマーの設計基準は次のとおりで ある。まず、プライマーの伸長により铸型核酸の相補鎖が合成された後に新たなブラ イマ一が効率よく同铸型核酸にアニーリングするためには、合成された相補鎖の 5 ' 末端におけるステム ループ構造形成により、铸型核酸上の前記配列 (A)の部分を 一本鎖とする必要がある。そのためには、配列 (Ac')の塩基数 Xと標的核酸配列中に おける前記配列 (A)と前記配列 (B)に挟まれた領域の塩基数 Yとの差 (X— Y)の、 X に対する割合 (X—Y) ZXが重要となる。ただし、铸型核酸上において配列 (Α)よりも 5'側に存在する、プライマーのハイブリダィズとは関係無い部分まで一本鎖とする必 要はない。また、新たなプライマーが効率よく铸型核酸にアニーリングするためには、 上述のステム—ループ構造形成を効率よく行なうことも必要となる。そして、効率の良 V、ステム ループ構造形成、すなわち、効率の良!、配列（Β')と配列（Be)とのハイブ リダィゼーシヨンには、前記配列（Β')と前記配列（Be)との間の距離 (X+Y)が重要と なる。一般に、プライマー伸長反応のための最適温度は最高でも 72°C付近であり、 そのような低い温度では、伸長鎖が長い領域にわたって解離することは困難である。 従って、配列（Β')が配列（Be)に効率よくハイブリダィズするためには、両配列の間の 塩基数は少ないほうが好ましいと考えられる。一方で、配列（Β')が配列（Be)にハイブ リダィズして铸型核酸上の前記配列 (A)の部分を一本鎖とするためには、配列（Β')と 配列（Be)との間の塩基数は多い方が好ましいと考えられる。 [0031] The design criteria for the first primer in a preferred embodiment of the present invention are as follows. First, in order for a new primer to efficiently anneal to the homologous nucleic acid after the complementary strand of the truncated nucleic acid is synthesized by extension of the primer, a stem loop structure is formed at the 5 'end of the synthesized complementary strand. The part of the sequence (A) on the vertical nucleic acid is Must be single stranded. For this purpose, the difference (X−Y) between the base number X of the sequence (Ac ′) and the base number Y of the region sandwiched between the sequence (A) and the sequence (B) in the target nucleic acid sequence, Ratio to X (X—Y) ZX is important. However, it is not necessary to make a single strand up to the part unrelated to the hybridization of the primer, which is present 5 ′ from the sequence (配 列) on the vertical nucleic acid. In addition, in order for a new primer to efficiently anneal to a vertical nucleic acid, it is necessary to efficiently perform the above-described stem-loop structure formation. In addition, in the hybridization between the array (Β ′) and the array (Be), the efficiency V, the formation of the stem loop structure, that is, the efficiency! The distance between (X + Y) is important. In general, the optimum temperature for the primer extension reaction is at most around 72 ° C, and at such a low temperature, it is difficult for the extended strand to dissociate over a long region. Therefore, in order for the sequence (Β ′) to hybridize efficiently to the sequence (Be), it is considered preferable that the number of bases between both sequences is small. On the other hand, in order for the sequence (Β ′) to hybridize to the sequence (Be) and the portion of the sequence (A) on the cage nucleic acid to be a single strand, the sequence (Β ′) and the sequence (Be) It is considered preferable that the number of bases between and is larger.
[0032] 以上のような観点から、本発明の好ましい実施態様による前記第一のプライマーは 、プライマーを構成する配列 (Ac )と配列 (Β')の間に介在配列が存在しない場合に おいて、（X— Υ) ΖΧが一 1. 00以上、好ましくは 0. 00以上、さらに好ましくは 0. 05 以上、さらに好ましくは 0. 10以上となり、また、 1. 00以下、好ましくは 0. 75以下、さ らに好ましくは 0. 50以下、さらに好ましくは 0. 25以下となるように設計される。さらに 、（Χ+Υ)は、好ましくは 15以上、さらに好ましくは 20以上、さらに好ましくは 30以上 とされ、また、好ましくは 50以下、さらに好ましくは 48以下、さらに好ましくは 42以下と される。 [0032] From the above viewpoint, the first primer according to a preferred embodiment of the present invention is the case where there is no intervening sequence between the sequence (Ac) and the sequence (Β ') constituting the primer. , (X—Υ) ΖΧ is 1.00 or more, preferably 0.00 or more, more preferably 0.05 or more, more preferably 0.10 or more, and 1.00 or less, preferably 0.75. In the following, it is designed to be more preferably 0.50 or less, and still more preferably 0.25 or less. Further, (Χ + Υ) is preferably 15 or more, more preferably 20 or more, more preferably 30 or more, and is preferably 50 or less, more preferably 48 or less, and even more preferably 42 or less.
[0033] また、プライマーを構成する配列 (Ac )と配列 (Β')の間に介在配列 (塩基数は Y' ) が存在する場合には、本発明の好ましい実施態様による前記第一のプライマーは、 { X— (Υ— Υ' ΜΖΧが一 1. 00以上、好ましくは 0. 00以上、さらに好ましくは 0. 05以 上、さらに好ましくは 0. 10以上となり、また、 1. 00以下、好ましくは 0. 75以下、さら に好ましくは 0. 50以下、さらに好ましくは 0. 25以下となるように設計される。さらに、 (Χ+Υ+Υ' )は、好ましくは 15以上、さらに好ましくは 20以上、さらに好ましくは 30 以上とされ、また、好ましくは 100以下、さらに好ましくは 75以下、さらに好ましくは 50 以下とされる。 [0033] When an intervening sequence (the number of bases is Y ') exists between the sequence (Ac) and the sequence (Β') constituting the primer, the first primer according to a preferred embodiment of the present invention {X— (Υ—Υ′ΜΖΧ is 1 1.00 or more, preferably 0.00 or more, more preferably 0.05 or more, more preferably 0.10 or more, and 1.00 or less, It is preferably designed to be 0.75 or less, more preferably 0.50 or less, and even more preferably 0.25 or less, and (。 + Υ + Υ ′) is preferably 15 or more, more preferably Is 20 or more, more preferably 30 Also, it is preferably 100 or less, more preferably 75 or less, and still more preferably 50 or less.
[0034] 前記第一のプライマーは、与えられた条件下で必要な特異性を維持しながら標的 核酸との塩基対結合を行うことができる程度の鎖長を有するものである。このプライマ 一の鎖長は、好ましくは 15〜100ヌクレオチド、より好ましくは 20〜60ヌクレオチドと する。また、前記第一のプライマーを構成する配列 (Ac')と配列 (Β')の長さは、それ ぞれ、好ましくは 5〜50ヌクレオチド、より好ましくは 7〜30ヌクレオチドである。また、 必要に応じて、配列 (Ac')と配列 (Β')の間に、反応に影響を与えない介在配列を挿 人してちょい。  [0034] The first primer has a chain length such that base pairing with a target nucleic acid can be carried out while maintaining necessary specificity under given conditions. The chain length of this primer is preferably 15 to 100 nucleotides, more preferably 20 to 60 nucleotides. The lengths of the sequence (Ac ′) and the sequence (Β ′) constituting the first primer are each preferably 5 to 50 nucleotides, more preferably 7 to 30 nucleotides. If necessary, insert an intervening sequence that does not affect the reaction between sequence (Ac ') and sequence (Β').
[0035] 前記第二のプライマーは、上述のように、前記標的核酸配列の相補配列 (第一の プライマーがハイブリダィズする鎖に対して反対側の鎖)の 3'末端部分の配列 (C)に ノ、イブリダィズする配列（Cc )を 3'末端部分に含んでなり、かつ相互にハイブリダィ ズする 2つの核酸配列を同一鎖上に含む折返し配列（D-Dc )を前記配列（Cc )の 5' 側に含んでなるものである。このような第二のプライマーの構造は、例えば、図 2に示 すようなものであるが、図 2に示される配列やヌクレオチド数に限定されるものではな い。第二のプライマーを構成する配列（Cc')の長さは、好ましくは 5〜50ヌクレオチド 、より好ましくは 10〜30ヌクレオチドである。また、前記折返し配列（D-Dc')の長さは 、好ましくは 2〜 1000ヌクレオチド、より好ましくは 2〜100ヌクレオチド、さらに好まし くは 4〜60ヌクレオチド、さらに好ましくは 6〜40ヌクレオチドであり、折返し配列の内 部におけるハイブリダィゼーシヨンによって形成される塩基対のヌクレオチド数は、好 ましく ίま 2〜500bp、より好ましく ίま 2〜50bp、さら【こ好ましく ίま 2〜30bp、さら【こ好ま しくは 3〜20bpである。折返し配列（D-Dc )のヌクレオチド配列は!、かなる配列であ つてもよく、特に限定されるものではないが、好ましくは標的核酸配列にハイブリダィ ズしない配列とされる。また、必要に応じて、配列（Cc')と折返し配列（D-Dc')の間に 、反応に影響を与えな ヽ介在配列を挿入してもよ ヽ。  [0035] As described above, the second primer is added to the sequence (C) of the 3 'end portion of the complementary sequence of the target nucleic acid sequence (the strand opposite to the strand to which the first primer hybridizes). A folded sequence (D-Dc) comprising two nucleic acid sequences hybridizing to each other on the same strand, comprising a sequence (Cc) to be hybridized (Cc) at the 3 ′ end. On the side. The structure of such a second primer is, for example, as shown in FIG. 2, but is not limited to the sequence and the number of nucleotides shown in FIG. The length of the sequence (Cc ′) constituting the second primer is preferably 5 to 50 nucleotides, more preferably 10 to 30 nucleotides. The length of the folded sequence (D-Dc ′) is preferably 2 to 1000 nucleotides, more preferably 2 to 100 nucleotides, further preferably 4 to 60 nucleotides, and further preferably 6 to 40 nucleotides. The number of nucleotides in the base pair formed by hybridization within the folded sequence is preferably 2 to 500 bp, more preferably 2 to 50 bp, more preferably 2 to 30 bp, more preferably 2 to 30 bp. Furthermore, it is preferably 3 to 20 bp. The nucleotide sequence of the folded sequence (D-Dc) may be! Or any sequence, and is not particularly limited, but is preferably a sequence that does not hybridize to the target nucleic acid sequence. If necessary, an intervening sequence that does not affect the reaction may be inserted between the sequence (Cc ′) and the folded sequence (D-Dc ′).
[0036] これら第一のプライマーおよび第二のプライマーによる核酸増幅反応について考え られる作用機序を、図 3 (図 3aおよび図 3b)を用いて説明する。なお、図 3では、説明 を簡略化するため、ハイブリダィズする 2つの配列を相互に相補的な配列として ヽる iS これにより本発明が限定されるものではない。まず、第一のプライマーが標的核 酸のセンス鎖にハイブリダィズし、該プライマーの伸長反応が起きる（図 3(a))。次い で、伸長鎖（一）上においてステム ループ構造が形成され、これにより一本鎖となつ た標的核酸センス鎖上の配列 (A)に新たな第一のプライマーがハイブリダィズし（図 3(b))、該プライマーの伸長反応が起きて、先に合成された伸長鎖（一）が脱離する。 次に、脱離した伸長鎖（-)上の配列 (C)に第二のプライマーがハイブリダィズし（図 3(c))、該プライマーの伸長反応が起き、伸長鎖（+ )が合成される（図 3(d))。生成し た伸長鎖（ + )の 3 '末端と伸長鎖（一）の 5 '末端ではステム ループ構造が形成され (図 3(e))、遊離型の 3 '末端である伸長鎖（+ )のループ先端力も伸長反応が起こると 同時に、前記伸長鎖（一）が脱離する（図 3(1))。ループ先端力 の前記伸長反応によ り、伸長鎖（ + )の 3 '側に配列 (A)および配列 (Be)を介して伸長鎖（ ）が結合した ヘアピン型の二本鎖核酸が生成し、その配列 (A)および配列（Be)に第一のプライマ 一がハイブリダィズし（図 3(g))、その伸長反応により伸長鎖（-)が生成する（図 3(h) および (0)。また、前記ヘアピン型二本鎖核酸の 3 '末端に存在する折返し配列によ つて遊離型の 3 '末端が提供され (図 3(h))、そこからの伸長反応により（図 3(0)、両端 に折返し配列を有し、第一および第二のプライマーに由来する配列を介して伸長鎖 ( + )と伸長鎖（一）とを交互に含む一本鎖核酸が生成する（図 3(j))。この一本鎖核酸 では、その 3 '末端に存在する折返し配列により遊離型の 3 '末端 (相補鎖合成起点） が提供されるため（図 3(k))、同様の伸長反応が繰り返され、 1回の伸長反応あたり 2 倍の鎖長となる（図 3(1)および (m))。また、図 3(0において脱離した第一のプライマー からの伸長鎖（-)では、その 3 '末端に存在する折返し配列により遊離型の 3 '末端（ 相補鎖合成起点）が提供されるため（図 3(n))、そこ力もの伸長反応により、両端にス テム—ループ構造が形成され、プライマーに由来する配列を介して伸長鎖（ + )と伸 長鎖（一）とを交互に含む一本鎖核酸が生成する（図 3(o))。この一本鎖核酸におい ても、 3 '末端におけるループ形成によって相補鎖合成起点が順次提供されるため、 そこからの伸長反応が次々に起こる。このようにして自動的に延長される一本鎖核酸 には、第一のプライマーおよび第二のプライマーに由来する配列が伸長鎖（+ )と伸 長鎖（-)との間に含まれているため、各プライマーがハイブリダィズして伸長反応を 起こすことが可能であり、これにより標的核酸のセンス鎖およびアンチセンス鎖が顕 著に増幅される。 [0036] A possible mechanism of action of the nucleic acid amplification reaction by the first primer and the second primer will be described with reference to Fig. 3 (Figs. 3a and 3b). In FIG. 3, in order to simplify the description, two sequences to be hybridized are referred to as complementary sequences. iS This does not limit the invention. First, the first primer hybridizes to the sense strand of the target nucleic acid, and the primer extension reaction occurs (FIG. 3 (a)). Next, a stem-loop structure is formed on the extended strand (one), and the new first primer hybridizes to the sequence (A) on the target nucleic acid sense strand that has become a single strand (Fig. 3 ( b)), the extension reaction of the primer occurs, and the previously synthesized extension strand (one) is released. Next, the second primer hybridizes to the sequence (C) on the released extended strand (-) (Fig. 3 (c)), and the extension reaction of the primer occurs, and the extended strand (+) is synthesized. (Figure 3 (d)). A stem-loop structure is formed between the 3 'end of the generated extended strand (+) and the 5' end of the extended strand (one) (Fig. 3 (e)), and the extended strand (+) that is the free 3 'end The extension force (1) is released at the same time as the elongation at the end of the loop occurs (Fig. 3 (1)). By the extension reaction of the loop tip force, a hairpin-type double-stranded nucleic acid in which the extension strand () is bound to the 3 ′ side of the extension strand (+) via the sequence (A) and the sequence (Be) is generated. The first primer hybridizes to the sequence (A) and sequence (Be) (Fig. 3 (g)), and the extension chain (-) is generated by the extension reaction (Figs. 3 (h) and (0)). In addition, the folded sequence present at the 3 ′ end of the hairpin double-stranded nucleic acid provides a free 3 ′ end (FIG. 3 (h)), and an extension reaction therefrom (FIG. 3 (0 ), A single-stranded nucleic acid having a folded sequence at both ends and containing an extended strand (+) and an extended strand (one) alternately through the sequences derived from the first and second primers (FIG. 3). (j)) In this single-stranded nucleic acid, the folded sequence present at the 3 'end provides a free 3' end (starting point of complementary strand synthesis) (Fig. 3 (k)). Anti The chain length is doubled per extension reaction (Fig. 3 (1) and (m)), and the extended strand from the first primer released in Fig. 3 (- ) Provides a free 3 'end (the complementary strand synthesis origin) due to the folding sequence present at the 3' end (Fig. 3 (n)). A loop structure is formed, producing a single-stranded nucleic acid containing an extended strand (+) and an extended strand (one) alternately via a sequence derived from the primer (Figure 3 (o)). In the case of nucleic acids as well, since the complementary strand synthesis origin is sequentially provided by the loop formation at the 3 ′ end, extension reactions occur one after another. Sequences derived from the first and second primers are included between the extended strand (+) and the extended strand (-). Because, each primer extension reaction was Haiburidizu This can significantly amplify the sense and antisense strands of the target nucleic acid.
[0037] 前記プライマーセットは、前記標的核酸配列またはその相補配列にハイブリダィズ する第三のプライマーであって、標的核酸配列またはその相補配列へのハイブリダィ ゼーシヨンについて他のプライマーと競合しない第三のプライマーをさらに含んでな るちのとしてちょい。  [0037] The primer set is a third primer that hybridizes to the target nucleic acid sequence or its complementary sequence, and a third primer that does not compete with other primers for hybridization to the target nucleic acid sequence or its complementary sequence. In addition, as a way to include.
[0038] 本発明にお 、て「競合しな!、」とは、そのプライマーが標的核酸にハイブリダィズす ることによって他のプライマーによる相補鎖合成起点の付与が妨げられないことを意 味する。  In the present invention, “do not compete!” Means that the primer does not interfere with the provision of the complementary strand synthesis starting point by hybridizing to the target nucleic acid.
[0039] 第一のプライマーおよび第二のプライマーにより標的核酸が増幅された場合には、 上述のように、増幅産物は標的核酸配列とその相補配列とを交互に有するものとなる 。その増幅産物の 3'末端には折返し配列またはループ構造が存在し、これにより提 供される相補鎖合成起点から次々に伸長反応が起こっている。第三のプライマーは 、このような増幅産物が部分的に一本鎖の状態になった時に、その一本鎖部分に存 在する標的配列にアニーリングすることができる。これにより、増幅産物中の標的核 酸配列内に新たな相補鎖合成起点が提供され、そこからの伸長反応が起こるため、 核酸増幅反応がより迅速に行われるようになる。  [0039] When the target nucleic acid is amplified by the first primer and the second primer, as described above, the amplification product has the target nucleic acid sequence and its complementary sequence alternately. There is a folded sequence or loop structure at the 3 ′ end of the amplified product, and extension reactions occur one after another from the complementary strand synthesis starting point provided. The third primer can anneal to the target sequence present in the single-stranded part when such an amplification product is partially in a single-stranded state. As a result, a new complementary strand synthesis origin is provided in the target nucleic acid sequence in the amplification product, and an extension reaction takes place therefrom, so that the nucleic acid amplification reaction is performed more rapidly.
[0040] 第三のプライマーは必ずしも 1種類に限定されるわけではなぐ核酸増幅反応の迅 速性および特異性を向上させるためには 2種類以上の第三のプライマーを同時に用 いてもよい。これら第三のプライマーは、典型的には第一のプライマーおよび第二の プライマーとは異なる配列力 なる力 これらのプライマーと競合しない限りにおいて 、部分的に重なる領域にハイブリダィズするものとしてもよい。第三のプライマーの鎖 長は、好ましくは 2〜： LOOヌクレオチド、より好ましくは 5〜50ヌクレオチド、さらに好ま しくは 7〜30ヌクレオチドとされる。  [0040] The third primer is not necessarily limited to one type, but two or more types of the third primer may be used simultaneously in order to improve the speed and specificity of the nucleic acid amplification reaction. These third primers typically have different sequence powers than the first primer and the second primer, and may hybridize to a partially overlapping region as long as they do not compete with these primers. The chain length of the third primer is preferably 2 to: LOO nucleotides, more preferably 5 to 50 nucleotides, and even more preferably 7 to 30 nucleotides.
[0041] 第三のプライマーは、第一のプライマーおよび第二のプライマーによる核酸増幅反 応をより迅速に進めるための補助的な働きをその主目的とするものである。従って、 第三のプライマーは、第一のプライマーおよび第二のプライマーの各 3，末端の Tmよ りも低い Tmを有するものとすることが好ましい。また、第三のプライマーの増幅反応 液への添加量は、第一のプライマーおよび第二のプライマーのそれぞれの添加量よ りも少ない方が好ましい。 [0041] The third primer mainly has an auxiliary function for proceeding more rapidly with the nucleic acid amplification reaction by the first primer and the second primer. Accordingly, it is preferable that the third primer has a Tm lower than the Tm of the third and third ends of the first primer and the second primer, respectively. The amplification reaction of the third primer The amount added to the solution is preferably smaller than the amount added to each of the first primer and the second primer.
[0042] 前記プライマーセットを利用して遺伝子変異の有無を判定するためには、変異部位 が前記配列 (A)、前記配列（B)または前記配列（C)に含まれるようにプライマーセット を設計することができる。これにより、増幅産物の有無を確認することによって前記変 異の有無を判定することが可能となり、また、増幅産物を定量することにより前記変異 を有する遺伝子の量を決定することが可能となる。  [0042] In order to determine the presence or absence of a gene mutation using the primer set, the primer set is designed so that the mutation site is included in the sequence (A), the sequence (B), or the sequence (C). can do. Accordingly, it is possible to determine the presence or absence of the mutation by confirming the presence or absence of the amplification product, and it is possible to determine the amount of the gene having the mutation by quantifying the amplification product.
[0043] 本発明の一つの実施態様によれば、前記プライマーセットは、変異に係るヌクレオ チド残基が前記配列 (A)に含まれるように設計される。この実施態様では、核酸試料 中に標的核酸配列が含まれている場合には、核酸増幅反応において第一のプライ マーが配列 (A)にアニーリングするため、増幅産物が得られる。核酸試料中に、変異 部位にぉ 、て標的核酸配列とは異なる核酸配列が含まれて 、る場合には、核酸増 幅反応において第一のプライマーが配列 (A)にアニーリングすることが困難となるた め、増幅産物が得られないか、または得られる増幅産物の量が著しく減少する。変異 に係るヌクレオチド残基は、好ましくは前記配列 (A)の 5 '末端 (第一のプライマーに おける 3'末端に対応する）に含まれるものとされる。また、第一のプライマーに含まれ る配列 (Ac')は、前記配列 (A)に相補的な配列とすることが好ましい。  [0043] According to one embodiment of the present invention, the primer set is designed such that a nucleotide residue related to mutation is included in the sequence (A). In this embodiment, when the target nucleic acid sequence is contained in the nucleic acid sample, the first primer is annealed to the sequence (A) in the nucleic acid amplification reaction, so that an amplification product is obtained. If the nucleic acid sample contains a nucleic acid sequence that is different from the target nucleic acid sequence at the mutation site, it is difficult for the first primer to anneal to the sequence (A) in the nucleic acid amplification reaction. Therefore, no amplification product is obtained or the amount of amplification product obtained is significantly reduced. The nucleotide residue involved in the mutation is preferably included in the 5 ′ end of the sequence (A) (corresponding to the 3 ′ end in the first primer). The sequence (Ac ′) contained in the first primer is preferably a sequence complementary to the sequence (A).
[0044] 本発明の他の実施態様によれば、前記プライマーセットは、変異に係るヌクレオチ ド残基が前記配列 (C)に含まれるように設計される。この実施態様では、核酸試料中 に標的核酸配列が含まれている場合には、核酸増幅反応において第二のプライマ 一が配列 (C)にアニーリングするため、増幅産物が得られる。核酸試料中に、変異部 位にお 、て標的核酸配列とは異なる核酸配列が含まれて 、る場合には、核酸増幅 反応において第二のプライマーが配列（C)にアニーリングすることが困難となるため 、増幅産物が得られないか、または得られる増幅産物の量が著しく減少する。変異に 係るヌクレオチド残基は、好ましくは前記配列 (C)の 5 '末端 (第二のプライマーにお ける 3'末端に対応する）に含まれるものとされる。また、第二のプライマーに含まれる 配列（Cc')は、前記配列（C)に相補的な配列とすることが好ましい。  [0044] According to another embodiment of the present invention, the primer set is designed such that a nucleotide residue related to mutation is included in the sequence (C). In this embodiment, when the target nucleic acid sequence is contained in the nucleic acid sample, the amplification product is obtained because the second primer anneals to the sequence (C) in the nucleic acid amplification reaction. If the nucleic acid sample contains a nucleic acid sequence that is different from the target nucleic acid sequence at the mutation site, it is difficult for the second primer to anneal to the sequence (C) in the nucleic acid amplification reaction. Therefore, no amplification product is obtained or the amount of amplification product obtained is significantly reduced. The nucleotide residue involved in the mutation is preferably contained in the 5 ′ end of the sequence (C) (corresponding to the 3 ′ end in the second primer). The sequence (Cc ′) contained in the second primer is preferably a sequence complementary to the sequence (C).
[0045] 本発明の他の実施態様によれば、前記プライマーセットは、変異に係るヌクレオチ ド残基が前記配列 (B)に含まれるように設計される。この実施態様では、核酸試料中 に標的核酸配列が含まれている場合には、核酸増幅反応において、第一のプライマ 一が配列 (A)にアニーリングして伸長反応が行なわれた後に該プライマーに含まれ る配列（Β')が伸長鎖上の配列（Be)にハイブリダィズするため、ステム ループ構造 が効率的に形成される。この効率的なステム ループ構造の形成により、他の第一 のプライマーが铸型にアニーリングすることが可能となり、図 1に示される作用機序が 効率的に進行するため、増幅産物が得られる。一方で、核酸試料中に、変異部位に ぉ 、て標的核酸配列とは異なる核酸配列が含まれて 、る場合には、核酸増幅反応 における上記ステム—ループ構造の形成が困難となるため、図 1に示される作用機 序が妨げられ、増幅産物が得られないか、または得られる増幅産物の量が著しく減 少する。第一のプライマーに含まれる配列 (Β')は、前記配列 (Β)と同一の配列とする ことが好ましい。 [0045] According to another embodiment of the present invention, the primer set may comprise a mutation-related nucleotide. It is designed such that a residue is included in the sequence (B). In this embodiment, when the target nucleic acid sequence is contained in the nucleic acid sample, in the nucleic acid amplification reaction, the first primer is annealed to the sequence (A) and the extension reaction is performed. Since the contained sequence (Β ') hybridizes to the sequence (Be) on the extended strand, a stem-loop structure is efficiently formed. The formation of this efficient stem loop structure allows the other first primer to anneal in a saddle shape, and the mechanism of action shown in FIG. 1 proceeds efficiently, resulting in an amplification product. On the other hand, if the nucleic acid sample contains a nucleic acid sequence that is different from the target nucleic acid sequence at the mutation site, it is difficult to form the stem-loop structure in the nucleic acid amplification reaction. The mechanism of action shown in 1 is hindered and no amplification product is obtained, or the amount of amplification product obtained is significantly reduced. The sequence (Β ') contained in the first primer is preferably the same sequence as the sequence (Β).
[0046] 上述の配列（Β)における変異の検出について、さらに詳細に説明する。図 1に示す 作用機序において、配列（Β')が配列（Be)にハイブリダィズする現象は、同一鎖上に 相補領域が存在することにより起こる。一般に、二本鎖核酸が一本鎖に解離するとき は、その末端あるいはそれ以外の比較的不安定な部分力 部分的な解離が始まる。 第一のプライマーによる伸長反応によって生成した二本鎖核酸は、比較的高温では 末端部分の塩基対は解離と結合の平衡状態にあり、全体としては二本鎖を保ってい る。そのような状態で末端の解離した部分に相補的な配列が同一鎖上に存在すると 、準安定な状態としてステム一ループ構造を形成することができる。しかし、このステ ムーループ構造は安定的には存在せず、特に、そのステムを形成する配列（Β')と配 列（Be)部分との間に相補的でないヌクレオチドが存在する場合には、非常に不安定 となり、あるいは、ステムが全く形成されない。この場合には、铸型上の配列 (A)とブラ イマ一中の配列（Ac')とのハイブリダィゼーシヨンの方が優位となり、配列（A)の部分 がー本鎖とならないため、次の第一のプライマーがアニーリングできなくなる。そのた め、図 1に示される連続した反応を起こすことが極めて困難となる。  [0046] The detection of the mutation in the above-described sequence (Β) will be described in more detail. In the mechanism of action shown in FIG. 1, the phenomenon that the sequence (Β ′) hybridizes to the sequence (Be) occurs due to the presence of complementary regions on the same strand. In general, when a double-stranded nucleic acid is dissociated into single strands, partial dissociation starts at its end or other relatively unstable partial force. In the double-stranded nucleic acid produced by the extension reaction with the first primer, the base pair of the terminal portion is in an equilibrium state of dissociation and binding at a relatively high temperature, and the double strand is maintained as a whole. In such a state, when a sequence complementary to the dissociated portion of the terminal is present on the same strand, a stem-and-loop structure can be formed as a metastable state. However, this stem loop structure does not exist stably, especially when there are non-complementary nucleotides between the sequence (Β ') and the sequence (Be) part that form the stem. Or the stem is not formed at all. In this case, the hybridization between the vertical sequence (A) and the sequence (Ac ') in the primer is more dominant, and the sequence (A) is not a single strand. The next first primer cannot be annealed. Therefore, it is extremely difficult to cause the continuous reaction shown in Fig. 1.
[0047] さらに、前記プライマーセットを利用して核酸配列における配列の欠失または挿入 の有無を検出するためには、欠失または挿入に係る部位力 配列 (A)、配列（B)もし くは配列（C)に含まれる力、または配列 (A)と配列（B)との間に配置されるようにブラ イマ一セットを設計することができる。これにより、増幅産物の有無を確認することによ つて配列の欠失または挿入の有無を判定することが可能となり、また、増幅産物を定 量することにより前記欠失または挿入を有する遺伝子の量を決定することが可能とな る。 [0047] Further, in order to detect the presence or absence of sequence deletion or insertion in the nucleic acid sequence using the primer set, the site force sequence (A) or sequence (B) related to the deletion or insertion Alternatively, a set of primers can be designed to be placed between the force contained in sequence (C) or between sequence (A) and sequence (B). This makes it possible to determine the presence or absence of a sequence deletion or insertion by confirming the presence or absence of an amplification product, and the amount of a gene having the deletion or insertion by quantifying the amplification product. Can be determined.
[0048] 本発明の一つの実施態様によれば、前記プライマーセットは、欠失または挿入に係 る部位が前記配列 (A)に含まれるように設計される。この実施態様では、核酸試料中 に標的核酸配列が含まれている場合には、核酸増幅反応において第一のプライマ 一が配列 (A)にアニーリングするため、増幅産物が得られる。核酸試料中に、欠失 Z 挿入により標的核酸配列とは異なる核酸配列が含まれている場合には、核酸増幅反 応において第一のプライマーが配列 (A)にアニーリングすることが困難となるため、 増幅産物が得られないか、または得られる増幅産物の量が著しく減少する。第一の プライマーに含まれる配列 (Ac )は、前記配列 (A)に相補的な配列とすることが好ま しい。  [0048] According to one embodiment of the present invention, the primer set is designed such that a site related to deletion or insertion is included in the sequence (A). In this embodiment, when the target nucleic acid sequence is contained in the nucleic acid sample, the first primer is annealed to the sequence (A) in the nucleic acid amplification reaction, so that an amplification product is obtained. If the nucleic acid sample contains a nucleic acid sequence that is different from the target nucleic acid sequence due to deletion Z insertion, it will be difficult for the first primer to anneal to the sequence (A) in the nucleic acid amplification reaction. No amplification product is obtained or the amount of amplification product obtained is significantly reduced. The sequence (Ac) contained in the first primer is preferably a sequence complementary to the sequence (A).
[0049] 本発明の他の実施態様によれば、前記プライマーセットは、欠失または挿入に係る 部位が前記配列 (C)に含まれるように設計される。この実施態様では、核酸試料中 に標的核酸配列が含まれている場合には、核酸増幅反応において第二のプライマ 一が配列 (c)にアニーリングするため、増幅産物が得られる。核酸試料中に、欠失 Z 挿入により標的核酸配列とは異なる核酸配列が含まれている場合には、核酸増幅反 応において第二のプライマーが配列（C)にアニーリングすることが困難となるため、 増幅産物が得られないか、または得られる増幅産物の量が著しく減少する。第二の プライマーに含まれる配列 (Cc )は、前記配列 (C)に相補的な配列とすることが好ま しい。  [0049] According to another embodiment of the present invention, the primer set is designed such that a site for deletion or insertion is included in the sequence (C). In this embodiment, when the target nucleic acid sequence is contained in the nucleic acid sample, the amplification product is obtained because the second primer anneals to the sequence (c) in the nucleic acid amplification reaction. If the nucleic acid sample contains a nucleic acid sequence that is different from the target nucleic acid sequence due to deletion Z insertion, it will be difficult for the second primer to anneal to the sequence (C) in the nucleic acid amplification reaction. No amplification product is obtained or the amount of amplification product obtained is significantly reduced. The sequence (Cc) contained in the second primer is preferably a sequence complementary to the sequence (C).
[0050] 本発明の他の実施態様によれば、前記プライマーセットは、欠失または挿入に係る 部位が前記配列 (B)に含まれるように設計される。この実施態様では、核酸試料中に 標的核酸配列が含まれている場合には、核酸増幅反応において、第一のプライマー が配列 (A)にアニーリングして伸長反応が行なわれた後に該プライマーに含まれる 配列（Β')が伸長鎖上の配列（Be)にハイブリダィズするため、ステム ループ構造が 効率的に形成される。この効率的なステム ループ構造の形成により、他の第一の プライマーが铸型にアニーリングすることが可能となり、図 1に示される作用機序が効 率的に進行するため、増幅産物が得られる。一方で、核酸試料中に、欠失 Z挿入に より標的核酸配列とは異なる核酸配列が含まれて 、る場合には、核酸増幅反応にお ける上記ステム—ループ構造の形成が困難となるため、図 1に示される作用機序が 妨げられ、増幅産物が得られないか、または得られる増幅産物の量が著しく減少する 。その詳細は、本発明による変異検出について上述したとおりである。また、第一の プライマーに含まれる配列（Β')は、前記配列（Β)と同一の配列とすることが好ましい。 [0050] According to another embodiment of the present invention, the primer set is designed such that a site related to deletion or insertion is included in the sequence (B). In this embodiment, when the nucleic acid sample contains the target nucleic acid sequence, the nucleic acid amplification reaction includes the first primer after annealing to the sequence (A) and the extension reaction, and then the primer is contained in the primer. Since the sequence (Β ') hybridizes to the sequence (Be) on the extended strand, the stem loop structure It is formed efficiently. The formation of this efficient stem-loop structure allows the other first primer to anneal in a saddle shape, and the mechanism of action shown in Fig. 1 proceeds efficiently, resulting in an amplified product. . On the other hand, if the nucleic acid sample contains a nucleic acid sequence that is different from the target nucleic acid sequence due to deletion Z insertion, formation of the stem-loop structure in the nucleic acid amplification reaction becomes difficult. The mechanism of action shown in FIG. 1 is hindered, and no amplification product is obtained, or the amount of amplification product obtained is significantly reduced. The details are as described above for the mutation detection according to the present invention. The sequence (配 列 ′) contained in the first primer is preferably the same sequence as the sequence (Β).
[0051] 本発明の好ましい実施態様によれば、前記プライマーセットは、欠失または挿入に 係る部位が前記配列 (Α)と前記配列（Β)との間に配置されるように設計される。この 実施態様では、核酸試料中に標的核酸配列が含まれている場合には、核酸増幅反 応において、第一のプライマーが配列 (Α)にアニーリングして伸長反応が行なわれた 後に該プライマーに含まれる配列 (Β')が伸長鎖上の配列 (Be)にハイブリダィズする ため、ステム ループ構造が効率的に形成される。この効率的なステム ループ構 造の形成により、他の第一のプライマーが铸型にアニーリングすることが可能となり、 図 1に示される作用機序が効率的に進行するため、増幅産物が得られる。一方で、 核酸試料中に、欠失 Z挿入により標的核酸配列とは異なる核酸配列が含まれて ヽる 場合には、第一のプライマーに含まれる配列 (Β')と伸長鎖上の配列 (Be)とが適切な 距離を維持して 、な 、ため、核酸増幅反応における上記ステム—ループ構造の形 成が困難となる。従って、この場合には、図 1に示される作用機序が妨げられ、増幅 産物が得られな 、か、または得られる増幅産物の量が著しく減少する。 [0051] According to a preferred embodiment of the present invention, the primer set is designed such that a site related to deletion or insertion is located between the sequence (Α) and the sequence (Β). In this embodiment, when the target nucleic acid sequence is contained in the nucleic acid sample, in the nucleic acid amplification reaction, the first primer is annealed to the sequence (Α) and the extension reaction is performed, and then the primer is added to the primer. Since the contained sequence (Β ') hybridizes to the sequence (Be) on the extended strand, a stem loop structure is efficiently formed. The formation of this efficient stem-loop structure allows the other first primer to anneal in a saddle shape, and the mechanism of action shown in Figure 1 proceeds efficiently, resulting in an amplified product. . On the other hand, if the nucleic acid sample contains a nucleic acid sequence that is different from the target nucleic acid sequence due to deletion Z insertion, the sequence (Β ') contained in the first primer and the sequence on the extended strand ( Therefore, it is difficult to form the stem-loop structure in the nucleic acid amplification reaction. Therefore, in this case, the mechanism of action shown in FIG. 1 is hindered and no amplification product is obtained, or the amount of amplification product obtained is significantly reduced.
[0052] 本発明の他の実施態様によれば、前記プライマーセットは、欠失または挿入に係る 部位が前記配列 (A)と前記配列 (C)との間に配置されるように設計される。この実施 態様では、核酸試料中に標的核酸配列が含まれている場合には、核酸増幅反応に おいて、第一のプライマーが配列 (A)にアニーリングして伸長反応が行なわれた後に 該プライマーに含まれる配列（Β')が伸長鎖上の配列（Be)にハイブリダィズするため 、ステム—ループ構造が効率的に形成される。この効率的なステム—ループ構造の 形成により、他の第一のプライマーが铸型にアニーリングすることが可能となり、図 1 に示される作用機序が効率的に進行するため、増幅産物が得られる。一方で、核酸 試料中に、欠失 z挿入により標的核酸配列とは異なる核酸配列が含まれて ヽる場合 には、増幅産物が得られないか、または得られる増幅産物の量が著しく減少する。例 えば、配列 (A)と配列（C)との間における長い配列の挿入により標的核酸配列とは異 なる核酸配列が核酸試料中に含まれて!/、る場合には、核酸増幅の速度 (効率)が著 しく低減されるため、増幅産物が得られないか、または得られる増幅産物の量が著し く減少する。また、配列 (A)と配列 (C)との間における配列の欠失により標的核酸配 列とは異なる核酸配列が核酸試料中に含まれており、かっこの欠失により配列（B)の 一部または全部が失われている場合には、第一のプライマーに含まれる配列（Β')が 伸長鎖上にハイブリダィズできな 、ため、ステム ループ構造の形成が不可能となる 力 または困難となるため、図 1に示される作用機序が妨げられ、増幅産物が得られ ないか、または得られる増幅産物の量が著しく減少する。さらに、配列 (Α)と配列 (C) との間における配列の欠失により標的核酸配列とは異なる核酸配列が核酸試料中に 含まれており、かっこの欠失による配列 (Β)の部分的欠失が生じない場合にも、核酸 増幅の速度 (効率)が低減されるため、増幅産物が得られないか、または得られる増 幅産物の量が著しく減少する。 [0052] According to another embodiment of the present invention, the primer set is designed such that a site for deletion or insertion is located between the sequence (A) and the sequence (C). . In this embodiment, when the target nucleic acid sequence is contained in the nucleic acid sample, the primer is annealed to the sequence (A) in the nucleic acid amplification reaction and the extension reaction is performed. Since the sequence (Β ′) contained in is hybridized with the sequence (Be) on the extended strand, a stem-loop structure is efficiently formed. The formation of this efficient stem-loop structure allows the other first primer to anneal in a saddle shape. Since the mechanism of action shown in (1) proceeds efficiently, an amplification product is obtained. On the other hand, if the nucleic acid sample contains a nucleic acid sequence that differs from the target nucleic acid sequence due to deletion z insertion, no amplification product is obtained or the amount of amplification product obtained is significantly reduced. . For example, when a long sequence is inserted between sequence (A) and sequence (C), a nucleic acid sequence that is different from the target nucleic acid sequence is included in the nucleic acid sample! Since (efficiency) is significantly reduced, no amplification product is obtained or the amount of amplification product obtained is significantly reduced. In addition, a nucleic acid sequence that differs from the target nucleic acid sequence is contained in the nucleic acid sample due to the deletion of the sequence between sequence (A) and sequence (C). If part or all is lost, the sequence (Β ') contained in the first primer cannot be hybridized on the extended strand, making it impossible or difficult to form a stem-loop structure. Therefore, the mechanism of action shown in FIG. 1 is hindered, and no amplification product is obtained, or the amount of amplification product obtained is significantly reduced. Furthermore, a nucleic acid sequence that differs from the target nucleic acid sequence due to the deletion of the sequence between sequence (Α) and sequence (C) is contained in the nucleic acid sample. Even if no deletion occurs, the speed (efficiency) of nucleic acid amplification is reduced, so that no amplification product is obtained or the amount of amplification product obtained is significantly reduced.
核酸増幅反応に用いられるポリメラーゼは、鎖置換 (strand displacement)活性 (鎖 置換能）を有するものであればよぐ常温性、中温性、もしくは耐熱性のいずれのもの も好適に使用できる。また、このポリメラーゼは、天然体もしくは人工的に変異を加え た変異体のいずれであってもよい。このようなポリメラーゼとしては、 DNAポリメラーゼ が挙げられる。さらに、この DNAポリメラーゼは、実質的に 5，→3，ェキソヌクレア一 ゼ活性を有しないものであることが好ましい。このような DNAポリメラーゼとしては、バ チルス 'ステア口サーモフィルス（Bacillus stearothermophilus、以下「B. st」という）、 バチルス.カルドテナックス（Bacillus caldotenax、以下「B. ca」という）等の好熱性バ チルス属細菌由来 DNAポリメラーゼの 5 '→3，ェキソヌクレアーゼ活性を欠失した変 異体、大腸菌 . coli)由来 DNAポリメラーゼ Iのタレノウフラグメント等が挙げられる 。核酸増幅反応において使用する DNAポリメラーゼとしては、さらに、 Vent DNAポリ メラーゼ、 Vent (Exo-) DNAポリメラーゼ、 DeepVent DNAポリメラーゼ、 DeepVent (Ex o-) DNAポリメラーゼ、 Φ 29ファージ DNAポリメラーゼ、 MS— 2ファージ DNAポリメ ラーゼ、 Z- Taq DNAポリメラーゼ、 Pfo DNAポリメラーゼ、 Pfo turbo DNAポリメラーゼ、 KOD DNAポリメラーゼ、 9° Nm DNAポリメラーゼ、 Therminater DNAポリメラーゼ等 が挙げられる。 As the polymerase used in the nucleic acid amplification reaction, any one having room temperature, medium temperature, or heat resistance can be suitably used as long as it has strand displacement activity (strand displacement ability). Further, this polymerase may be either a natural body or a mutant having an artificial mutation. An example of such a polymerase is DNA polymerase. Further, it is preferable that this DNA polymerase has substantially no 5 → 3 exonuclease activity. Such DNA polymerases include thermophilic Bacillus such as Bacillus stearothermophilus (hereinafter referred to as “B. st”), Bacillus caldotenax (hereinafter referred to as “B. ca”), and the like. 5 ′ → 3 of DNA polymerase derived from genus bacteria, mutants lacking exonuclease activity, and Tarenow fragment of DNA polymerase I derived from E. coli. Examples of DNA polymerases used in nucleic acid amplification reactions include Vent DNA polymerase, Vent (Exo-) DNA polymerase, DeepVent DNA polymerase, DeepVent (Ex o-) DNA polymerase, Φ 29 phage DNA polymerase, MS-2 phage DNA polymerase, Z-Taq DNA polymerase, Pfo DNA polymerase, Pfo turbo DNA polymerase, KOD DNA polymerase, 9 ° Nm DNA polymerase, Therminater DNA polymerase, etc. Can be mentioned.
[0054] 核酸増幅反応に用いられるその他の試薬としては、例えば、塩化マグネシウム、酢 酸マグネシウム、硫酸マグネシウム等の触媒、 dNTPミックス等の基質、トリス塩酸バッ ファー、トライシンバッファー、リン酸ナトリウムバッファー、リン酸カリウムバッファ一等 の緩衝液を使用することができる。さらに、ジメチルスルホキシド (dimethyl sulfoxide) やべタイン（Ν,Ν,Ν- trimethylglycine)等の添加物、国際公開第 99/54455号パンフ レットに記載の酸性物質、陽イオン錯体等を使用してもよい。  [0054] Examples of other reagents used in the nucleic acid amplification reaction include catalysts such as magnesium chloride, magnesium acetate, and magnesium sulfate, substrates such as dNTP mix, tris hydrochloride buffer, tricine buffer, sodium phosphate buffer, A buffer such as potassium phosphate buffer can be used. Furthermore, additives such as dimethyl sulfoxide and betaine (Ν, Ν, Ν-trimethylglycine), acidic substances and cation complexes described in WO 99/54455 pamphlets may be used. .
[0055] 核酸増幅反応において、核酸の増幅効率を高めるために、融解温度調整剤を反 応溶液中に添加することができる。核酸の融解温度 (Tm)は、一般的に、核酸中の二 本鎖形成部分の具体的なヌクレオチド配列によって決定される。反応溶液中に融解 温度調整剤を添加することにより、この融解温度を変化させることができ、従って、一 定の温度下では、核酸における二本鎖形成の強度を調整することが可能となる。一 般的な融解温度調整剤は、融解温度を下げる効果を有する。このような融解温度調 整剤を添加することにより、 2本の核酸の間の二本鎖形成部分の融解温度を下げるこ とができ、換言すれば、その二本鎖形成の強度を下げることが可能となる。従って、 前記核酸増幅反応においてこのような融解温度調整剤を反応溶液中に添加すると、 強固な二本鎖を形成する GCの豊富な核酸領域や複雑な二次構造を形成する領域 において効率的に二本鎖部分を一本鎖とすることが可能となり、これにより、プライマ 一による伸長反応が終わった後に次のプライマーが目的領域にハイブリダィズしゃ すくなるため、核酸の増幅効率を上げることができる。本発明において用いられる融 解温度調整剤およびその反応溶液中での濃度は、ハイブリダィゼーシヨン条件に影 響を与える他の反応条件、例えば塩濃度、反応温度等を考慮して、当業者により適 切に選択される。従って、融解温度調整剤は特に制限されるものではないが、好まし くはジメチルスルホキシド（DMSO)、ベタイン、ホルムアミドもしくはグリセロール、ま たはこれらの任意の組み合わせとされ、より好ましくはジメチルスルホキシド（DMSO )とされる。 In the nucleic acid amplification reaction, a melting temperature adjusting agent can be added to the reaction solution in order to increase the nucleic acid amplification efficiency. The melting temperature (Tm) of a nucleic acid is generally determined by the specific nucleotide sequence of the double stranded portion in the nucleic acid. By adding a melting temperature adjusting agent to the reaction solution, this melting temperature can be changed. Therefore, under a certain temperature, it is possible to adjust the intensity of double strand formation in the nucleic acid. A general melting temperature adjusting agent has an effect of lowering the melting temperature. By adding such a melting temperature adjusting agent, it is possible to lower the melting temperature of the duplex forming part between two nucleic acids, in other words, to reduce the strength of the duplex formation. Is possible. Therefore, when such a melting temperature adjusting agent is added to the reaction solution in the nucleic acid amplification reaction, it is efficiently used in a GC-rich nucleic acid region that forms a strong double strand or a region that forms a complex secondary structure. The double-stranded portion can be made into a single strand, whereby the next primer becomes hybridized to the target region after the extension reaction by the primer is completed, so that the nucleic acid amplification efficiency can be increased. The concentration of the melting temperature adjusting agent used in the present invention and the concentration thereof in the reaction solution is determined based on other reaction conditions that affect the hybridization conditions such as salt concentration, reaction temperature, and the like. Is selected appropriately. Therefore, the melting temperature adjusting agent is not particularly limited, but is preferably dimethyl sulfoxide (DMSO), betaine, formamide or glycerol, or any combination thereof, more preferably dimethyl sulfoxide (DMSO). ).
[0056] さらに、核酸増幅反応において、酵素安定化剤を反応溶液中に添加することもでき る。これにより、反応液中の酵素が安定ィ匕されるため、核酸の増幅効率を高めること が可能となる。本発明において用いられる酵素安定化剤は、グリセロール、ゥシ血清 アルブミン、糖類などの、当技術分野において知られているいかなるものであってもよ ぐ特に制限されない。  [0056] Further, in the nucleic acid amplification reaction, an enzyme stabilizer can be added to the reaction solution. As a result, the enzyme in the reaction solution is stabilized, so that the nucleic acid amplification efficiency can be increased. The enzyme stabilizer used in the present invention is not particularly limited and may be any one known in the art such as glycerol, urine serum albumin, saccharides and the like.
[0057] さらに、核酸増幅反応において、 DNAポリメラーゼ、逆転写酵素などの酵素の耐熱 性を増強するための試薬を、酵素安定化剤として反応溶液中に添加することもできる 。これにより、反応液中の酵素が安定化されるため、核酸の合成効率および増幅効 率を高めることが可能となる。このような試薬は当技術分野にぉ 、て知られて 、る!/ヽ 力なるものであってもよぐ特に制限されないが、好ましくは糖類、より好ましくは単糖 またはオリゴ糖、さらに好ましくはトレハロース、ソルビトールもしくはマン-トール、ま たはこれらの 2種以上の混合物とされる。  [0057] Furthermore, in the nucleic acid amplification reaction, a reagent for enhancing the heat resistance of an enzyme such as DNA polymerase or reverse transcriptase can be added to the reaction solution as an enzyme stabilizer. As a result, the enzyme in the reaction solution is stabilized, so that the nucleic acid synthesis efficiency and amplification efficiency can be increased. Such reagents are well known in the art and may be rugged, but are not particularly limited, but are preferably saccharides, more preferably monosaccharides or oligosaccharides, more preferably It can be trehalose, sorbitol or mannitol, or a mixture of two or more of these.
[0058] 本発明の好ましい実施態様によれば、核酸増幅反応はミスマッチ認識タンパク質の 存在下で行なわれ、これにより、より正確に変異を検出することが可能となる。  [0058] According to a preferred embodiment of the present invention, the nucleic acid amplification reaction is carried out in the presence of a mismatch recognition protein, which makes it possible to detect mutations more accurately.
[0059] 細菌や酵母等には、 DNAの 2本鎖において部分的に対合できない（ミスマッチ)塩 基対が生じたときに、これを修復するための機構があることが既に知られている。この 修復は「ミスマッチ結合タンパク質」（「ミスマッチ認識タンパク質」とも称される）と呼ば れるタンパク質によって行なわれるものであり、 MutSタンパク質（特表平 9 50469 9号公報）、 MutMタンパク質（特開 2000— 300265号公報）、 GFP (Green Fluores cence Protein)に結合した MutSタンパク質（国際公開第 99/06591号パンフレット )などの様々なミスマッチ結合タンパクの使用が報告されている。さらに、近年、ミスマ ツチ結合タンパク質を利用してミスマッチを検出する遺伝子診断法が開発されている (M. Gotoh et al.， Genet. Anal, 14, 47-50, 1997) 0核酸中における特定のヌクレオ チドにおける多型および突然変異の検出法としては、例えば、変異のない対照核酸 と、変異が存在することが疑われる被検核酸とをハイブリダィズさせ、そこにミスマッチ 認識タンパク質を導入することによりミスマッチを検出する方法が知られている。 [0059] It is already known that bacteria, yeast, and the like have a mechanism for repairing a mismatched base pair that is not partially matched in the double strand of DNA. . This repair is performed by a protein called “mismatch binding protein” (also referred to as “mismatch recognition protein”). MutS protein (Japanese Patent Publication No. 9 50469 9), MutM protein (Japanese Patent Laid-Open No. 2000-2000) No. 300265) and various mismatch binding proteins such as MutS protein (International Publication No. 99/06591 pamphlet) bound to GFP (Green Fluores cence Protein) have been reported. Furthermore, in recent years, Misuma Tutsi gene diagnostic method to detect mismatches utilizes a binding protein have been developed (M. Gotoh et al., Genet . Anal, 14, 47-50, 1997) specific in 0 in a nucleic acid As a method for detecting polymorphisms and mutations in a nucleotide, for example, a mismatched control nucleic acid is hybridized with a test nucleic acid suspected of having a mutation, and a mismatch recognition protein is introduced into the mismatched nucleic acid. There are known methods for detecting.
[0060] 本発明にお！/、て「ミスマッチ」とは、アデニン (A)、グ了ニン (G)、シトシン（C)、およ びチミン (T) (RNAの場合はゥラシル (U) )から選択される一組の塩基対が正常な塩 基対 (Aと Tの組み合わせ、または Gと Cの組み合わせ)ではないことを意味する。ミス マッチには、 1つのミスマッチのみならず、複数の連続したミスマッチ、 1または複数の 塩基の挿入および Zまたは欠失により生じるミスマッチ、ならびにそれらの組み合わ せが含まれる。 [0060] In the present invention! / “Mismatch” means adenine (A), gyonin (G), cytosine (C), and Means that one base pair selected from thymine (T) (uracil (U) in the case of RNA) is not a normal base pair (A and T combination or G and C combination) . Mismatches include not only one mismatch but also multiple consecutive mismatches, mismatches caused by insertion and Z or deletion of one or more bases, and combinations thereof.
[0061] 遺伝子変異の検出のための核酸増幅反応においても、これらのミスマッチ結合タン ノ^質を利用することにより、その特異性 (正確さ）を向上させることができる。核酸増 幅反応にぉ 、ては、相互に相補的でな 、2つのヌクレオチド配列の間で少量のへテ ロニ本鎖構造が生ずることがある。本発明において「ヘテロ二本鎖構造」とは、実質 的には相補的な二本鎖構造である力 1または複数のミスマッチを有することにより非 相補的な領域を含む二本鎖構造を意味する。このようなヘテロ二本鎖構造により、本 来的には生成しないはずの誤った増幅産物力あたらされる。そこで、核酸増幅反応 に用いられる反応液中にミスマッチ結合タンパク質を添加しておけば、上記のような ヘテロ二本鎖構造にこのミスマッチ結合タンパク質が結合し、その後の増幅反応が妨 げられる。従って、ミスマッチ結合タンパク質を利用することにより、誤った増幅産物の 生成を防ぐことが可能となる。  [0061] The specificity (accuracy) can be improved also in a nucleic acid amplification reaction for detecting a gene mutation by using these mismatched binding proteins. In the case of a nucleic acid amplification reaction, a small amount of a heterozygous double-stranded structure may occur between two nucleotide sequences that are not complementary to each other. In the present invention, the term “heteroduplex structure” means a double-stranded structure containing a non-complementary region by having one or more mismatches, which is a substantially complementary double-stranded structure. . Such a heteroduplex structure results in a false amplification product that should not be produced by nature. Therefore, if a mismatch binding protein is added to the reaction solution used in the nucleic acid amplification reaction, the mismatch binding protein binds to the heteroduplex structure as described above, and the subsequent amplification reaction is prevented. Therefore, by using a mismatch binding protein, it is possible to prevent the generation of an erroneous amplification product.
[0062] 本発明に用いられるミスマッチ結合タンパク質は、二本鎖核酸におけるミスマッチを 認識し、そのミスマッチの部位に結合することが可能なタンパク質であればよぐ例え ば、当業者に公知のいずれのものであってもよい。また、本発明に用いられるミスマツ チ結合タンパク質は、二本鎖核酸中のミスマッチを認識しうる限り、野生型タンパク質 のアミノ酸配列において 1または複数のアミノ酸が置換、欠失、付加、および/または 挿入されたアミノ酸配列力もなるタンパク質 (変異体)であってもよ!/、。このような変異 体は、自然界において生じることもある力 人為的に作製することも可能である。タン ノ ク質にアミノ酸変異を導入する方法としては、多くの方法が知られている。例えば、 部位特異的変異導入法としては、 W.P. Dengと J.A. Nickoloffの方法（Anal. Biochem. , 200, 81, 1992)、 Κ丄. Makamayeと F. Ecksteinの方法（Nucleic Adids Res., 14, 9679 -9698, 1986)などが知られており、ランダム変異導入法としては、基本的な修復系を 欠損した大腸菌 XLl-Red株 (Stratagene社)を用いる方法、亜硝酸ナトリウム等を用 いて化学的に塩基を修飾する方法 (J.- J. Diaz et al.， BioTechnique, 11, 204-211, 1 991)などが知られている。このようなミスマッチ結合タンパク質としては、 MutM、 Mut Sおよびそれらの類似体など、多くのものが知られている（Radman,M.et al.Annu.Rev .Genet.20:523-538(1986);Radaman,M.etal.,Sci.Amer.,August 1988,pp40-46;Modric h,P.,J.Biol.Chem.264:6597-6600(1989) ; Lahue.R.S. et al, Science 245:160-164(198 8);Jiricny,J.et al'.Nucl. Acids Res.16:7843- 7853(1988);Su,S.S.et al.,J.Biol.Chem.263; 6829-6835(1988);Lahue,R.S.et al.'Mutat.Res.198:37- 43(1988);Dohet,C.et al.Mol.G en.Gent.206: 181-184(1987); Jones.M.et al.'Gentics 115:605—610(1987) ; Salmonella typhimuriumの Muts (Lu,A.L.,Genetics 118:593- 600(1988);HaberL.T. et al.J.Bact eriol.l70:197-202(1988);Pang,P.P.et al.J.Bacteriol.163: 1007- 1015(1985))；および P riebe S.D.et al.,J.Bacterilo.l70:190- 196(1988))。本発明に用いられるミスマッチ結合 タンパク質は、好ましくは MutS、 MutH、 MutL、または酵母に由来するものとされ、 より好ましくは MutS、 MutH、または MutLとされる。 [0062] The mismatch binding protein used in the present invention may be any protein known to those skilled in the art as long as it is a protein capable of recognizing a mismatch in a double-stranded nucleic acid and binding to the mismatch site. It may be a thing. In addition, the mismatch-binding protein used in the present invention has one or more amino acid substitutions, deletions, additions, and / or insertions in the amino acid sequence of the wild-type protein as long as the mismatch in the double-stranded nucleic acid can be recognized. It may be a protein (mutant) that also has an amino acid sequence ability! /. Such mutants can also be created artificially by forces that may occur in nature. Many methods are known for introducing amino acid mutations into proteins. For example, site-directed mutagenesis methods include WP Deng and JA Nickoloff's method (Anal. Biochem., 200, 81, 1992), Κ 丄. Makamaye and F. Eckstein's method (Nucleic Adids Res., 14, 9679). -9698, 1986), etc., and the random mutagenesis method uses a method using Escherichia coli XLl-Red strain (Stratagene) lacking the basic repair system, sodium nitrite, etc. And methods for chemically modifying bases (J.-J. Diaz et al., BioTechnique, 11, 204-211, 1 991) are known. Many such mismatch binding proteins are known, such as MutM, Mut S and their analogs (Radman, M. et al. Annu. Rev. Genet. 20: 523-538 (1986)). Radaman, M. etal., Sci. Amer., August 1988, pp40-46; Modric h, P., J. Biol. Chem. 264: 6597-6600 (1989); Lahue.RS et al, Science 245: 160-164 (198 8); Jiricny, J. et al '. Nucl. Acids Res. 16: 7843-7853 (1988); Su, SSet al., J. Biol. Chem. 263; 6829-6835 (1988) Lahue, RSet al. 'Mutat. Res. 198: 37-43 (1988); Dohet, C. et al. Mol. G en. Gent. 206: 181-184 (1987); Jones.M. Et al. 'Gentics 115: 605-610 (1987); Muts of Salmonella typhimurium (Lu, AL, Genetics 118: 593-600 (1988); HaberL.T. Et al.J.Bact eriol.l70: 197-202 (1988) Pang, PPet al. J. Bacteriol. 163: 1007-1015 (1985)); and Pribebe SD et al., J. Bacterilo. L70: 190-196 (1988)). The mismatch binding protein used in the present invention is preferably derived from MutS, MutH, MutL, or yeast, and more preferably MutS, MutH, or MutL.
[0063] ミスマッチ結合タンパク質は、一本鎖核酸にも結合することがあり、このようなミスマツ チ結合タンパク質の一本鎖核酸への結合は、一本鎖結合タンパク質により阻害され ることが知られている。従って、核酸増幅反応においてミスマッチ結合タンパク質を用 いる場合には、一本鎖結合タンパク質を併用することが好ましい。また、ミスマッチ結 合タンパク質は、ミスマッチを含まない二本鎖核酸にも結合することがあり、このような ミスマッチ結合タンパク質の誤った結合は、あら力じめ活性剤を用いてミスマッチ結合 タンパク質を活性ィ匕しておくことにより阻害されることが知られている。従って、核酸増 幅反応においてミスマッチ結合タンパク質を用いる場合には、活性剤によりあらかじ め活性ィ匕されたものを用いることが好まし 、。  [0063] A mismatch binding protein may also bind to a single-stranded nucleic acid, and it is known that binding of such a mismatch binding protein to a single-stranded nucleic acid is inhibited by the single-stranded binding protein. ing. Therefore, when a mismatch binding protein is used in the nucleic acid amplification reaction, it is preferable to use a single-stranded binding protein in combination. Mismatch-binding proteins can also bind to double-stranded nucleic acids that do not contain mismatches. Such misbinding of mismatch-binding proteins can be activated by using a activating agent to activate the mismatch-binding proteins. It is known that it is inhibited by keeping it. Therefore, when using a mismatch binding protein in a nucleic acid amplification reaction, it is preferable to use a protein that has been activated in advance by an activator.
[0064] 一本鎖核酸にミスマッチ結合タンパク質が結合するのを阻害するために使用する 一本鎖結合タンパク質 (SSB)は、当技術分野において公知の任意の SSBとすること ができる。好ましい SSBとしては、ェシエリキア'コリ、ショウジヨウバエ、およびアフリカ ッメガエルに由来する一本鎖結合タンパク質、および T4バタテリオファージ由来の遺 伝子 32タンパク質、ならびに他の種に由来するこれらの相当物が挙げられる。この場 合に使用されるミスマッチ結合タンパク質としては、 MutS、 MutH、 MutL, HexA、 MSH1〜6、 Rep3、 RNaseA、ゥラシルー DNAグリコシダーゼ、 T4エンドヌクレア一 ゼ VII、レゾルバーゼなどが挙げられ、好ましくは MutS、 MSH2もしくは MSH6、ま たはこれらの 2種以上の混合物とされ、より好ましくは MutSとされる。 [0064] The single-stranded binding protein (SSB) used to inhibit the binding of the mismatch binding protein to the single-stranded nucleic acid can be any SSB known in the art. Preferred SSBs include single-stranded binding proteins from Escherichia coli, Drosophila, and Xenopus, and gene 32 protein from T4 butteriophage, and their equivalents from other species. Can be mentioned. The mismatch binding proteins used in this case are MutS, MutH, MutL, HexA, MSH1-6, Rep3, RNaseA, uracil-DNA glycosidase, T4 endonuclease VII, resolvase, etc., preferably MutS, MSH2 or MSH6, or a mixture of two or more of these, more preferably MutS Is done.
[0065] ミスマッチ結合タンパク質を活性ィ匕するための活性剤は、当業者であれば適宜選 択することができるため、特に限定されないが、好ましくは、 ATP (アデノシン 5'—三リ ン酸)、 ADP (アデノシン 5'—二リン酸)、 ATP- γ 3(ァデノシン5' 0 (3—チォ 三リン酸))、 AMP— PNP(ァデノシン5' [ J8 , γ イミド]三リン酸)などの化合物とさ れ、あるいは、ミスマッチ結合タンパク質に結合できるヌクレオチドの一つとされる。ミ スマッチ結合タンパク質の活性ィ匕は、ミスマッチ結合タンパク質と活性剤とを、室温で 数秒間から数分間インキュベートすることにより行うことができる。 [0065] The active agent for activating the mismatch binding protein can be appropriately selected by those skilled in the art, and is not particularly limited, but preferably ATP (adenosine 5'-triphosphate). , ADP (adenosine 5'-diphosphate), ATP-γ 3 (adenosine 5 '0 (3-thio triphosphate)), AMP-PNP (adenosine 5' [ J 8, γ imido] triphosphate), etc. Or one of the nucleotides that can bind to the mismatch binding protein. The activity of the mismatch binding protein can be performed by incubating the mismatch binding protein and the active agent at room temperature for several seconds to several minutes.
[0066] 核酸増幅法によって得られた増幅産物の存在は、多くのあらゆる方法により検出が 可能である。一つの方法は、一般的なゲル電気泳動による特定のサイズの増幅産物 の検出である。この方法では、例えば、ェチジゥムブロマイドやサイバーグリーン等の 蛍光物質により検出できる。他の方法としては、ピオチンのような標識を有する標識 プローブを用い、これを増幅産物にハイブリダィズさせることにより検出することもでき る。ピオチンは、蛍光標識されたアビジン、ペルォキシダーゼのような酵素に結合し たアビジン等との結合により検出可能である。さらに別の方法としては、免疫クロマト グラフを用いる方法がある。この方法では、肉眼で検出可能な標識を利用したクロマ トグラフ媒体を用いることが考案されて 、る (ィムノクロマトグラフィー法)。上記増幅断 片と標識プローブとをハイブリダィズさせ、該増幅断片のさらに異なる配列とハイプリ ダイズ可能な捕捉用プローブをクロマト媒体に固定しておけば、その固定した部分で トラップすることができ、クロマト媒体での検出が可能となる。その結果、肉眼的にシン プルな検出が可能となる。さらに、本発明者らによる核酸増幅法では、核酸増幅反応 における増幅効率が非常に高いため、増幅の副産物としてピロリン酸が生じることを 利用して、増幅産物を間接的に検出することもできる。このような方法としては、例え ば、ピロリン酸が反応溶液中のマグネシウムと結合することによりピロリン酸マグネシゥ ムの白色沈澱が生じることを利用して、反応溶液の白濁を目視で観察する方法があ る。また、他の方法としては、ピロリン酸がマグネシウムなどの金属イオンと強く結合し て不溶性塩を形成することにより、反応溶液中のマグネシウムイオン濃度が著しく減 少することを利用する方法がある。この方法では、マグネシウムイオン濃度に応じて 色調が変化する金属指示薬（例えば、 Eriochrome Black T、 Hydroxy Naphthol Blue 等）を反応溶液に添加しておくことにより、反応溶液の色の変化を目視で観察するこ とにより、増幅の有無を検出することが可能となる。さらに、 Calceinなどを利用すること により、増幅反応に伴う蛍光の増大を目視で観察することができるため、リアルタイム での増幅産物の検出が可能となる。 [0066] The presence of the amplification product obtained by the nucleic acid amplification method can be detected by many various methods. One method is the detection of amplification products of a specific size by general gel electrophoresis. In this method, for example, it can be detected by a fluorescent substance such as ethidium bromide or cyber green. As another method, detection can be performed by using a labeled probe having a label such as piotin and hybridizing it to the amplification product. Piotin can be detected by binding to fluorescently labeled avidin, avidin bound to an enzyme such as peroxidase, and the like. Yet another method is to use immunochromatography. In this method, it is devised to use a chromatographic medium using a label detectable with the naked eye (Immunochromatography method). If the amplified fragment and the labeled probe are hybridized and a further different sequence of the amplified fragment and a hybridizable capture probe are fixed to the chromatographic medium, the trapped portion can be trapped. Detection with is possible. As a result, simple detection with the naked eye is possible. Furthermore, in the nucleic acid amplification method by the present inventors, the amplification efficiency in the nucleic acid amplification reaction is very high, so that the amplification product can also be detected indirectly by using the fact that pyrophosphate is generated as an amplification byproduct. As such a method, for example, there is a method of visually observing the white turbidity of the reaction solution by utilizing the fact that pyrophosphoric acid binds to magnesium in the reaction solution to cause white precipitation of magnesium pyrophosphate. The Another method is that pyrophosphate binds strongly to metal ions such as magnesium. There is a method that utilizes the fact that the magnesium ion concentration in the reaction solution is significantly reduced by forming an insoluble salt. In this method, a metal indicator whose color tone changes according to the magnesium ion concentration (for example, Eriochrome Black T, Hydroxy Naphthol Blue, etc.) is added to the reaction solution, and the color change of the reaction solution is visually observed. This makes it possible to detect the presence or absence of amplification. Furthermore, by using Calcein, etc., the increase in fluorescence accompanying the amplification reaction can be observed visually, so that amplification products can be detected in real time.
[0067] 本発明による検出法では、次いで、第一の検体から検出された遺伝子変異と第二 の検体から検出された遺伝子変異とが比較される。この比較において、検出されたこ れらの遺伝子変異が相互に同一である場合には、第一の検体に含まれる疾患関連 細胞またはこれに由来する細胞が第二の検体力 検出されたことが示される。  [0067] In the detection method according to the present invention, the gene mutation detected from the first specimen is then compared with the gene mutation detected from the second specimen. In this comparison, if these detected gene mutations are identical to each other, it indicates that a disease-related cell contained in the first sample or a cell derived therefrom was detected in the second sample. It is.
[0068] 本発明による検出法によれば、目的とする疾患の治療前に採取された検体を第一 の検体とし、該疾患の治療中または治療後に採取された検体を第二の検体とするこ とにより、その治療効果を評価することが可能となる。従って、本発明によれば、被験 者における疾患の治療の効果を評価する方法が提供され、該方法は、（a)被験者か ら前記疾患の治療前に採取した第一の検体に含まれるゲノムにぉ 、て、前記疾患に 関連する遺伝子変異を検出する工程、（b)被験者から前記疾患の治療中または治 療後に採取した第二の検体に含まれるゲノムにおいて、前記遺伝子変異を検出する 工程、ならびに (c)第一の検体力 検出された遺伝子変異と第二の検体力 検出さ れた遺伝子変異とを比較する工程を含んでなる。この方法では、検出された遺伝子 変異が相互に異なるか、または第二の検体において前記遺伝子変異が検出されな い場合に、治療の効果が高いものと評価される。前記治療は、目的とする疾患に応じ て当業者により適宜選択されるが、例えば、薬物療法、放射線療法、温熱療法、ホル モン療法、免疫療法、外科的治療等が挙げられる。  [0068] According to the detection method of the present invention, the sample collected before the treatment of the target disease is the first sample, and the sample collected during or after the treatment of the disease is the second sample. This makes it possible to evaluate the therapeutic effect. Therefore, according to the present invention, there is provided a method for evaluating the effect of treatment of a disease in a subject, the method comprising: (a) a genome contained in a first sample collected from a subject before the treatment of the disease. And (b) detecting a gene mutation in a genome contained in a second specimen collected from the subject during or after the treatment of the disease. And (c) comparing a first gene force detected gene mutation and a second sample force detected gene mutation. In this method, when the detected gene mutation is different from each other or when the gene mutation is not detected in the second specimen, it is evaluated that the therapeutic effect is high. The treatment is appropriately selected by those skilled in the art depending on the target disease, and examples thereof include drug therapy, radiation therapy, hyperthermia, hormone therapy, immunotherapy, and surgical treatment.
[0069] また、本発明による検出法によれば、目的とする疾患の病巣部から採取された検体 を第一の検体とし、他の部位から採取された検体を第二の検体とすることにより、該 疾患の原発巣力 他の部位への転移 (例えば、癌転移)を評価することが可能となる 。従って、本発明によれば、被験者における疾患の原発巣力 他の部位への転移を 評価する方法が提供され、該方法は、（a)被験者の前記疾患の原発巣から採取した 第一の検体に含まれるゲノムにおいて、前記疾患に関連する遺伝子変異を検出する 工程、（b)被験者の他の部位力も採取した第二の検体に含まれるゲノムにおいて、 前記遺伝子変異を検出する工程、ならびに (c)第一の検体から検出された遺伝子変 異と第二の検体力 検出された遺伝子変異とを比較する工程を含んでなる。この方 法では、検出された遺伝子変異が相互に同一である場合に、疾患の原発巣から他の 部位への転移があるものと評価される。本発明の好ましい実施態様によれば、前記 疾患は癌または腫瘍とされ、これらの他の部位への転移が評価される。また、原発巣 の癌組織または腫瘍組織を第一の検体とし、原発巣の周辺部の組織を第二の検体 とすることにより、癌または腫瘍の浸潤を評価することもできる。さらに、原発巣の癌組 織を第一の検体とし、他の部位に存在する癌組織を第二の検体とすることにより、そ の部位に存在する癌組織が前記原発巣からの転移癌であるか否かを評価することが できる。 [0069] Further, according to the detection method of the present invention, the sample collected from the lesion of the target disease is used as the first sample, and the sample collected from other sites is used as the second sample. It becomes possible to evaluate the primary focus of the disease and metastasis to other sites (for example, cancer metastasis). Therefore, according to the present invention, the primary lesion force of the disease in the subject is transferred to other sites. A method of evaluating, comprising: (a) detecting a genetic mutation associated with the disease in a genome contained in a first specimen collected from the primary lesion of the disease of the subject, (b) the subject A step of detecting the genetic mutation in the genome contained in the second specimen from which the other site forces were also collected, and (c) the genetic mutation detected from the first specimen and the second specimen force. Comparing the mutation. In this method, if the detected gene mutations are identical to each other, it is evaluated that there is metastasis from the primary lesion site to another site. According to a preferred embodiment of the present invention, the disease is cancer or tumor, and metastasis to these other sites is evaluated. Further, cancer or tumor infiltration can be evaluated by using a cancer tissue or tumor tissue in the primary lesion as the first specimen and a tissue in the periphery of the primary lesion as the second specimen. Further, by setting the cancer tissue of the primary lesion as the first specimen and the cancer tissue present in the other part as the second specimen, the cancer tissue present in that part is metastatic cancer from the primary lesion. It can be evaluated whether or not there is.
実施例  Example
[0070] 以下、本発明を実施例により具体的に説明するが、本発明の範囲はこれら実施例 に限定されるものではない。  [0070] Hereinafter, the present invention will be specifically described by way of examples. However, the scope of the present invention is not limited to these examples.
[0071] 実施例 ι : Ό53遺伝子の栾異を指標 した大腸癌の原発病 部 その周 i刀組織の分 近 [0071] Example ι : Ό Primary lesion of colorectal cancer indexed by 53 gene differences
本実施例では、大腸癌患者力 得た組織を検体として用いて、核酸増幅法によりヒ ト p53遺伝子 (配列番号 1)中の変異を調べた。検査の対象とする変異は、同遺伝子 中の第 175コドン、第 213コドン、第 245コドン、第 248コドン、および第 273コドン（ 図 4中の囲み部分）におけるヌクレオチド置換とした。  In this example, using the tissue obtained from colon cancer patients as a specimen, mutations in the human p53 gene (SEQ ID NO: 1) were examined by nucleic acid amplification. The mutations to be examined were nucleotide substitutions at the 175th, 213rd, 245th, 248th, and 273rd codons (boxed in Fig. 4).
[0072] 核酸増幅反応には、下記の配列を有するプライマーを用いた。  [0072] Primers having the following sequences were used in the nucleic acid amplification reaction.
[0073] フォワードプライマー：  [0073] Forward primer:
175F-W: 5'-GGCAGCGCCTTCTACAAGCAGTCACAGCAC-3' (配列番号 2)； 175F-M： 5 -GGCAGTGCCTTCTACAAGCAGTCACAGCAC-3' (配列番号 3)； 213F-W： 5 '-ATGTCGAAAAAATTTGCGTGTGGAGTATTT-3 ' (配列番号 4)； 213F-M： 5 -ATGTCAAAAAAATTTGCGTGTGGAGTATTT-3' (配列番号 5)； 245F- W: 5し GATGCCGCCCATCCACTACAACTACATGTG- 3' (配列番号 6)； 245F-M1： 5 -GATGTCGCCCATCCACTACAACTACATGTG-3' (配列番号 7)； 245F-M2： 5 -GATGCTGCCCATCCACTACAACTACATGTG-3' (配列番号 8)； 248F-W： 5 -CCTCCGGTTCAACTACATGTGTAACAGTTC-3' (配列番号 9)； 248F-M1： 5 -CCTCTGGTTCAACTACATGTGTAACAGTTC-3' (配列番号 10)； 248F-M2： 5 '-CCTCCAGTTCAACTACATGTGTAACAGTTC-3 ' (配列番号 11)； 273F-W： 5 -AAACACGCACTGGTAATCTACTGGGACGGA-3' (配列番号 12)； 273F-M1： 5 -AAACATGCACTGGTAATCTACTGGGACGGA-3' (配列番号 13)； 273F-M2： 5 ' -AAAC ACGC ACTGGTAATCTACTGGGACGGA-3 ' (配列番号 14)。 175F-W: 5'-GGCAGCGCCTTCTACAAGCAGTCACAGCAC-3 '(SEQ ID NO: 2); 175F-M: 5 -GGCAGTGCCTTCTACAAGCAGTCACAGCAC-3' (SEQ ID NO: 3); 213F-W: 5 '-ATGTCGAAAAAATTTGCGTGTGGAGTATTT-3' (SEQ ID NO: 4); 213F-M: 5 -ATGTCAAAAAAATTTGCGTGTGGAGTATTT-3 '(SEQ ID NO: 5); 245F-W: 5 GATGCCGCCCATCCACTACAACTACATGTG-3 '(SEQ ID NO: 6); 245F-M1: 5 -GATGTCGCCCATCCACTACAACTACATGTG-3' (SEQ ID NO: 7); 245F-M2: 5 -GATGCTGCCCATCCACTACAACTACATGTG-3 '(SEQ ID NO: 8); W: 5 -CCTCCGGTTCAACTACATGTGTAACAGTTC-3 '(SEQ ID NO: 9); 248F-M1: 5 -CCTCTGGTTCAACTACATGTGTAACAGTTC-3' (SEQ ID NO: 10); 248F-M2: 5 '-CCTCCAGTTCAACTACATGTGTAACAGTTC-3' (SEQ ID NO: 11); 273F-W : 5 -AAACACGCACTGGTAATCTACTGGGACGGA-3 '(SEQ ID NO: 12); 273F-M1: 5 -AAACATGCACTGGTAATCTACTGGGACGGA-3' (SEQ ID NO: 13); 273F-M2: 5 '-AAAC ACGC ACTGGTAATCTACTGGGACGGA-3' (SEQ ID NO: 14).
[0074] リバースプライマー： [0074] Reverse primer:
R1： 5'— GGATATATATATATCCAGATTCTCTTCCTCTGTGCG— 3' (配列番号 15)； R2： 5'— GGATATATATATATCCCTGGAGTCTTCCAGTGTGAT— 3' (配列番号 16)； R3： 5し GGATATATATATATCCCTCAGGCGGCTCATAGGGCA- 3' (配列番号 17)  R1: 5'—GGATATATATATATCCAGATTCTCTTCCTCTGTGCG—3 ′ (SEQ ID NO: 15); R2: 5′—GGATATATATATATCCCTGGAGTCTTCCAGTGTGAT—3 ′ (SEQ ID NO: 16); R3: 5 GGATATATATATATCCCTCAGGCGGCTCATAGGGCA-3 ′ (SEQ ID NO: 17)
R4： 5'— GGATATATATATATCCCATCGCTATCTGAGCAGCGC— 3' (配列番号 18) R4: 5'— GGATATATATATATCCCATCGCTATCTGAGCAGCGC— 3 '(SEQ ID NO: 18)
[0075] フォワードプライマーの名称の最初に付された番号は、検査の対象となる p53遺伝 子中のコドン番号を示している。また、フォワードプライマーの配列中、下線を付した 箇所は検査対象の変異に力かるヌクレオチド残基である。さらに、「F」はフォワードプ ライマーを示し、「R」はリバースプライマーを示す。また、「W」はそのプライマーが野 生型の配列を含むことを意味し、「M」はそのプライマーが変異型の配列を含むことを 意味する。 [0075] The number attached to the beginning of the name of the forward primer indicates the codon number in the p53 gene to be examined. The underlined portion of the forward primer sequence is a nucleotide residue that can be used for the mutation to be examined. Furthermore, “F” indicates a forward primer, and “R” indicates a reverse primer. “W” means that the primer contains a wild type sequence, and “M” means that the primer contains a mutant type sequence.
[0076] フォワードプライマーは、その 3，末端側にある配列 (約 20mer)が铸型にァユーリン グし、 5'末端側にある配列（lOmer)がそのプライマーによる伸長鎖上の、該プライマ 一の 3'末端残基の 16塩基下流力 始まる領域にハイブリダィズするように設計され ている。リバースプライマーは、その 3，末端側にある配列（20mer)が铸型にァニーリ ングし、 5'末端側にある配列（16mer)がその領域内で折り畳まれる構造をとるように 設計されて 、る。フォワードプライマーは変異に力かるヌクレオチド残基を含んで 、る ため、核酸増幅反応による増幅産物の有無によって、変異に力かる残基がそのフォ ワードプライマーに含まれているもの力否かを判定することが可能となる。 [0076] In the forward primer, the sequence at the 3, terminal side (about 20 mer) is ringed in a cage shape, and the sequence at the 5 'end (lOmer) is on the extension strand of the primer. It is designed to hybridize to the region starting 16 bases downstream of the 3 'terminal residue. The reverse primer is designed to have a structure in which the sequence at the 3rd end (20mer) anneals in a cage shape and the sequence at the 5 'end (16mer) folds within that region. . The forward primer contains nucleotide residues that are resistant to mutation Therefore, it is possible to determine whether or not a residue that can be mutated is included in the forward primer depending on the presence or absence of an amplification product by the nucleic acid amplification reaction.
[0077] 2名の大腸癌患者 (症例 1および症例 2)力 手術によって摘出されたヒト大腸癌組 織の原発病巣中心部、近縁部、遠隔部、転移部などの部位から生検針を用いて癌 組織を採取し、それぞれの組織を lmlの水に懸濁して 98°Cで 5分間加熱した。これ らのサンプルにっき、それぞれ 1 μ 1の懸濁液を核酸増幅反応に用いた。  [0077] Two patients with colorectal cancer (Case 1 and Case 2) Using a biopsy needle from the central lesion, close part, remote part, metastasis, etc. of a human colon cancer tissue removed by surgery Cancer tissues were collected, and each tissue was suspended in 1 ml of water and heated at 98 ° C. for 5 minutes. A 1 μl suspension of each sample was used for the nucleic acid amplification reaction.
[0078] 上述の組織懸濁液（1 μ 1)を、次の組成を有する反応液（24 L)： Tris—HCl (20 mM, pH8. 8)、 KCl (lOmM)ゝ (NH ) SO (lOmM)ゝ MgSO (8mM)、DMSO  [0078] The above-described tissue suspension (1 μ 1) was added to a reaction solution (24 L) having the following composition: Tris—HCl (20 mM, pH 8.8), KCl (lOmM) ゝ (NH) SO ( lOmM) ゝ MgSO (8mM), DMSO
4 2 4 4  4 2 4 4
(3%)、 Triton X— 100 (1%)、 dNTP (1. 4mM)、 MutS (1 μ g)、それぞれ 2000 nMのプライマー対、サイバーグリーン（0. 01 μ 1/ml)、および 8Uの Bst DNAポリ メラーゼ（NEW ENGLAND BioLabs)を含有；に添カ卩し、これを 60°Cで 26分間ィ ンキュペートした。この増幅反応は、リアルタイム PCR装置 (ストラタジーン社製)を用 いて行った。  (3%), Triton X—100 (1%), dNTP (1.4 mM), MutS (1 μg), 2000 nM primer pair, Cyber Green (0.01 μ1 / ml), and 8 U Contains Bst DNA polymerase (NEW ENGLAND BioLabs); and incubated at 60 ° C for 26 minutes. This amplification reaction was carried out using a real-time PCR apparatus (Stratagene).
[0079] 図 5に示すグラフは、症例 1の原発巣中心部の組織における増幅反応の経時変化 を示す。下記の表 1および表 2は、それぞれ症例 1および症例 2について、増幅反応 を行った結果をまとめたものである。これらの表において、増幅反応の結果は、増幅 産物の増加速度が速いもの（グラフにおいて曲線の立ち上がりが速いもの）から順に 、 + + + , + + , +,および士とし、全く増幅が見られなかったものを一とした。  [0079] The graph shown in FIG. 5 shows the time course of the amplification reaction in the tissue at the center of the primary lesion in case 1. Tables 1 and 2 below summarize the results of amplification reactions for Case 1 and Case 2, respectively. In these tables, the amplification reaction results are +++, ++, +, and king, starting from the one with the highest increase rate of the amplification product (the one with the fastest rise of the curve in the graph). What did not exist was taken as one.
[0080] [表 1] [0080] [Table 1]
表 1 ：症例 1についての増幅反応の結果Table 1: Results of amplification reaction for case 1
a 織 フォヮ一ド リバース 増幅の結果 プライマ一 プライマー  a Woven Ford Reverse Amplification Result Primer Primer
原発病巣中心部 175F-W R4 + + +  Central focus 175F-W R4 + + +
175F-M R4 ―  175F-M R4 ―
213F- R3 + + + 213F- R3 + + +
213F-M R3 ― 213F-M R3 ―
245F-W R2 + + + 245F-W R2 + + +
245F-M1 R2 ― 245F-M1 R2 ―
245F-M2 R2 ―  245F-M2 R2 ―
248F- R2 土  248F- R2 Sat
248F-M1 R2 + +  248F-M1 R2 + +
248F-M2 R2 ―  248F-M2 R2 ―
273F-W R1 + + + 273F-W R1 + + +
273F- 1 R1 一 273F- 1 R1
273F- 2 R1 一  273F- 2 R1
近縁部 ' 175F-W R4 + + +  Proximity '' 175F-W R4 + + +
175F-M R4 ―  175F-M R4 ―
213F-W R3 + + + 213F-W R3 + + +
213F-M R3 ― 213F-M R3 ―
245F-W R2 + + + 245F-W R2 + + +
245F-M1 R2 ― 245F-M1 R2 ―
245F-M2 R2 ―  245F-M2 R2 ―
248F-W R2 + + + 248F-W R2 + + +
248F-M1 R2 + 248F-M1 R2 +
248F-M2 R2 ―  248F-M2 R2 ―
273F-W R1 + + + 273F-W R1 + + +
273F-M1 R1 ― 273F-M1 R1 ―
273F-M2 R1 ―  273F-M2 R1 ―
遠隔部 175F-W R4 + + +  Remote part 175F-W R4 + + +
175F-M R4 ―  175F-M R4 ―
213F-W R3 + + + 213F-W R3 + + +
213F- R3 ― 213F- R3 ―
245F-W R2 + + + 245F-W R2 + + +
245F-M1 R2 ― 245F-M1 R2 ―
245F- 2 R2 ―  245F- 2 R2 ―
248F-W R2 + + + 248F-W R2 + + +
248F-M1 R2 ― 248F-M1 R2 ―
248F- 2 R2 ―  248F-2 R2 ―
273F-W R1 + + + 273F-W R1 + + +
273F-M1 R1 ― 273F-M1 R1 ―
273F-M2 R1 ― 表 2 ：症例 2についての増幅反応の結果 273F-M2 R1 ― Table 2: Results of amplification reaction for case 2
糸且 織 フォワード リバース 増幅の結果 プライマー プライマー  Yarn and Weave Forward Reverse Amplification Result Primer Primer
原発病巣中心部 175F-W R4 + + +  Central focus 175F-W R4 + + +
175F-M R4 ―  175F-M R4 ―
213F-W R3 + + + 213F-W R3 + + +
213F-M R3 ― 213F-M R3 ―
245F-W R2 + +  245F-W R2 + +
245F- 1 R2 + +  245F- 1 R2 + +
245F- 2 R2 ―  245F- 2 R2 ―
248F-W R2 + + + 248F-W R2 + + +
248F- 1 R2 ― 248F- 1 R2 ―
248F-M2 R2 ―  248F-M2 R2 ―
273F-W R1 + + + 273F-W R1 + + +
273F-M1 R1 ― 273F-M1 R1 ―
273F-M2 R1 ―  273F-M2 R1 ―
近縁部 175F-W R4 + + +  Proximity 175F-W R4 + + +
175F-M R4 ―  175F-M R4 ―
213F-W R3 + + + 213F-W R3 + + +
213F-M R3 ― 213F-M R3 ―
245F-W R2 + + + 245F-W R2 + + +
245F-M1 R2 + 245F-M1 R2 +
245F-M2 R2 ―  245F-M2 R2 ―
248F-W R2 + + + 248F-W R2 + + +
248F-M1 R2 ― 248F-M1 R2 ―
248F-M2 R2 ―  248F-M2 R2 ―
273F-W R1 + + + 273F-W R1 + + +
273F-M1 R1 ― 273F-M1 R1 ―
273F-M2 R1 ―  273F-M2 R1 ―
転移部 175F-W R4 + + +  Transition 175F-W R4 + + +
175F-M R4 ―  175F-M R4 ―
213F-W R3 + + + 213F-W R3 + + +
213F-M R3 一 213F-M R3
245F-W R2 + + + 245F-W R2 + + +
245F-M1 R2 ― 245F-M1 R2 ―
245F-M2 R2 ―  245F-M2 R2 ―
248F-W R2 + + + 248F-W R2 + + +
248F-M1 R2 一 248F-M1 R2
248F-M2 R2 +  248F-M2 R2 +
273F-W R1 + + + 273F-W R1 + + +
273F-M1 R1 ― 273F-M1 R1 ―
273F-M2 R1 ― 表 2 ：症例 2についての増幅反応の結果 （つづき） 273F-M2 R1 ― Table 2: Results of amplification reaction for case 2 (continued)
Figure imgf000032_0001
Figure imgf000032_0001
[0082] 症例 1では、癌病巣の中心部には第 248コドン（アルギニンをコードするコドン）に点 突然変異が見つかり、病巣近縁部からも同様の突然変異が見つ力つたことから、近 縁部への癌細胞の浸潤があると考えられた。遠隔部には癌細胞を示す突然変異は 観察されなかった。 [0082] In Case 1, a point mutation was found at the 248th codon (codon encoding arginine) in the center of the cancer lesion, and a similar mutation was found in the vicinity of the lesion. It was thought that there was invasion of cancer cells to the edge. No mutations indicating cancer cells were observed in the remote area.
[0083] 症例 2では、主な病巣と近縁部に第 245コドン (グリシンをコードするコドン）に点突 然変異が見つ力つた力 すこし離れた位置にあった転移巣と思われる組織からはこ の変異は検出されず、第 248コドンに変異が見つ力つた。このことから、転移巣の癌 組織は原発巣力 の転移ではなぐ由来が異なる癌細胞であることが示唆された。  [0083] In Case 2, the force of the point mutation was found in the 245th codon (codon encoding glycine) in the immediate vicinity of the main lesion. This mutation was not detected, and a strong mutation was found at codon 248. This suggests that the cancer tissue of the metastatic lesion is a cancer cell with a different origin from the metastasis of the primary focal force.

Claims

請求の範囲 The scope of the claims
[1] 被験者力 の第一の検体に含まれる疾患に関連する細胞またはこれに由来する細 胞を、第二の検体において検出する方法であって、  [1] A method for detecting in a second specimen a cell related to a disease contained in the first specimen of subject power or a cell derived therefrom,
(a)第一の検体に含まれるゲノムにおいて、前記疾患に関連する遺伝子変異を検出 する工程、  (a) detecting a genetic mutation associated with the disease in the genome contained in the first specimen;
(b)第二の検体に含まれるゲノムにおいて、前記遺伝子変異を検出する工程、なら びに  (b) a step of detecting the genetic mutation in the genome contained in the second specimen, and
(c)第一の検体力 検出された遺伝子変異と第二の検体力 検出された遺伝子変異 とを比較する工程  (c) Step of comparing the first specimen force detected gene mutation and the second specimen force detected gene mutation
を含んでなり、検出された遺伝子変異が相互に同一である場合に、第一の検体に含 まれる疾患関連細胞またはこれに由来する細胞が第二の検体力 検出されたことが 示される、方法。  When the detected gene mutations are identical to each other, it is indicated that the disease-related cells contained in the first specimen or cells derived therefrom are detected in the second specimen force. Method.
[2] 第一の検体が前記疾患の治療前に採取されたものであり、第二の検体が前記疾患 の治療中または治療後に採取されたものである、請求項 1に記載の方法。  [2] The method according to claim 1, wherein the first specimen is collected before the treatment of the disease, and the second specimen is collected during or after the treatment of the disease.
[3] 前記治療が、薬物療法、放射線療法、温熱療法、ホルモン療法、免疫療法、およ び外科的治療力もなる群より選択されるものである、請求項 2に記載の方法。  [3] The method according to claim 2, wherein the treatment is selected from the group consisting of drug therapy, radiation therapy, hyperthermia, hormone therapy, immunotherapy, and surgical therapeutic power.
[4] 第一の検体が前記疾患の病巣部力 採取されたものであり、第二の検体が他の部 位力 採取されたものである、請求項 1に記載の方法。  [4] The method according to claim 1, wherein the first specimen is obtained by collecting the force of the lesion of the disease, and the second specimen is obtained by collecting another force of the part.
[5] 前記疾患関連細胞が、癌細胞または類癌腫細胞である、請求項 1に記載の方法。  5. The method according to claim 1, wherein the disease-related cell is a cancer cell or a carcinoma cell.
[6] 遺伝子変異の検出が核酸増幅法によって行なわれる、請求項 1に記載の方法。  [6] The method according to claim 1, wherein the detection of the gene mutation is performed by a nucleic acid amplification method.
[7] 前記核酸増幅法が、等温下での反応によって標的核酸の増幅を可能とするもので ある、請求項 6に記載の方法。  [7] The method according to claim 6, wherein the nucleic acid amplification method enables amplification of a target nucleic acid by a reaction under isothermal conditions.
[8] 核酸増幅法に用いられるプライマーセットに含まれる第一のプライマーが、標的核 酸配列の 3 '末端部分の配列 (A)にハイブリダィズする配列 (Ac )を 3，末端部分に含 んでなり、かつ前記標的核酸配列にお 、て前記配列 (A)よりも 5 '側に存在する配列 (B)の相補配列（Be)にハイブリダィズする配列（Β')を前記配列 (Ac )の 5，側に含ん でなるものである、請求項 7に記載の方法。  [8] The first primer included in the primer set used in the nucleic acid amplification method comprises a sequence (Ac) that hybridizes to the sequence (A) at the 3 'end of the target nucleic acid sequence, and 3 at the end. In addition, in the target nucleic acid sequence, a sequence (ハ イ ブ リ ′) that hybridizes to a complementary sequence (Be) of the sequence (B) existing 5 ′ to the sequence (A) is represented by 5 in the sequence (Ac). The method according to claim 7, comprising:
[9] 核酸増幅法に用いられるプライマーセットが、標的核酸配列を増幅しうる少なくとも 2種のプライマーを含んでなるプライマーセットであって、 [9] At least the primer set used in the nucleic acid amplification method can amplify the target nucleic acid sequence. A primer set comprising two kinds of primers,
前記プライマーセットに含まれる第一のプライマーが、標的核酸配列の 3'末端部分 の配列 (A)にノ、イブリダィズする配列 (Ac')を 3'末端部分に含んでなり、かつ前記標 的核酸配列にぉ 、て前記配列 (A)よりも 5'側に存在する配列（B)の相補配列（Be) にハイブリダィズする配列（Β')を前記配列 (Ac )の 5'側に含んでなるものであり、 前記プライマーセットに含まれる第二のプライマーカ、前記標的核酸配列の相補配 列の 3 '末端部分の配列 (C)にハイブリダィズする配列 (Cc')を 3，末端部分に含んで なり、かつ相互にノ、イブリダィズする 2つの核酸配列を同一鎖上に含む折返し配列（ D-Dc )を前記配列（Cc )の 5'側に含んでなるものである、請求項 7に記載の方法。  The first primer included in the primer set comprises a sequence (Ac ′) that is to be hybridized to the sequence (A) at the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and the target nucleic acid sequence. The sequence comprises a sequence (Β ') that hybridizes to the complementary sequence (Be) of the sequence (B) existing 5' to the sequence (A) on the 5 'side of the sequence (Ac). A second primer included in the primer set, a sequence (Cc ') that hybridizes to a sequence (C) of the 3' end portion of the complementary sequence of the target nucleic acid sequence, and 3 at the end portion. And a folded sequence (D-Dc) containing two nucleic acid sequences that are mutually and non-hybridized on the same strand, on the 5 'side of the sequence (Cc). Method.
[10] 前記プライマーセットが、前記標的核酸配列またはその相補配列にハイブリダィズ する第三のプライマーであって、標的核酸配列またはその相補配列へのハイブリダィ ゼーシヨンについて他のプライマーと競合しない第三のプライマーをさらに含んでな るものである、請求項 9に記載の方法。  [10] The primer set is a third primer that hybridizes to the target nucleic acid sequence or its complementary sequence, and a third primer that does not compete with other primers for hybridization to the target nucleic acid sequence or its complementary sequence. 10. The method of claim 9, further comprising:
[11] 核酸増幅法において鎖置換能を有するポリメラーゼが使用される、請求項 7に記載 の方法。  [11] The method according to claim 7, wherein a polymerase having strand displacement ability is used in the nucleic acid amplification method.
[12] 核酸増幅法が融解温度調整剤の存在下で行われる、請求項 7に記載の方法。  [12] The method according to claim 7, wherein the nucleic acid amplification method is performed in the presence of a melting temperature adjusting agent.
[13] 融解温度調整剤が、ジメチルスルホキシド、ベタイン、ホルムアミドもしくはグリセ口 ール、またはこれらの 2種以上の混合物である、請求項 12に記載の方法。 [13] The method according to claim 12, wherein the melting temperature adjusting agent is dimethyl sulfoxide, betaine, formamide or glycerol, or a mixture of two or more thereof.
[14] 核酸増幅反応がミスマッチ結合タンパク質の存在下で行われる、請求項 7に記載の 方法。 [14] The method of claim 7, wherein the nucleic acid amplification reaction is performed in the presence of a mismatch binding protein.
[15] 被験者における疾患の治療の効果を評価する方法であって、  [15] A method for assessing the effect of treatment of a disease in a subject comprising:
(a)被験者力 前記疾患の治療前に採取した第一の検体に含まれるゲノムにお！、て 、前記疾患に関連する遺伝子変異を検出する工程、  (a) Subject power The genome contained in the first sample collected before treatment of the disease! Detecting a genetic mutation associated with the disease,
(b)被験者力 前記疾患の治療中または治療後に採取した第二の検体に含まれる ゲノムにおいて、前記遺伝子変異を検出する工程、ならびに  (b) subject power a step of detecting the gene mutation in a genome contained in a second specimen collected during or after treatment of the disease, and
(c)第一の検体力 検出された遺伝子変異と第二の検体力 検出された遺伝子変異 とを比較する工程  (c) Step of comparing the first specimen force detected gene mutation and the second specimen force detected gene mutation
を含んでなり、検出された遺伝子変異が相互に異なるか、または第二の検体におい て前記遺伝子変異が検出されない場合に、治療の効果が高いものと評価される、方 法。 And the detected genetic variation is different from each other or in the second specimen If the gene mutation is not detected, the method is evaluated as having a high therapeutic effect.
[16] 前記治療が、薬物療法、放射線療法、温熱療法、ホルモン療法、免疫療法、およ び外科的治療力もなる群より選択されるものである、請求項 15に記載の方法。  [16] The method of claim 15, wherein the treatment is selected from the group consisting of drug therapy, radiation therapy, hyperthermia, hormonal therapy, immunotherapy, and surgical therapeutic power.
[17] 遺伝子変異の検出が核酸増幅法によって行なわれる、請求項 15に記載の方法。  [17] The method according to claim 15, wherein the detection of the gene mutation is performed by a nucleic acid amplification method.
[18] 前記核酸増幅法が、等温下での反応によって標的核酸の増幅を可能とするもので ある、請求項 17に記載の方法。  [18] The method according to claim 17, wherein the nucleic acid amplification method enables amplification of a target nucleic acid by a reaction under isothermal conditions.
[19] 核酸増幅法に用いられるプライマーセットに含まれる第一のプライマーが、標的核 酸配列の 3 '末端部分の配列 (A)にハイブリダィズする配列 (Ac )を 3，末端部分に含 んでなり、かつ前記標的核酸配列にお 、て前記配列 (A)よりも 5 '側に存在する配列 (B)の相補配列（Be)にハイブリダィズする配列（Β')を前記配列 (Ac )の 5，側に含ん でなるものである、請求項 18に記載の方法。  [19] The first primer included in the primer set used in the nucleic acid amplification method comprises a sequence (Ac) that hybridizes to the sequence (A) at the 3 'end of the target nucleic acid sequence, and 3 at the end. In addition, in the target nucleic acid sequence, a sequence (ハ イ ブ リ ′) that hybridizes to a complementary sequence (Be) of the sequence (B) existing 5 ′ to the sequence (A) is represented by 5 in the sequence (Ac). 19. The method of claim 18, wherein the method comprises:
[20] 核酸増幅法に用いられるプライマーセットが、標的核酸配列を増幅しうる少なくとも 2種のプライマーを含んでなるプライマーセットであって、  [20] The primer set used in the nucleic acid amplification method is a primer set comprising at least two kinds of primers capable of amplifying the target nucleic acid sequence,
前記プライマーセットに含まれる第一のプライマーが、標的核酸配列の 3'末端部分 の配列 (A)にノ、イブリダィズする配列 (Ac')を 3'末端部分に含んでなり、かつ前記標 的核酸配列にぉ 、て前記配列 (A)よりも 5'側に存在する配列（B)の相補配列（Be) にハイブリダィズする配列（Β')を前記配列 (Ac )の 5'側に含んでなるものであり、 前記プライマーセットに含まれる第二のプライマーカ、前記標的核酸配列の相補配 列の 3 '末端部分の配列 (C)にハイブリダィズする配列 (Cc')を 3，末端部分に含んで なり、かつ相互にノ、イブリダィズする 2つの核酸配列を同一鎖上に含む折返し配列（ D-Dc )を前記配列（Cc )の 5'側に含んでなるものである、請求項 18に記載の方法。  The first primer included in the primer set comprises a sequence (Ac ′) that is to be hybridized to the sequence (A) at the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and the target nucleic acid sequence. The sequence comprises a sequence (Β ') that hybridizes to the complementary sequence (Be) of the sequence (B) existing 5' to the sequence (A) on the 5 'side of the sequence (Ac). A second primer included in the primer set, a sequence (Cc ') that hybridizes to a sequence (C) of the 3' end portion of the complementary sequence of the target nucleic acid sequence, and 3 at the end portion. 19. A folded sequence (D-Dc) comprising two nucleic acid sequences that are mutually and non-hybridized on the same strand on the 5 ′ side of the sequence (Cc). Method.
[21] 前記プライマーセットが、前記標的核酸配列またはその相補配列にハイブリダィズ する第三のプライマーであって、標的核酸配列またはその相補配列へのハイブリダィ ゼーシヨンについて他のプライマーと競合しない第三のプライマーをさらに含んでな るものである、請求項 20に記載の方法。  [21] The primer set is a third primer that hybridizes to the target nucleic acid sequence or its complementary sequence, and a third primer that does not compete with other primers for hybridization to the target nucleic acid sequence or its complementary sequence 21. The method of claim 20, further comprising:
[22] 核酸増幅法において鎖置換能を有するポリメラーゼが使用される、請求項 18に記 載の方法。 [22] The method according to claim 18, wherein a polymerase having strand displacement ability is used in the nucleic acid amplification method.
[23] 核酸増幅法が融解温度調整剤の存在下で行われる、請求項 18に記載の方法。 [23] The method according to claim 18, wherein the nucleic acid amplification method is performed in the presence of a melting temperature adjusting agent.
[24] 融解温度調整剤が、ジメチルスルホキシド、ベタイン、ホルムアミドもしくはグリセ口 ール、またはこれらの 2種以上の混合物である、請求項 23に記載の方法。 [24] The method according to claim 23, wherein the melting temperature adjusting agent is dimethyl sulfoxide, betaine, formamide or glycerol, or a mixture of two or more thereof.
[25] 核酸増幅反応がミスマッチ結合タンパク質の存在下で行われる、請求項 18に記載 の方法。 [25] The method according to claim 18, wherein the nucleic acid amplification reaction is performed in the presence of a mismatch binding protein.
[26] 被験者における疾患の原発巣力 他の部位への転移を評価する方法であって、  [26] A method for evaluating the primary focus of disease in a subject to metastasis to another site,
(a)被験者の前記疾患の原発巣力 採取した第一の検体に含まれるゲノムにおいて 、前記疾患に関連する遺伝子変異を検出する工程、  (a) the primary focal force of the disease of the subject in the genome contained in the collected first specimen, detecting a genetic mutation associated with the disease,
(b)被験者の他の部位力 採取した第二の検体に含まれるゲノムにぉ 、て、前記遺 伝子変異を検出する工程、ならびに  (b) another site force of the subject, detecting the gene mutation in the genome contained in the second sample collected, and
(c)第一の検体力 検出された遺伝子変異と第二の検体力 検出された遺伝子変異 とを比較する工程  (c) Step of comparing the first specimen force detected gene mutation and the second specimen force detected gene mutation
を含んでなり、検出された遺伝子変異が相互に同一である場合に、疾患の原発巣か ら他の部位への転移があるものと評価される、方法。  And wherein the detected genetic mutation is identical to each other, the method is evaluated as having metastasis from the primary lesion site to another site.
[27] 前記疾患が癌または腫瘍である、請求項 26に記載の方法。 [27] The method of claim 26, wherein the disease is cancer or tumor.
[28] 遺伝子変異の検出が核酸増幅法によって行なわれる、請求項 26に記載の方法。 [28] The method according to claim 26, wherein the detection of the gene mutation is performed by a nucleic acid amplification method.
[29] 前記核酸増幅法が、等温下での反応によって標的核酸の増幅を可能とするもので ある、請求項 28に記載の方法。 [29] The method according to claim 28, wherein the nucleic acid amplification method enables amplification of a target nucleic acid by a reaction under isothermal conditions.
[30] 核酸増幅法に用いられるプライマーセットに含まれる第一のプライマーが、標的核 酸配列の 3 '末端部分の配列 (A)にハイブリダィズする配列 (Ac )を 3，末端部分に含 んでなり、かつ前記標的核酸配列にお 、て前記配列 (A)よりも 5 '側に存在する配列[30] The first primer included in the primer set used in the nucleic acid amplification method comprises a sequence (Ac) that hybridizes to the sequence (A) at the 3 'end of the target nucleic acid sequence, and 3 at the end. And a sequence present 5 'to the target nucleic acid sequence from the sequence (A)
(B)の相補配列（Be)にハイブリダィズする配列（Β')を前記配列 (Ac )の 5，側に含ん でなるものである、請求項 29に記載の方法。 30. The method according to claim 29, comprising a sequence (Β ′) that hybridizes to the complementary sequence (Be) of (B) on the 5th side of the sequence (Ac).
[31] 核酸増幅法に用いられるプライマーセットが、標的核酸配列を増幅しうる少なくとも[31] The primer set used in the nucleic acid amplification method is at least capable of amplifying the target nucleic acid sequence.
2種のプライマーを含んでなるプライマーセットであって、 A primer set comprising two kinds of primers,
前記プライマーセットに含まれる第一のプライマーが、標的核酸配列の 3'末端部分 の配列 (A)にノ、イブリダィズする配列 (Ac')を 3'末端部分に含んでなり、かつ前記標 的核酸配列にぉ 、て前記配列 (A)よりも 5'側に存在する配列（B)の相補配列（Be) にハイブリダィズする配列（Β')を前記配列 (Ac )の 5'側に含んでなるものであり、 前記プライマーセットに含まれる第二のプライマーカ、前記標的核酸配列の相補配 列の 3 '末端部分の配列 (C)にハイブリダィズする配列 (Cc')を 3，末端部分に含んで なり、かつ相互にノ、イブリダィズする 2つの核酸配列を同一鎖上に含む折返し配列（ D-Dc )を前記配列（Cc )の 5'側に含んでなるものである、請求項 29に記載の方法。 The first primer included in the primer set comprises a sequence (Ac ′) that is to be hybridized to the sequence (A) at the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and the target nucleic acid sequence. Complementary sequence (Be) of sequence (B) existing 5 ′ from sequence (A) A sequence (Β ′) that hybridizes to the 5 ′ side of the sequence (Ac), the second primer included in the primer set, and the 3 ′ end of the complementary sequence of the target nucleic acid sequence. The sequence (Cc ') that hybridizes to the sequence (C) of the part 3 and the folded sequence (D-Dc) that contains two nucleic acid sequences on the same strand, which are included in the end part and mutually cross and hybridize. 30. The method according to claim 29, comprising 5 'of the sequence (Cc).
[32] 前記プライマーセットが、前記標的核酸配列またはその相補配列にハイブリダィズ する第三のプライマーであって、標的核酸配列またはその相補配列へのハイブリダィ ゼーシヨンについて他のプライマーと競合しない第三のプライマーをさらに含んでな るものである、請求項 31に記載の方法。  [32] The primer set is a third primer that hybridizes to the target nucleic acid sequence or a complementary sequence thereof, and a third primer that does not compete with other primers for hybridization to the target nucleic acid sequence or the complementary sequence. 32. The method of claim 31, further comprising:
[33] 核酸増幅法において鎖置換能を有するポリメラーゼが使用される、請求項 29に記 載の方法。  [33] The method according to claim 29, wherein a polymerase having strand displacement ability is used in the nucleic acid amplification method.
[34] 核酸増幅法が融解温度調整剤の存在下で行われる、請求項 29に記載の方法。  [34] The method according to claim 29, wherein the nucleic acid amplification method is performed in the presence of a melting temperature adjusting agent.
[35] 融解温度調整剤が、ジメチルスルホキシド、ベタイン、ホルムアミドもしくはグリセ口 ール、またはこれらの 2種以上の混合物である、請求項 34に記載の方法。 [35] The method according to claim 34, wherein the melting temperature adjusting agent is dimethyl sulfoxide, betaine, formamide or glycerol, or a mixture of two or more thereof.
[36] 核酸増幅反応がミスマッチ結合タンパク質の存在下で行われる、請求項 29に記載 の方法。 [36] The method according to claim 29, wherein the nucleic acid amplification reaction is performed in the presence of a mismatch binding protein.
PCT/JP2005/020963 2004-11-15 2005-11-15 Diagnostic method using nucleic acid amplification WO2006051990A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US62718104P 2004-11-15 2004-11-15
US60/627,181 2004-11-15
JP2005-129830 2005-04-27
JP2005129830A JP2008029205A (en) 2005-04-27 2005-04-27 Diagnostic method using nucleic acid amplification

Publications (1)

Publication Number Publication Date
WO2006051990A1 true WO2006051990A1 (en) 2006-05-18

Family

ID=36336655

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2005/020963 WO2006051990A1 (en) 2004-11-15 2005-11-15 Diagnostic method using nucleic acid amplification

Country Status (1)

Country Link
WO (1) WO2006051990A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008136436A (en) * 2006-12-04 2008-06-19 Fujifilm Corp Method for detecting variation of nucleic acid by using protein binding single-stranded dna
WO2009107816A1 (en) * 2008-02-29 2009-09-03 独立行政法人理化学研究所 Method for increasing enzymatic reactivity

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NOMOTO SHIMETSUGU ET AL: "Suigan ni okeru K-ras Gan Idenshi no ten Totsuzen Hen'i to Bishoten Isu no Kensaku.", IGAKU NO AYUMI., vol. 186, no. 6,7, 15 August 1998 (1998-08-15), pages 392 - 393, XP002998449 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008136436A (en) * 2006-12-04 2008-06-19 Fujifilm Corp Method for detecting variation of nucleic acid by using protein binding single-stranded dna
WO2009107816A1 (en) * 2008-02-29 2009-09-03 独立行政法人理化学研究所 Method for increasing enzymatic reactivity
JP5515121B2 (en) * 2008-02-29 2014-06-11 独立行政法人理化学研究所 How to improve enzyme reactivity

Similar Documents

Publication Publication Date Title
EP1712618B1 (en) Method of amplifying nucleic acid and method of detecting mutated nucleic acid using the same
CN108138209B (en) Method for preparing cell-free nucleic acid molecules by in situ amplification
US20150126376A1 (en) Compositions and methods for sensitive mutation detection in nucleic acid molecules
CN110719957B (en) Methods and kits for targeted enrichment of nucleic acids
KR20070011354A (en) Detection of strp, such as fragile x syndrome
JPH09512428A (en) Detection of mutations by resorbase cleavage
JP6100933B2 (en) Allelic ladder locus
US6335165B1 (en) Methods and kits for characterizing GC-rich nucleic acid sequences
JP2014507950A (en) Kits and methods for sequencing target DNA in mixed populations
JP4216266B2 (en) High-sensitivity known mutant gene detection method and EGFR mutant gene detection method
JP4743787B2 (en) Methods for methylation and detection of cytosine in DNA
KR20130099092A (en) Method for inhibiting nucleic acid amplification using light and highly sensitive method for selective nucleic acid amplification
WO2000043531A9 (en) Methods and kits for characterizing gc-rich nucleic acid sequences
JP3942627B2 (en) Mutant nucleic acid detection method
JPWO2017170644A1 (en) Gene mutation detection method
JP2008161165A (en) Method for detecting gene using competing oligonucleotide
JP2006254784A (en) Method for decreasing nonspecific amplification from primer dimer
WO2019062614A1 (en) A method of amplifying a target nucleic acid
WO2006051990A1 (en) Diagnostic method using nucleic acid amplification
JP2007319096A (en) Nucleic acid amplification method utilizing nicking activity of endonuclease
EP4074840A1 (en) Pcr method and pcr kit for increasing allelic discrimination
JP2008029205A (en) Diagnostic method using nucleic acid amplification
WO2006051991A1 (en) Method of amplifying and detecting nucleic acid
JP2008161164A (en) Method for detecting gene with primer containing artificial mismatched nucleic acid
WO2018079579A1 (en) Method for detecting target base sequence, method for designing and producing probe, and kit

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LC LK LR LS LT LU LV LY MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 05806932

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP