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WO2001012849A1 - Procede de distinction d'acides nucleiques et kits pour l'analyse d'acides nucleiques - Google Patents

Procede de distinction d'acides nucleiques et kits pour l'analyse d'acides nucleiques Download PDF

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Publication number
WO2001012849A1
WO2001012849A1 PCT/JP2000/005286 JP0005286W WO0112849A1 WO 2001012849 A1 WO2001012849 A1 WO 2001012849A1 JP 0005286 W JP0005286 W JP 0005286W WO 0112849 A1 WO0112849 A1 WO 0112849A1
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Prior art keywords
nucleic acid
labeled
label
stranded
strand
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PCT/JP2000/005286
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English (en)
Japanese (ja)
Inventor
Akio Yamane
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Wakunaga Pharmaceutical Co., Ltd.
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Application filed by Wakunaga Pharmaceutical Co., Ltd. filed Critical Wakunaga Pharmaceutical Co., Ltd.
Priority to AU63204/00A priority Critical patent/AU6320400A/en
Publication of WO2001012849A1 publication Critical patent/WO2001012849A1/fr

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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer

Definitions

  • the present invention relates to a method for identifying a nucleic acid and a test kit, and more particularly, to a simple method that does not require a solid-liquid separation operation from a sample by determining the presence and proportion of a nucleic acid having a gene mutation or polymorphism from a sample.
  • the present invention relates to a method for identifying a nucleic acid which can be directly detected and quantified in a short time by a simple operation, and a test kit for performing the identification method.
  • a detection method using an oligonucleotide probe Proc. Nat 1. Acad. Sci. 80, 278, ( 198 3)
  • a method using restriction enzyme fragment length polymorphism analysis RFLP method
  • RFLP method restriction enzyme fragment length polymorphism analysis
  • a method for cleaving a single base mismatch in a hybrid of RNA and DNA using ribonuclease Science 230, 1243 (19895)
  • Natl. Acad. Sci. 88, 189 (1991) Anal. Bioche m. 1886, 64-4-6 8 (1990)).
  • SSCP single-strand conformation ionpolymorphism
  • DGGE single-strand conformation ionpolymorphism
  • ddF ddF
  • any of the above methods has a problem that the reproducibility is low and the electrophoresis method is used for the detection, so that the method lacks quickness and is not practical.
  • a method of determining the base sequence of the target nucleic acid by using an automatic sequencer and detecting the mutation can be considered, but it is difficult to detect when the sample is a mixture or the ratio of the mutant nucleic acid is small. It was possible. In recent years, microchip-type measurement technology using a very small amount of pitting technology has been developed, and is expected to be applied to new drug research and gene polymorphism analysis.
  • This method detects mutated nucleic acids by immobilizing tens of thousands of DNA probes on a fine carrier surface and hybridizing with a labeled sample (Nucleic Acids Res. 2Q). 6, 4975-4982 (19998)), which can process a large number of specimens at once, which can save a great deal of labor, but has low reproducibility and special equipment There is a problem when the cost is high because of the use of
  • the applicant of the present invention has proposed a method of adding an excessive amount of unlabeled standard DNA to a labeled sample DNA and performing cooperative competitive hybridization.
  • a method that can detect the presence or absence of a gene mutation or polymorphism regardless of the type of nucleic acid and can easily calculate the abundance ratio hereinafter abbreviated as PCR-PHFA method.
  • PCR-PHFA PCR-PHFA method
  • it is possible to reliably detect or quantify a mutation or polymorphism gene that is present only in a small amount in a normal gene or even when the type of mutation or polymorphism is unspecified.
  • it is very effective in detecting nucleic acids having genetic mutations and polymorphisms such as genes related to genetic diseases or cancer-related genes (British J. Hemato 1 ogy 95, 198— 203).
  • the PCR-PHFA method requires complicated solid-liquid separation work after competitive hybridization, and gene mutation or multiplication by simple operations at the forefront medical site. It has not been able to adequately respond to the demand for rapid detection of nucleic acids having a form.
  • the relationship between genetic mutations and diseases is very complex and diverse, and the rare cases in which a single genetic mutation found at the outset causes disease are relatively rare and unexpectedly many. It is becoming clear that the gene mutations involved in the onset of the disease are evident.To investigate the gene mutations that cause a certain disease, it is necessary to check many mutations for multiple genes without fail. Therefore, development of a nucleic acid detection method that can automatically and easily process a large amount of a sample more easily and quickly is desired.
  • the present invention has been made in view of the above circumstances, and the presence and the ratio of nucleic acid having a gene mutation or polymorphism can be determined in a short time directly by a simple operation that does not require a solid-liquid separation operation from a sample.
  • An object of the present invention is to provide a nucleic acid identification method that can be accurately detected and quantified and that can be automated, and a test kit for performing the identification method.
  • a first nucleic acid and a second nucleic acid are mixed and subjected to competitive hybridization, and the degree of substitution of complementary strand between the two nucleic acids is measured.
  • At least one of two types of labels capable of energy transfer to each other is introduced into the 3 ′ end of one strand of the first nucleic acid, which is a double-stranded nucleic acid, and the other strand of the first nucleic acid is
  • the first nucleic acid is labeled by introducing the other label at the 5 'end of the nucleic acid, and the labeled first nucleic acid is mixed with the unlabeled second nucleic acid, which is a single-stranded or double-stranded nucleic acid, and mixed.
  • first nucleic acid prepared by introducing one of at least two kinds of labels capable of energy transfer to each other into the first nucleic acid, and label prepared by introducing the other label into the second nucleic acid
  • the second nucleic acid is mixed with the following combinations (a) to (c) to perform competitive hybridization, and the degree of energy change due to the energy transfer between the labels is measured. By doing so, the degree of replacement of the complementary strand generated between the first nucleic acid and the second nucleic acid can be measured.
  • Both the first nucleic acid and the second nucleic acid are double-stranded nucleic acids, and a labeled first nucleic acid having one label introduced at the 3 ′ end of one strand, and a label introduction of the labeled first nucleic acid A combination with a labeled second nucleic acid in which the other label is introduced at the 5 'end of the strand to be hybridized with the strand,
  • the PCR-PHFA method proposed by the present applicant can amplify a specific region of a target nucleic acid in a sample to prepare a double-stranded sample nucleic acid, and bind to one of the strands with a solid phase carrier.
  • Competitive hybridization is carried out by adding an excess amount of the sample nucleic acid to a labeled standard nucleic acid comprising a double-stranded nucleic acid having a unique site and a detectable label on the other strand, and as a result, By detecting the reconstituted labeled standard nucleic acid using the detectable label and the label having a site capable of binding to a solid support,
  • a nucleic acid having a gene mutation or polymorphism is detected.
  • this method uses a detectable label and a label having a site capable of binding to a solid phase carrier, a cumbersome solid-liquid separation operation is required, and the operation is simple. It has not been widely adopted in the medical field where quick and accurate detection is required.
  • the above-described identification method of the present invention at least two kinds of labels capable of energy transfer with each other are used, and the degree of energy change due to the energy transfer between the two labels is measured. Since the degree of displacement of the complementary strand due to the hybridization is measured, the first nucleic acid and the first nucleic acid can be quickly and easily collected without complicated work such as solid-liquid separation. (2) The identity with the nucleic acid can be identified, and both labels are introduced at the 3 'end and the 5' end which are close to each other, so that the degree of displacement of the complementary strand can be accurately and accurately determined.
  • the degree of substitution of the complementary strand can be accurately and accurately measured with good sensitivity at all times, and the identity of the nucleic acids can be determined. Accurate and Ru 3D in what can and this identifies the Joteki.
  • one of the first nucleic acid and the second nucleic acid is used as a sample nucleic acid containing a target nucleic acid, and the other is used as a standard nucleic acid for identifying the identity of the sample nucleic acid.
  • nucleic acid having a gene mutation or polymorphism in the sample is extremely small, or if the normal nucleic acid differs from the mutant nucleic acid (wild-type gene and mutant gene) by only one base, it is accurate. It can be detected and quantified by simple operations.
  • the 5 'end and the 3' end indicate a range within 30 bases from the 5 'end and the 3' end of the nucleic acid chain, respectively. .
  • energy transfer is more likely to occur closer to the 5 'end and the 3' end, so it is preferably within 10 bases from each end, and most preferably the 5 'end and the 3' end. The end.
  • the present invention provides a kit for performing the identification method of the present invention
  • a nucleic acid having a trace amount of a mutation or a polymorphic gene in a sample can be rapidly and reliably detected by a simple operation according to the identification method of the present invention, and automation can be achieved. , At the forefront of medical care
  • FIG. 1 is a schematic diagram showing a case where the method for identifying a nucleic acid according to the first invention is performed, wherein (A) shows a normal nucleic acid and (B) shows a nucleic acid having a mutation.
  • FIG. 2 is a schematic diagram showing a case where the method for identifying a nucleic acid according to the second invention is performed.
  • (A) shows a normal nucleic acid
  • (B) shows a nucleic acid having a mutation.
  • 2 is a fluorescence vector showing the state of energy transfer between the two types of labels shown in FIG.
  • FIG. 4 is an explanatory diagram showing the conventional method of Ge1fand. BEST MODE FOR CARRYING OUT THE INVENTION
  • the method for identifying a nucleic acid comprises the steps of: mixing a first nucleic acid and a second nucleic acid; performing implicit hybridization; and measuring the degree of occurrence of complementary strand displacement.
  • the method for identifying the identity between the first nucleic acid and the second nucleic acid at least two labels capable of energy transfer to each other are introduced into one or both of the first nucleic acid and the second nucleic acid.
  • Two types of labels for example, a donor label that generates fluorescence upon excitation and an receptor label that absorbs the fluorescence
  • a competitive hybridization is performed. (Complementary strand displacement reaction).
  • one of the two types of labels is introduced as a double-stranded nucleic acid at the 3 ′ end of one strand of the first nucleic acid, and the other strand of the first nucleic acid is The other label is introduced into the 5 ′ end of the nucleic acid to prepare a labeled first nucleic acid, and the labeled first nucleic acid is mixed with a double-stranded or single-stranded unlabeled second nucleic acid to form a compound.
  • the first nucleic acid can be obtained by performing pettive hybridization and measuring the degree of change in energy transfer between the labels.
  • the degree of substitution of the complementary strand generated between 20 and the second nucleic acid is measured to discriminate the identity between the first nucleic acid and the second nucleic acid.
  • Fig. 1 (A) two types of labels D and A capable of binary energy rearrangement (donor label D that generates energy and energy from the donor label are absorbed) Axceptor label A)
  • the unlabeled second nucleic acid 2 (which is a double-stranded nucleic acid in FIG. 1 but may be a single-stranded nucleic acid) which is homologous to the labeled first nucleic acid 1 is mixed and denatured, and then annealed.
  • FIG. 1 (B) a similar hybrid-stranded first nucleic acid 1 is mixed with an unlabeled second nucleic acid 2 ′ having a mutation X to perform a competitive hybridization. Since the complementary strand displacement reaction does not occur, the proportion of nucleic acids that cause energy translocation does not change (as is).
  • the degree of displacement of the complementary strand generated between the first nucleic acid 1 and the second nucleic acid 2 or 2 ′ is determined.
  • the identity between the first nucleic acid 1 and the second nucleic acid 2 or 2 ′ can be identified.
  • One of the first nucleic acid 1 and the second nucleic acid 2 or 2 ′ is used as a sample nucleic acid containing the target nucleic acid, and the other is used as a standard nucleic acid for identifying the identity of the sample nucleic acid.
  • the presence / absence of the gene mutation in the sample nucleic acid and the ratio thereof can be measured.
  • a nucleic acid having a known mutation as the first nucleic acid 1 was prepared, and the first nucleic acid 1 having this mutation and the second nucleic acid 2 or 2 ′ were mixed to form a competitive nucleic acid.
  • the presence or absence of the mutation in the gene in the second nucleic acid 2 or 2 ′ and the ratio thereof can be similarly detected.
  • the second method of the identification method of the present invention comprises the steps of: combining a label first nucleic acid prepared by introducing at least one of two kinds of labels capable of energy transfer with each other into the first nucleic acid;
  • the labeled nucleic acid prepared by introduction into the second nucleic acid The two nucleic acids are mixed with the following combinations (a) to (c) to perform a competitive hybridization, and the degree of energy change due to energy-translocation between the labels is measured.
  • the degree of substitution of the complementary strand generated between the first nucleic acid and the second nucleic acid is measured to discriminate the identity between the first nucleic acid and the second nucleic acid.
  • Both the first nucleic acid and the second nucleic acid are double-stranded nucleic acids, and a labeled first nucleic acid in which one label is introduced into the 3 ′ end of one strand, and a label of the labeled first nucleic acid A combination of the introduced strand and a labeled second nucleic acid in which the other label is introduced at the 5 'end of the strand to be hybridized.
  • the two types of energy transferable labels D and A are replaced with the 3 ′ end of one strand of the first nucleic acid 3 and the second nucleic acid 4 ′ having a mutation X.
  • Competitive hybridization is performed by mixing and denaturing the labeled first nucleic acid 3 and the labeled second nucleic acid 4 ', followed by annealing. In this case, since no complementary strand displacement reaction occurs, no nucleic acid in which the two types of labels D and A are close to each other is generated, and no energy transfer occurs.
  • the degree of substitution of the complementary strand generated between the first nucleic acid 3 and the second nucleic acid 4 or 4 ′ can be determined.
  • the identity between the first nucleic acid 3 and the second nucleic acid 4 or 4 ′ can be identified.
  • one of the first nucleic acid 3 and the second nucleic acid 4 or 4 ′ is used as a sample nucleic acid containing the target nucleic acid, and the other is used as a standard nucleic acid for identifying identity with the sample nucleic acid. It can measure the presence or absence of the gene mutation in the sample nucleic acid and its ratio.
  • a nucleic acid having a known mutation was prepared as the first nucleic acid 3, and the first nucleic acid 3 having the mutation was mixed with the second nucleic acid 4 or 4 ′, and The presence or absence of the mutation in the gene in the second nucleic acid 4 or 4 ′ and the ratio thereof can be similarly detected by performing the competitive hybridization.
  • one of the nucleic acids of the first nucleic acid 3 and the second nucleic acids 4 and 4 ′ in FIG. 2 is a single-stranded nucleic acid.
  • the principle is the same as that of the double-stranded nucleic acid shown in FIG.
  • the nucleic acid identification method of the present invention directly measures the degree of change in energy transfer between at least two kinds of labels capable of performing energy transfer. It is possible to detect the presence or absence of the nucleic acid having the gene mutation or polymorphism in the sample and the ratio thereof quickly and reliably by a simple operation without the necessity.
  • both labels are introduced at the 3 'end and the 5' end which are close to each other, even when the degree of substitution of the complementary strand is small, a sufficient energy change that can be easily detected is generated, and correct
  • the degree of substitution can be accurately and reliably measured with good sensitivity even if the first or second nucleic acid is a gene fragment having a long chain.
  • one of the first nucleic acid and the second nucleic acid is used as a sample nucleic acid containing a target nucleic acid, and the other is used as a standard nucleic acid for identifying identity with the sample nucleic acid. It is preferably used for detecting the presence or absence of a nucleic acid having a gene mutation or polymorphism and its ratio.
  • a specific region of the target nucleic acid in the sample is amplified to prepare a sample nucleic acid, and a standard nucleic acid having the same region as the specific region of the target nucleic acid is prepared.
  • the target nucleic acid to be detected includes cancer-related genes, genes related to genetic diseases, virus genes, bacterial genes, and polymorphisms called disease risk factors. And the like.
  • examples of the cancer-related gene include a k-ras gene, an N-ras gene, a p53 gene, a BRCA1 gene, a BRCA2 gene, and an APC gene.
  • Genes related to genetic diseases include various inborn errors of metabolism.
  • examples of viruses and bacterial genes include hepatitis C virus and hepatitis B virus.
  • Genes that show polymorphism are genes that have different nucleotide sequences depending on individuals that are not directly related to the cause of the disease, such as HLA (Human Leukocyte Antigen) and blood type genes.
  • HLA Human Leukocyte Antigen
  • blood type genes Alternatively, there are genes that are considered to be involved in the onset of hypertension, diabetes and the like. These genes are usually present on the chromosome of the host, but may be encoded by the mitochondrial gene.
  • specimens containing the target nucleic acid include pathogens such as bacteria and viruses, blood, saliva, tissue debris isolated from living bodies, and excrement such as manure.
  • pathogens such as bacteria and viruses
  • fetal cells present in amniotic fluid or a part of dividing egg cells in a test tube can be used as a sample.
  • these samples may be centrifuged directly or as necessary. After being concentrated as a sediment by a separation operation or the like, it is possible to use, for example, an enzyme treatment, a heat treatment, a surfactant treatment, an ultrasonic treatment, or a cell treatment that has been subjected to cell destruction treatment by a combination thereof in advance. it can.
  • the cell destruction treatment is performed for the purpose of revealing the DNA derived from the target tissue.
  • the specific method of the cell disruption treatment is as follows: PCR protocol / less Academic Press Ink pl 4, p 352 (1990) (PCRPROTOCOLSAcademic Press Inc., pl 4 , P 352 (1990)), etc., according to known methods described in literatures. It is preferable that the total amount of DNA in the sample is about 1 to 10 g, but amplification can be sufficiently performed with 1 ⁇ g or less. ⁇
  • the sample nucleic acid and the standard nucleic acid are obtained by a known PCR (Polymerase Chain Reaction) method, L-R (lgasechain Reaction) 3 ⁇ 4 3 SR (Self-sustained S) prepared by the equence Rep 1 ication) method, SDA (Strand D isp 1 acement Amp 1) method, etc. (Manak, DNA Probes 2nd Editionp 255 to 291, Stokton Press) (1993)), PCR is particularly preferred.
  • the primer for amplifying the sample nucleic acid and the standard nucleic acid is, as long as the sample nucleic acid and the standard nucleic acid are present, a primer extension.
  • a gene amplification reaction based on the reaction occurs.
  • the extension reaction of the primers was carried out with four or five nucleotide triphosphates (deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate, and thymidine triphosphate).
  • the reaction is carried out by incorporating an acid or dextrinidine triphosphate (a mixture of these as dNTP, or a mixture thereof)) into the primer as a substrate.
  • an amplification reaction reagent containing the above-mentioned unit nucleic acid and nucleic acid elongation enzyme is usually used to amplify the nucleic acid chain.
  • the nucleic acid elongation enzyme is E. coli DNA polymerase. Any DNA polymerase, such as Klenow fragment of E. coli DNA polymerase I, or DNA polymerase 4 can be used.
  • thermostable DNA polymerase such as th DNA polymerase and Vent DNA polymerase, which eliminates the need to add a new enzyme for each cycle. This makes it possible to automatically repeat the cycle, and furthermore, it is possible to set the annealing temperature to 50 to 60 ° C, thereby improving the specificity of the target base sequence recognition by the primer. Rapid and specific gene amplification (For details, refer to JP-A-11-314965 and JP-A-252230).
  • oil can be added to prevent evaporation of water in the reaction solution.
  • the oil may be any oil that can be distributed with water and has a lower specific gravity than water, and specific examples include silicone oil and mineral oil.
  • some gene amplification devices do not require such a medium, and a primer extension reaction can be performed using such a gene amplification device.
  • the target nucleic acid in the sample can be efficiently amplified and the sample nucleic acid can be prepared in a large amount.
  • a large amount of a standard nucleic acid whose identity with the target nucleic acid is to be identified can be similarly prepared.
  • Specific methods such as conditions for carrying out the gene amplification reaction are described in Experimental Medicine, Yodosha, 8, No. 9 (1990), and PCR Technology Stockton. Pressing can be performed according to a known method described in a literature such as a press (PCR Technology Locktonpress (1989)).
  • nucleic acid may be directly cut out enzymatically, or the amplified normal nucleic acid may be added to a vector selected from a plasmid vector, a phage vector, or a plasmid and phage chimera vector. It can also be prepared in large quantities by integration into a host such as Escherichia coli, Bacillus subtilis, or any other proliferable host such as yeast (gene Cloung). Further, in some cases, it can be prepared by chemical synthesis. Examples of chemical synthesis include the ester method and the phosphite method. These methods are based on a liquid phase method or a solid phase synthesis method using an insoluble carrier. 392)), a large amount of single-stranded DNA can be prepared, and then annealing can be performed to prepare double-stranded DNA.
  • One of the sample nucleic acid and the standard nucleic acid thus prepared in a large amount as described above is used as the first nucleic acid, and the other is used as the second nucleic acid, and one or both of the first and second nucleic acids are used as energy. Introduce at least two transposable labels.
  • the energy transfer between the labels in the present invention means that at least two types of labels, a donor label for generating energy and an axebuta label for absorbing energy generated from the donor label, are mutually separated.
  • the transfer of energy from a donor label to an acceptor label when in close proximity when the two types of labels are fluorescent labels, the fluorescent label generated by exciting the donor label is absorbed by the receptor label, and the force for measuring the fluorescence emitted by the receptor label, or the fluorescence generated by exciting the donor label is generated.
  • the quenching of the donor label caused by the absorption of the fluorescence by the receptor label can be measured (PCRM ethodsandapp 1 ications 4, 35 7 — 3 62 (1995), Nature).
  • the at least two kinds of labels are not particularly limited as long as they are capable of energy transfer in a state close to each other. Among them, a fluorescent substance and a delayed fluorescent substance are preferable, and in some cases, a chemiluminescent substance, Bioluminescent materials can also be used.
  • Such label combinations include fluororesin and its derivatives (for example, fluorescein isothiosinate) and rhodamine and its derivatives (for example, tetramethyl rhodamin isothiosinate, tetramethyl rhodamine). 5 — (and — 6-) hexanoic acid acid, etc.), fluorescein and dabsir, and any combination can be selected from these (Nonisotopic DNAP robe ⁇ echniques. Academic Press 1992) No.
  • a general method of introducing a label into a nucleic acid may be employed. It can. For example, a method for directly introducing a labeled substance into a nucleic acid (Biotechniques 24, 484-489 (1998)), a DNA polymerase reaction or an RNA polymerase reaction. Method of introducing labeled mononucleotides (Science 238, 336-33341 (19897)), and performing a PCR reaction using a primer into which the labeled substance has been introduced. (PCRM ethodsand Applications 2, 34-40 (1992)).
  • the position where the label is introduced into the sample nucleic acid and the standard nucleic acid is the position where energy transfer occurs or disappears due to the complementary strand displacement reaction, that is, the 3 ′ of the nucleic acid strand.
  • each nucleic acid strand when a large number of labels are introduced into the base portion that hybridizes with the complementary strand, Since the substitution of about a group may become undetectable, it is preferable to introduce only into the end of each nucleic acid strand.
  • one of the two types of labels is introduced at the 5 'end (3' end) of one nucleic acid strand, and the 3 'end (5' end) of the other nucleic acid strand complementary thereto is introduced. If the other label is introduced into the second strand, both nucleic acid strands undergo energy transfer or disappear by the complementary strand displacement reaction without affecting the hybridization reaction.
  • a method in which a PCR reaction is performed using a primer having a label at the 5' end (PCRM ethodsand Applications 2, 34) -40 (1992)) or a method in which a linker having a label introduced at the 5 'end and an arbitrary nucleic acid strand are bound by ligase (Nuc 1 eic Acids Res. , 9 2 2 — 9 2 3 (1 9 9 7)).
  • a linker having a label introduced at the 3 ′ end and an arbitrary nucleic acid strand are prepared in the same manner as when the label is introduced at the 5 ′ end.
  • the nucleic acid chain is RNA instead of DNA, or when the 3 ′ end of DNA is RNA, it is generated by selectively opening the sugar (ribose) portion of the RNA at that end.
  • Labeling can also be carried out using an aldehyde group:
  • the labeled mononucleotide triphosphate can be used to terminate the nucleic acid strand by the action of terminal hydroxy nucleotidyl transferase. It is also possible to introduce a marker at the 3 'end (Biotechniques 15, 486-496 (1993)).
  • the labeled nucleic acid can be prepared by direct chemical synthesis. (Nucleic A cids Res. 16, 2659-2669 (1988), Bioconjug. Chem. 3, 85-87 (1992)) c Next, competitive hybridization is performed using the sample nucleic acid and the standard nucleic acid (the first nucleic acid and the second nucleic acid) into which the label has been introduced as described above.
  • the competitive hybridization in the present invention refers to between a double-stranded nucleic acid having a homologous base sequence and a single-stranded nucleic acid, or between a double-stranded nucleic acid having a homologous base sequence and a double-stranded nucleic acid.
  • a competitive nucleic acid strand displacement reaction that takes place between denaturing and annealing single- and double-stranded nucleic acids, or multiple double-stranded nucleic acids.
  • the sample nucleic acid and Z or the standard nucleic acid first nucleic acid and / or second nucleic acid
  • the time when the sample nucleic acid and the standard nucleic acid are mixed may be either immediately before the denaturation or after the denaturation.
  • the temperature conditions of the competitive hybridization are appropriately set according to the length and base sequence of the nucleic acid to be hybridized, but are usually in the range of 98 to 50 ° C for 3 to 10 minutes.
  • the temperature can be reduced at a rate of 1 ° C, more preferably in the range of 98-70 ° C, at a rate of 1 ° C in 10 minutes.
  • the method for identifying a nucleic acid according to the present invention comprises at least two kinds of targets as described above.
  • a competitive hybridization is performed to gradually lower the temperature from a high temperature.
  • a nucleic acid having a gene mutation or a polymorphism is detected by performing annealing and measuring the degree of change in energy transfer between the labels.
  • a nucleic acid having a completely complementary base sequence forms a double strand more preferentially than a nucleic acid having a gene mutation or a polymorphism, and accordingly, the energy change between the labels.
  • the degree of change in energy due to the displacement that is, the degree of change in energy transposition that occurs or disappears due to the complementary strand displacement reaction, is measured using an arbitrary detector, so that gene mutation can be detected.
  • the presence or absence of the nucleic acid having the polymorphism and the ratio thereof can be detected.
  • gene mutation or multiple mutations can be measured by measuring the fluorescence spectrum at a specific wavelength with a spectrofluorometer, fluorescence blade reader, or the like.
  • the presence or absence of a nucleic acid having a type can be easily detected.
  • At least two types of labels that can be energy-transferred in close proximity to each other are used.
  • One of the two labels is introduced into the 3 ′ end of one strand of the first nucleic acid, which is a double-stranded nucleic acid, and the other label is attached to the 5 ′ end of the other strand of the first nucleic acid.
  • a double-stranded or single-stranded unlabeled second nucleic acid is mixed with the labeled first nucleic acid to perform competitive hybridization, and the above-described labeling is performed.
  • the degree of substitution of the complementary strand generated between the first nucleic acid and the second nucleic acid is measured to determine the identity between the first nucleic acid and the second nucleic acid.
  • the identity of the standard nucleic acid and the sample nucleic acid, Presence and proportion thereof of nucleic acids having a mutation or polymorphism of the child can be a detection child a.
  • the labeled first nucleic acid prepared by introducing the one label into the first nucleic acid and the labeled second nucleic acid prepared by introducing the other label into the second nucleic acid are as follows.
  • the above-mentioned first nucleic acid is obtained by mixing in a combination of (a) to (c), performing a competitive hybridization, and measuring the degree of energy change due to energy-transposition between the labels.
  • the degree of displacement of the complementary strand generated between the first nucleic acid and the second nucleic acid is measured to identify the identity between the first nucleic acid and the second nucleic acid, that is, the identity between the standard nucleic acid and the sample nucleic acid, and It is possible to detect the presence or absence of a nucleic acid having a gene mutation or polymorphism and its ratio.
  • the first nucleic acid and the second nucleic acid are both double-stranded nucleic acids, and a labeled first nucleic acid having one label introduced at the 3 ′ end of one strand; and a label introduction of the labeled first nucleic acid. Combination with a labeled second nucleic acid having the other label introduced at the 5 'end of the strand to be hybridized with the strand.
  • the degree of energy transfer is significantly changed according to the ratio of the wild-type gene and the mutant gene in the sample. If a standard curve is prepared in advance and determined, the ratio of the wild-type gene to the mutant gene can be easily known.
  • the test kit for nucleic acid identification of the present invention is a test kit for detecting the presence and proportion of nucleic acid having a gene mutation or polymorphism according to the above-described identification method of the present invention.
  • a sample nucleic acid is prepared from a specific region of the target nucleic acid of the sample that has been subjected to pretreatment such as cell destruction treatment.
  • a standard nucleic acid having a base sequence complementary to the sample nucleic acid is prepared, and the sample nucleic acid and the standard nucleic acid are used as the first nucleic acid and the second nucleic acid, respectively, and at least one of which can be energy-transferred to one or both of them.
  • the complementation is achieved. It measures the extent to which strand displacement has occurred.
  • a sample nucleic acid amplification reagent for amplifying a specific region of the target nucleic acid in the sample to prepare a sample nucleic acid, and a standard for preparing a standard nucleic acid for identifying the identity of the sample nucleic acid and the target nucleic acid Identification of the nucleic acid of the present invention by combining a nucleic acid amplification reagent and a reagent for introducing at least two kinds of labels capable of energy transfer into one or both of a sample nucleic acid and a standard nucleic acid, And a quantitative inspection kit.
  • the cell disrupting reagent for sample pretreatment the washing solution for washing the amplification reaction product, the oil for preventing the evaporation of the water in the reaction solution, and two kinds of labels described in the above-described nucleic acid identification method of the present invention.
  • a reagent or the like for measuring the degree of change in energy transfer between the two can be used, and in combination therewith, the inspection kit of the present invention can be used.
  • CF i 'R cysticfibrosis transmembrane conductance regulator
  • cystic fibrosis cystic fibrosis
  • PCR A chain reaction
  • the PCR was performed according to a conventional method [denaturation: 30 sec at 94 ° C., annealing: 54. The cycle of 30 sec at C and extension: 6 sec at 72 ° C] was repeated 35 times. After the reaction, the PCR reaction solution was measured by 3% agarose gel electrophoresis. In the wild type PCR reaction solution, a band was detected at a length of 44 bases. In the PCR reaction solution of the ⁇ 508 mutant, a band was detected at a length of 41 bases.
  • TFA-amino link CE phosphoramidite manufactured by PerkinElmer Japan
  • TAMRA was added.
  • NHS Perkin-Elmer Japan
  • TAMRA group Tetramethyllodin-1-5— (and—6—) hexanoyctic acid
  • Oligonucleotides into which these fluorescent labels (FITC, TAMRA) were introduced were purified using high performance liquid chromatography (HPLC) and used as labeled oligonucleotides.
  • HPLC high performance liquid chromatography
  • oligonucleotides with amino groups introduced were purified using polyacrylamide gel electrophoresis. And used as unlabeled oligonucleotide. Labeled and unlabeled oligonucleotides are named as follows and are shown together with their nucleotide sequences.
  • a double-stranded DNA was prepared using the above-mentioned labeled and unlabeled oligonucleotide by the following method.
  • Wild-type labeled oligonucleotide CF 10 WSN 1 Dissolves TAMRA (30 nmol) and CF 10 WSN 2-FITC (30 nmo 1) (10 mM MTris-HC 1 p . H 8 0 5 0 m MN a C 1 1 m MEDTA l O ng / ⁇ ⁇ herring DNA) was dissolved in 1 0 0 mu 1, di - N'anpu PCR system 9 6 0 0 (Pas one rk (p e rkin The temperature was lowered continuously to 98 ° C and 60 ° C over 4 hours using a commercial product (Elmer), and the resulting mixture was subjected to arranging. The wild-type labeled double-stranded DNA (A) described below was used. Was prepared.
  • No. 1 has 10 equivalents of 10 (A) complementary to each strand of the wild-type labeled double-stranded DNA.
  • No. 2 is 10 equivalents of ⁇ 508 non-labeled DNA to the wild-type labeled double-stranded DNA of (A). Is added to perform a complementary strand displacement reaction.
  • No.1 the nucleotide sequences of labeled and unlabeled DNA are exactly the same.
  • wild-type unlabeled DNA and ⁇ 508 mutant can be obtained by adding wild-type and ⁇ 508 mutant unlabeled DNA to wild-type labeled double-stranded DNA and performing complementary strand displacement reaction. It was confirmed that it could be distinguished from unlabeled DNA. Also, when comparing No. 3 and No. 4, the labeled double-stranded DNA
  • Example 2 As shown in Table 2, an experiment similar to that in Example 1 was performed by reducing the amount of labeled double-stranded DNA and the amount of unlabeled DNA to 1 Z10 equivalent. Using a fluorescence plate reader (fluorester (BMG Labtechnologies GmbH), excitation filter: 485 nm, fluorescence filter: 538 nm) for microtiter array The fluorescence intensity was measured. In addition, it measured as a control without adding unlabeled DNA. Table 2 shows the results.
  • a plasmid containing the nucleotide sequence of wild-type exon 10 (pCF10-3) and a plasmid containing the nucleotide sequence of the ⁇ 508 mutant exon 10 (PCF508-1) ) was used as a template to perform a polymerase chain reaction (PCR) using the following pair of primers (CF10 ETP, CF10 ETM).
  • the PCR was performed according to a conventional method [denaturation: 30 sec at 94 C, annealing ring: 54]. A cycle of 30 sec at C and elongation: 60 sec at 72 ° C] was repeated 35 times. After the reaction, the PCR reaction solution was measured by 3% agarose gel electrophoresis. In the reaction solution from the plasmid containing the nucleotide sequence of wild-type exon 10, a band was detected at a length of 44 bases. In addition, in the reaction solution from the plasmid containing the base sequence of exon 10 of the ⁇ 508 mutant, a band was detected at a length of 41 bases.
  • a complementary strand displacement reaction was performed between the PCR reaction solution prepared above and the labeled double-stranded DNAs (A) and ( ⁇ ) prepared in Example 1, and a fluorescent plate reader was prepared in the same manner as in Example 3.
  • the fluorescence intensity was measured using [Bolenostar (BMGL abtechno 1 ogies GmbH), excitation filter: 485 nm, fluorescence filter: 538 nm]. Table of results Shown in 3
  • No. 6 using the ⁇ 508 mutant-labeled double-stranded DNA complementary to the ⁇ 508 mutant PCR reaction solution is a complementary strand displacement reaction.
  • the fluorescence intensity is higher than that of No. 5 using ⁇ 508 mutant double-stranded DNA that differs from the wild-type PCR reaction solution by 3 bases.
  • nucleic acids having a small amount of gene mutation or polymorphism in a sample can be easily and easily treated in a homogeneous system that does not require complicated solid-liquid separation. Has many mutations or polymorphisms in the sample It can simultaneously detect and quantify nucleic acids in a short time.
  • a nucleic acid having a mutation or a polymorphism of a trace gene in a sample can be rapidly and reliably detected by a simple operation according to the identification method of the present invention. Automation is also possible, which is extremely useful in medical settings.
  • a small amount of mutation or polymorphism of a gene contained in a specimen can be reliably detected, and a gene showing the mutation or polymorphism can be quantified. It is extremely useful for early detection, diagnosis and treatment of cancer and specific viruses and bacterial infections in the field, and determination of the success or failure of bone marrow transplantation and the presence / absence of rejection.

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Abstract

L'invention concerne un procédé de détection et de quantification précises et rapides d'un changement ou d'un polymorphisme dans plusieurs gènes contenus dans plusieurs spécimens, par une opération pratique permettant la détermination de la présence/absence d'un acide nucléique présentant une modification ou un polymorphisme et de la proportion de celle-ci/celui-ci dans les gènes contenu dans une faible quantité de ces spécimens. Ledit procédé consiste à mélanger, dans un système homogène ne nécessitant pas de procédures de séparation compliquées solide/liquide, d'un premier acide nucléique contenant l'acide nucléique cible avec un deuxième acide nucléique possédant une séquence de base complémentaire d'un domaine spécifique de l'acide nucléique cible, de sorte qu'une hybridation compétitive soit assurée ; à mesurer l'ampleur de la substitution du brin complémentaire entre ces acides nucléiques, et à distinguer ainsi l'identité du premier acide nucléique de celle du deuxième acide nucléique. Dans ledit procédé, au moins deux marqueurs capables de transférer mutuellement de l'énergie sont introduits dans l'un des acides nucléiques ou les deux, et l'ampleur du changement d'énergie induit par le transfert d'énergie entre les deux marqueurs conjointement avec la substitution de brin complémentaire susmentionnée, est mesurée, ce qui permet l'évaluation de l'ampleur de la substitution de brin complémentaire.
PCT/JP2000/005286 1999-08-12 2000-08-07 Procede de distinction d'acides nucleiques et kits pour l'analyse d'acides nucleiques WO2001012849A1 (fr)

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JP5504676B2 (ja) * 2009-03-31 2014-05-28 凸版印刷株式会社 遺伝子型の識別方法
JP5568935B2 (ja) * 2009-09-30 2014-08-13 凸版印刷株式会社 標的塩基配列の識別方法
WO2011122501A1 (fr) 2010-03-29 2011-10-06 凸版印刷株式会社 Méthode de distinction d'une séquence de bases cible

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010113452A1 (fr) * 2009-03-31 2010-10-07 凸版印刷株式会社 Procédé pour différencier des génotypes
CN102369297A (zh) * 2009-03-31 2012-03-07 凸版印刷株式会社 识别基因型的方法
JP5720564B2 (ja) * 2009-03-31 2015-05-20 凸版印刷株式会社 遺伝子型の識別方法
CN102369297B (zh) * 2009-03-31 2016-07-06 凸版印刷株式会社 识别基因型的方法
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