[go: up one dir, main page]

WO2004003199A1 - Methode d'obtention d'un acide nucleique ou d'un gene - Google Patents

Methode d'obtention d'un acide nucleique ou d'un gene Download PDF

Info

Publication number
WO2004003199A1
WO2004003199A1 PCT/JP2003/008311 JP0308311W WO2004003199A1 WO 2004003199 A1 WO2004003199 A1 WO 2004003199A1 JP 0308311 W JP0308311 W JP 0308311W WO 2004003199 A1 WO2004003199 A1 WO 2004003199A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
primer probe
obtaining
reaction
species
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/JP2003/008311
Other languages
English (en)
Japanese (ja)
Inventor
Shinya Kurata
Yoichi Kamagata
Yuji Sekiguchi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
National Institute of Advanced Industrial Science and Technology AIST
Kankyo Engineering Co Ltd
Original Assignee
National Institute of Advanced Industrial Science and Technology AIST
Kankyo Engineering Co Ltd
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
Application filed by National Institute of Advanced Industrial Science and Technology AIST, Kankyo Engineering Co Ltd filed Critical National Institute of Advanced Industrial Science and Technology AIST
Publication of WO2004003199A1 publication Critical patent/WO2004003199A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present invention relates to a method for searching for and obtaining a novel useful nucleic acid or gene existing in one system in nature. Specifically, using a consensus base sequence for a nucleic acid species having a known identical function, the nucleic acid in the system is amplified, and a new or identically functional nucleic acid species is searched for from the amplified nucleic acid.
  • the present invention relates to a method for obtaining a novel useful nucleic acid (including a gene) from among them. Background art
  • an object of the present invention is to detect and measure a plurality of useful nucleic acid groups having the same function simultaneously, quickly, simply, and with high sensitivity from all nucleic acids existing in one system in nature. And next!
  • the present invention provides a method for selectively and quickly and easily obtaining only those novel and useful nucleic acids by using (1) / (2).
  • the present inventors conducted nucleic acid amplification using a nucleic acid probe labeled with a substance such as a fluorescent substance or a quencher substance and having a consensus base sequence to a known nucleic acid, and performed the nucleic acid concentration or amplification before amplification. Copy number measurement, analysis of temperature dissociation curve of amplification products, It was found that the above problems could be solved by combining type analysis, base sequence analysis, anchor PCR method, and the like. The present invention has been completed based on strong knowledge.
  • a method for obtaining a nucleic acid comprising at least the following steps:
  • nucleotide sequence is decoded and determined based on the nucleic acid species preferentially in concentration or the broad nucleic acid species specified as a new or the same functional nucleic acid species in the above 2), and identified as the new or the same functional nucleic acid species.
  • a new or identically functional nucleic acid species is specified from among the amplified nucleic acid species thus obtained.
  • nucleic acid species containing at least a partial nucleotide sequence or a full-length nucleotide sequence of a nucleic acid species of the same or new function is obtained.
  • a method for obtaining a nucleic acid comprising at least the following steps:
  • linker 11 and linker 12 At least one kind of linker pair (hereinafter, referred to as linker 11 and linker 12) is added to the above-mentioned restriction enzyme-treated product 1), and a ligation reaction is performed. 3) Divide the reaction product described in 2) into two (one is A and the other is B), and perform the following reaction.
  • A For A, perform the following reactions (a) and (b) simultaneously or sequentially: (a) At least one antisense primer probe is added to A, and a nucleic acid extension reaction is performed.
  • a nucleic acid extension reaction is performed by adding at least one kind of antisense adapter primer probe to A or the reaction product of the above (a).
  • At least one kind of sense primer probe is added to B, and a nucleic acid extension reaction is performed.
  • At least one kind of sense adapter primer probe is added to B or the reaction product of the above (c) to perform a nucleic acid extension reaction.
  • nucleic acid treated with the restriction enzyme is a nucleic acid treated with the restriction enzyme for the polymorphism analysis according to the above 1,
  • An acquisition method is provided.
  • FIG. 1 is a diagram showing a state where a restriction enzyme fragment of a nucleic acid and a linker are bound.
  • FIG. 2 shows a temperature dissociation curve of a QP-PCR product.
  • FIG. 3 is a diagram showing a curve obtained by differentiating the temperature dissociation curve of FIG. -O- No. 1 -m- No. 4 -0- No. 7
  • Fig. 4 shows the T-RFLP pattern of sample (sample) No. 4.
  • FIG. 5 is a diagram showing a T-RFLP pattern of sample (sample) No. 5.
  • At least one type means one or more types.
  • nucleic acid or gene is collectively referred to simply as “nucleic acid”.
  • nucleic acid species refers to a group of nucleic acids and / or genes having identical functions.
  • the “function” means biologically significant. And it has the function of nucleic acid (DNA, RNA) itself and its expression product (mRNA, protein).
  • mRNA, protein examples of the function of the nucleic acid itself include, in the case of DNA, a base sequence other than structural genes such as a promoter, a regulator, a terminator, and the like.
  • RNA the base sequence of the active site of an enzyme such as ribozyme can be mentioned.
  • Wear In the case of an expression product, for example, a base sequence that controls the structure of the protein and a base sequence that controls the enzyme activity and the like can be mentioned. These are merely examples of some of the functions to explain them. Therefore, the present invention is not limited by these examples.
  • Concentration-preferred nucleic acid species '' means, when multiple nucleic acid species are present, the highest (high) or relatively high concentration in terms of concentration based on the electrophoresis electrophoresis pattern or as a result of quantification. (Approximately 1 to 5 from the ranking) It is a nucleic acid species. It does not distinguish between before and after nucleic acid amplification. -In the present invention, the “nucleic acid contained in a sample” may be simply referred to as a target nucleic acid or a target nucleic acid.
  • the term "measuring nucleic acid” or “measuring nucleic acid concentration” refers to not only quantifying the concentration of a target nucleic acid, but also performing quantitative detection, qualitative detection, It simply means the ability to simply measure or simply monitor the fluorescence intensity of the nucleic acid amplification system.
  • the data thus obtained was analyzed by the method of Kurata et al. (EP Patent Publication, EP1 046 717 A9), and the concentration (copy number, etc.) existing in one system was analyzed. ) Shall be included.
  • nucleic acid contained in the sample” of the present invention is not limited to a specific nucleic acid for the purpose of detection and measurement, but refers to an unspecified nucleic acid that can be unintentionally detected by the method of the present invention. Shall be included. Of course, it includes genes and the like. These nucleic acids may be mixed. Regardless of the concentration. That is, specific and unspecified detectable nucleic acids that are considered to be useful in the present and future, present in one system. Nucleic acids include DNA, RNA and their modifications. In the present invention, these nucleic acids may be referred to as target nucleic acids.
  • the consensus primer probe means that the nucleotide sequence of the primer probe hybridizing to the complementary nucleotide sequence is part or all of a consensus nucleotide sequence site recognized from a group of known nucleic acid species having the same function. Let's look at the primer probe that hybridizes.
  • the “consensus sequence” is defined as defined in molecular biology and includes consensus sequences with known nucleic acids, as well as consensus sequences with known nucleic acids.
  • linkers linker 1 and linker 1
  • an antisense primer probe an antisense primer probe
  • sense primer probe an antisense primer probe
  • sense Or specifically, the antisense adapter primer probe
  • optical character refers to various absorption spectra or fluorescence emission spectra of fluorescent substances and quencher substances that label primer probes, and their absorption intensity, polarization, fluorescence emission, fluorescence intensity, fluorescence lifetime. Characteristics such as fluorescence, polarization and anisotropy! /, U (when collectively referred to as "fluorescence intensity", there is power S.) It also refers to the property obtained by comprehensively evaluating the measurement values of at least one fluorescent substance or the like, which is labeled on a primer probe, at at least one or more measurement wavelengths. For example, a fluorescence intensity curve of a nucleic acid denaturation reaction is one of them.
  • the term “from the change or the amount of change in the fluorescence intensity” refers to a homogenous solution labeled with a fluorescent substance and Z or toluene on the amplified nucleic acid, not only the change in the fluorescence intensity based on the amplified nucleic acid of the present invention.
  • the system nucleic acid probe is hybridized, the change or the amount of change in the fluorescence intensity before and after the hybridization is included.
  • a complex of a primer probe and a nucleic acid by hybridization of a primer probe and a corresponding nucleic acid is referred to as a hybrid, a hybrid complex, or simply a nucleic acid-primer complex or a primer. It is called a nucleic acid complex.
  • Nucleic acid polymerases have the ability to synthesize nucleic acids. Typical examples are DNA polymerases, RNA polymerases, reverse transcriptases (reverse
  • DNA polymerase it may or may not have exonuclease activity. It may be either purified or unpurified crude enzyme.
  • the origin of the enzyme is not particularly limited. Preferably, those having heat resistance are preferred.
  • the amino acid sequence of the enzyme may be modified by genetic engineering, and then included.
  • Preferred examples include ent (exo-) DNA Polymerase (from Thermococcus litoralis, Tgo (exo-) D Polymerase ⁇ ThermoSequenase DNA
  • the fluorescent substance (sometimes referred to as a fluorescent dye) in the present invention is a kind of fluorescent substance generally used for measurement and detection of nucleic acid, which is labeled on a nucleic acid probe.
  • fluorescein or derivatives thereof for example, fluorescein isothiocyanate (FITC) or derivatives thereof
  • Alexa 488 Alexa 532
  • Cy3, Cy5, 6-joe EDANS
  • Rhodamine 6G R6G
  • tetramethylrhodamine TMR
  • tetramethylrinorhodamine isothiocyanate tetramethylrhodamine
  • BODIPY isothiocyanate
  • x- rhodamine x- rhodamine
  • Texas Red Texas red
  • Bodepi BODIPY
  • FL trade name: Morekiyura 1 probe (Molecular Probes) Co., Ltd., the United States; BODIPY tips Rere The same applies hereinafter.
  • BODIPY BODIPY FL / OS ⁇ BODIPY (BODIPY) FL / ce.
  • BODIPY PODIPY
  • TAMRA TAMRA
  • AMRA AMRA
  • FITC EDANS, Texas Red, 6-joe, TMR, Alexa 488, Alexa 532, BODIPY FL / C3, BODIPY R6G, BODIPY (BODIPY) FL, BODEPY ( BODIPY) FL C6, BODIPY (TMR), TMD, BODIPY (BODIPY) 5-FAM, BODIPY (BODIPY) 493,503, BODIPY (BODIPY) 564, BODIPY (BODIPY) 581, Cy3, Cy5, x-Rhodamine, etc. It can be cited as suitable.
  • a quencher substance is a substance that acts on the fluorescent substance to suppress or extinguish its light emission.
  • the luminescence of the fluorescent substance is subjected to a quenching effect by the quencher substance.
  • the present invention has four inventions, and is characterized by a combination of a nucleic acid amplification method, a Tm value analysis method, a polymorphism analysis method, and an anchor PCR method.
  • the sample containing at least one kind of nucleic acid referred to in the present invention can survive in any place in nature, for example, sewage, paddy fields, uplands, mountains, swamps, animal bodies, and various organs of animals. It is collected at a location or when it survives vigorously and cannot be specifically limited. It also includes various cells of various organisms. It may be one containing all the nucleic acids in the system or one containing-part. The nucleic acid may or may not be subjected to nucleic acid extraction. Nucleic acid is as defined above.
  • nucleic acid amplification reaction For each of these samples, perform a nucleic acid amplification reaction on at least one type of nucleic acid to confirm nucleic acid amplification. In this case, it is preferable to measure the concentration or copy number of the amplified nucleic acid before and / or after amplification at the same time as the amplification reaction.
  • the nucleic acid amplification method refers to a method for amplifying a nucleic acid in vitro.
  • PCR method LCR method (ligase chain reaction) s ⁇
  • AS force method AS force method
  • ICAN Isothermal and Chimeric primer-initiated Amplification of Nucleic acids
  • LAMP method LAMP method
  • NASR A method NASR A method
  • RCA method TAMA method
  • UCAN method Shall be included.
  • the PCR method may be of any type. For example, quantitative PCR methods, Anotime quantitative PCR method, RT-PCR, RNA-primed PCR, Stretch PCR, reverse PCR, PCR using Alu sequence, multiplex PCR, mixed primer probe PCR, and PCR using PNA can be performed.
  • the PCR method also includes a method of analyzing a melting curve or a method of analyzing nuclear sensitivity amplified by PCR.
  • a quantitative PCR method or a real-time quantitative PCR method is employed.
  • any primer can be used as long as the present invention can be achieved, and at least one type of forward-type and reverse-type primer probes can be used. It is preferable to use a consensus primer probe that also provides a mold force.
  • a nucleic acid amplification reaction is performed on the assumption that a nucleic acid having a consensus base sequence (a nucleic acid having the same function) exists in a sample.
  • the consensus primer probe is a fluorescent substance-labeled consensus primer probe labeled with at least one fluorescent substance in a forward type and / or a reverse type.
  • the at least one fluorescent substance-labeled consensus primer probe it is preferable to use at least one Q probe or sunrise probe. The details of the primer probe will be described later.
  • the present invention also relates to a fluorescent substance-labeled consensus primer probe comprising at least one forward-type or reverse-type fluorescent substance, each of which has a different consensus base sequence and a different fluorescent level.
  • a method for obtaining a nucleic acid using a fluorescent substance-labeled consensus primer probe labeled with a fluorescent substance that gives a luminescent color is also a method for obtaining a nucleic acid using a fluorescent substance-labeled consensus primer probe labeled with a fluorescent substance that gives a luminescent color.
  • the nucleic acid to be amplified may be a plurality of nucleic acid species having the same function.
  • nucleic acid species that have a A function a nucleic acid species having a B functions may be simultaneously amplified (where, A and B are not equal.) 0
  • a plurality of fluorescent substance-labeled consensus primer probes each having a different consensus base sequence and labeled with a fluorescent substance giving a different fluorescent emission color, a plurality of the same functional nucleic acid species are subjected to one reaction. It may be amplified by the system.
  • the concentration or copy of the amplified nucleic acid before and after amplification or after amplification can be measured irrespective of the type of nucleic acid in the sample, which is also preferable.
  • the amplified nucleic acid species a concentration-priority nucleic acid species before or after amplification is specified. That is, the amplified nucleic acid species amplified by using one consensus primer probe is not necessarily one species! / ⁇ . Thus, it is preferred to identify high-level nucleic acid species at high concentrations.
  • the amplification product is electrophoresed using a known 0.5% (w / v) to 1.5% (w / v) agarose gel or 6 to 8% (w / v) polyacrylamide gel ( Bioengineering Experiments, 97-163, 1993, edited by The Society of Biotechnology, Japan, published by Baifukan), or easily analyzed or analyzed by the general HPLC method (with various fillers) or the sequencer method. Can be identified.
  • the electrophoresis method, the HPLC method, and the sequencer method are preferably performed under conventionally known conditions! Examples of the sequencer include ABI 373A (ABI's sequencer),
  • ABI377 (Sequencer of ABI), Biofocus 3000 (Biorat) and the like can be mentioned.
  • This procedure involves gradually increasing the temperature of the amplified nucleic acid (eg, from 50 ° C to 95 ° C) until the nucleic acid is completely denatured at low temperatures, in which a short time interval (eg, (The interval corresponding to a temperature rise of 2 ° C to 0.5 ° C). Also in this case, it is preferable to use a fluorescent substance-labeled consensus primer probe. Appropriate data can be obtained. This is specifically shown in the following and Examples.
  • Tm value a specific temperature dissociation curve
  • Tm value a nucleic acid species showing a specific temperature dissociation curve
  • the identification method can be easily performed by analyzing or analyzing the amplified product by a known electrophoresis method, HPLC method, sequencer method or the like as described above.
  • the polymorphism analysis method may be any method as long as the object of the present invention can be achieved, and is preferably performed by a known method.
  • the object of the present invention can be achieved by using the T-RFLP method, the SSCP method, the CFLP method, the ARMS method, the direct-sequence method, and the MPH method.
  • the T-RFLP method can be suitably used in the present invention. Therefore, the method will be described below as one example of the present invention.
  • the gene is amplified by a quantitative PCR method using the Q probe as a primer probe, particularly a real-time quantitative PCR method, and the initial gene amount before amplification is measured. Then, the polymorphism is analyzed by the T-RFLP method based on the amplified nucleic acid.
  • the nucleic acid amplified using the Q probe as a primer probe has its 5 'end labeled with the fluorescent substance of the present invention.
  • the procedure includes the following steps (a) to (c).
  • the amplified nucleic acid is treated (digested) with a restriction enzyme.
  • restriction enzyme is not particularly limited.
  • the concentration used is also not particularly limited since it varies depending on the kind of the target restriction enzyme. As an example, 0.01 to 10 units / ⁇ l, preferably (here, 0.1 to 1. Ounits /; U1).
  • the above-mentioned processed product is analyzed and analyzed for gene fragments.
  • the polymorphism analysis method of the present invention only a gene fragment labeled with a fluorescent substance is analyzed and analyzed by an electrophoresis method, an HPLC method, a sequencer method, or the like. That is, each band and each peak are detected based on the fluorescence intensity value. Detection can be performed using conventional analytical instruments currently available on the market. For example, ABI 373A (sequencer from ABI), ABI 377 (sequencer from ABI), Biofocus 3000 (Biorat) and the like can be mentioned.
  • the appearance of a plurality of bands or a plurality of peaks in the analysis means that a polymorphism exists.
  • polymorphism does not exist.
  • the ratio of the fluorescence intensity value of each band or each peak is the abundance ratio of the polymorphism.
  • the amount of the target gene before PCR may be measured in some cases.Therefore, by multiplying the measured value by the ratio of the polymorphism, the initial presence of the polymorphism can be determined. The quantity will be required.
  • This polymorphism analysis' analysis 0 0 If because without using electrophoresis method can identify a new or the same functional nucleic acid species concentration Assured nucleic acid species in the amplified nucleic acid
  • the above procedure that is, the procedure may proceed to the next procedure only by any one of the items 1) to 3), but it is preferable to extend over a plurality of items.
  • these procedures make it possible to identify a new or the same functional nucleic acid species in the amplified nucleic acid.
  • the nucleic acid fragment obtained by the polymorphism analysis is partially decoded and determined.
  • cycle sequence reaction reagent kit
  • thermoSequenase (Amersham), AmpliTaqKS (Per km Elmer)) and a sequence determination reaction by a cycle sequence method (fluorescent substance-labeled primer probe method, fluorescent substance-labeled terminator method). It is convenient to carry out the reaction using a commercially available automatic fluorescence sequencer (row A; JBI, ABI3739 / 377 (PerkinElmer)). And the sequencer (such as PJ9600,
  • nucleotide sequence information obtained from the 'Decoding' of 4 above a new or identically functional nucleic acid species is specified from among the amplified nucleic acid species.
  • nucleic acid species having the same function is the same as a known nucleic acid species only based on information on the nucleotide sequence of the nucleic acid species having the same function determined in this procedure. This is because the amplified nucleic acid does not necessarily amplify the entire base sequence of the same functional nucleic acid species.
  • the primer probe may be in the same form as described in 1 above. The details are described later.
  • the amplification method can be performed in the same manner as in the above 1.
  • novel or functional nucleic acid species is ligated to a vector, and a clone library of the new or functional nucleic acid can be prepared.
  • a probe labeled with a fluorescent substance it is preferable to use a probe labeled with a fluorescent substance! This is because the product labeled with the fluorescent substance does not become a substrate for ligation with the vector, which is indispensable in the cloning and sequence steps.
  • Microbial cells eg, Escherichia coli, yeast, etc.
  • animal or plant cultured cells may be transformed with the novel or functional nucleic acid (gene) linked to the vector, and gene expression may be examined. Then, the expression level and / or the function of the expression product (eg, a physiological activity such as an enzyme activity) are measured.
  • the expression level and / or the function of the expression product eg, a physiological activity such as an enzyme activity
  • the nucleic acid may be directly inserted into the cell.
  • suitable vectors include YAC, BAC and the like.
  • the method of ligation to the vector may be performed by blunt-end cloning, TA cloning, clawing using a restriction enzyme, or the like.
  • the ligation method shown in the second invention You may do the same.
  • a host bacterium such as Escherichia coli can be used to prepare a clone library by a conventional method.
  • the procedure of the first invention has the following features.
  • the invention is preferably carried out in the following order with respect to the nucleic acid species contained in the sample.
  • the procedure 4 is performed in the order of higher nucleic acid species before or after amplification of the amplified nucleic acid.
  • the procedure 4 is performed in the order of higher nucleic acid species before and after amplification of the amplified nucleic acid having a specific temperature dissociation curve (Tm value).
  • the procedure 4 is performed in the order of nucleic acid species having higher concentrations before or after amplification of the amplified nucleic acid specified as a new or target nucleic acid.
  • the invention is carried out in the following order for a sample containing a nucleic acid.
  • the procedure 4 is performed in the order of samples containing nucleic acid species having a high concentration of the amplified nucleic acid before amplification.
  • the amplified nucleic acids identified as new or identically functional nucleic acid species are identified in the order of samples containing nucleic acid species with high concentrations before amplification, or as newly or identically functional nucleic acid species.
  • Step 4 is performed in the order of the samples containing a large number of amplified nucleic acid species.
  • Primer probes that can be suitably used in the PCR method of the present invention are as follows, for example.
  • Fluorescent substance whose fluorescence intensity changes when hybridized to the complementary base sequence of the target nucleic acid
  • Any type of probe can be used as long as it is a quality-labeled nucleic acid probe and can be used as a primer for PCR.
  • a consensus primer probe is suitable. Therefore, in the present invention, they are also referred to as consensus primer probes and consensus probes.
  • this probe can amplify many genes if the consensus base sequence is a promoter region.
  • the nucleotide sequence has a consensus or is common to the nucleotide sequence encoding the S protein of the probe, all the genes having the same function can be amplified.
  • primer probe examples include an oligonucleotide force, a free OH group at the 3-position of the terminal ribose or deoxyribose, and a phase of the target nucleic acid.
  • This is a fluorescent substance-labeled primer probe formed from an oligonucleotide containing a base sequence that hybridizes to a capture base sequence, and having the property of changing an optical character by hybridizing to a complementary base sequence.
  • the S primer probe one labeled with a fluorescent substance and / or a quencher substance, preferably an interaction between the fluorescent substances, preferably between the fluorescent substances, or between the fluorescent substance and G (Guayun) ), which are labeled with a fluorescent substance and Z or quencher, which interact with each other.
  • the Q probe KURATA et al., Ucleic acids Research, 2001, vol. 29, o. 6 e34
  • the optical character change is a fluorescence quenching phenomenon
  • the optical character change increases the fluorescence emission
  • Ucleic acid Research, 1292-1305 page, vol. 30, o. 6, 2002 azarenko et al., Ucleic acids Res., 25, 2516-2521, 1997.
  • a preferred example can be mentioned.
  • the present invention is not limited to this example.
  • the PCR method using the Q probe is called QP-PCR. Quenching primer or label means probe.
  • the preferred form of the Q probe has at least the following characteristics.
  • the whole primer probe may hybridize with the target nucleic acid. It is used as a primer for PCR by the hybridization.
  • the base sequence at the 5 ′ terminal site hybridizes with or does not hybridize with the complementary base sequence, and contains the base sequence.
  • the labeling position of the fluorescent substance may be either the sugar moiety on the fifth side and / or its phosphate moiety or base moiety.
  • the site is not necessarily the terminal or the site as long as it is on the 5 'side. Preferably it is the 5 'end.
  • a probe having a basic force C (cytosine) at the labeling site is desirable.
  • C basic force
  • the Q probe and the target nucleic acid hybridize they are separated from the base of the target nucleic acid complementary to the labeled base by 1 to 3 bases (the base of the target nucleic acid complementary to the labeled base is 1
  • G guanine
  • a G base may be present.
  • the C need not hybridize to the target nucleic acid in the sample. This is because G complementary to the C is synthesized during the nucleic acid amplification reaction.
  • An arbitrary base sequence may be interposed between C and the hybridizing base sequence.
  • the intervening base sequence has the number of bases;! To 30, preferably 1 to 20.
  • the nucleotide sequence may be any, but Suitably contains C.
  • the fluorescence intensity can be reduced by the above phenomenon when hybridized with the PCR amplification product. The decrease is proportional to the concentration of the corresponding nucleic acid.
  • the sunrise probe consists of a single-stranded oligonucleotide whose terminal or terminal site (preferably at the 5 'end) is a single fluorescent substance, and which itself emits fluorescence in another region (in the chain).
  • a quencher substance and an acceptor fluorescent substance (in this case, the terminal or the fluorescent substance at the terminal part is called a donor fluorescent substance)
  • a donor fluorescent substance is generally labeled.
  • the nucleic acid is not hybridized to the corresponding nucleic acid, a stem-loop structure is formed between the labeled bases due to the homology of the base sequence in the probe molecule. Due to the formation of the structure, the fluorescent substance and the quencher substance are arranged at positions close to each other.
  • the FRET phenomenon occurs, and the fluorescence emission of the fluorescent substance is suppressed.
  • the probe hybridizes to the corresponding nucleic acid, the stem 'loop structure is broken. Then, the FRET phenomenon is eliminated and the fluorescence emission of the light substance increases.
  • the concentration of the corresponding nucleic acid is proportional to the increase in the fluorescence intensity of the fluorescent substance in the measurement system.
  • a PCR method using the probe is called a sunrise PCR method.
  • the probe is designed so that at least the base sequence at the 3′-terminal site hybridizes to the target nucleic acid, and the 3′-terminal OH group of the 3′-terminal ribose or deoxyribose is free.
  • the whole primer probe may hybridize with the target nucleic acid. It is used as a primer for PCR by the hybridization.
  • part or all of the nucleotide sequence hybridizes to the complementary nucleotide sequence of the target nucleic acid.
  • any nucleotide sequence may be used as long as it contains a nucleotide sequence that hybridizes to a part or all of a consensus nucleotide sequence recognized by a group of known nucleic acid species having the same function.
  • the concentration of the primer probe used is not particularly limited since various concentrations are changed depending on the purpose, that is, the concentration of the nucleic acid contained in the sample.
  • concentration of the nucleic acid contained in the sample 0.01-1. 01 ⁇ , preferably 0.1-1.
  • the target nucleic acid to be amplified is labeled with a fluorescent substance.
  • the primer probe of the present invention can be prepared by a conventionally known method.
  • Species of primer probe oligonucleotides are suitable for use with commercially available nucleic acid synthesizers (lane; tJ, ABI394 (Perkin Elmer, USA)).
  • any of the conventionally known labeling methods can be used (Applied and
  • n 3 to 8, preferably 6.
  • a labeled oligonucleotide can be synthesized by binding the labeling substance having SH group reactivity or a derivative thereof to the spacer.
  • the oligonucleotide labeled with the labeling substance thus synthesized can be purified by reverse phase chromatography or the like to obtain the target nucleic acid primer and nucleic acid probe of the present invention.
  • the 3 ′ terminal base of the oligonucleotide can be labeled.
  • a phosphate group is introduced, and for example,-(CH 2 ) n -SH is introduced as a spacer to the OH group of the phosphate group.
  • n is 3-8, preferably 4-7.
  • a labeled oligonucleotide can be synthesized by binding the above-mentioned labeling substance having a reactivity to an amino group or SH group or a derivative thereof to the spacer.
  • bases in the oligonucleotide strand can be labeled.
  • the amino group or the ⁇ H group of the base is labeled with the labeling substance of the present invention in the same manner as in the method of the 5 'or 3' end (ANALYTICAL BIOCHEMISTRY 225, pp. 32-38 (1998)).
  • kit reagents for example, Unilink
  • the labeling substance molecule can be bound to the oligoliponucleotide according to a conventional method.
  • the oligonucleotide synthesized in this manner can be purified by chromatography such as reverse phase or the like to obtain the target nucleic acid primer probe of the present invention.
  • a nucleic acid amplification method particularly a PCR method, or a real-time quantitative PCR method using the above-described primer probe, and a data analysis method for determining the concentration or copy number of an amplified nucleic acid before amplification at the same time as nucleic acid amplification.
  • the annealing reaction, nucleic acid extension reaction, and nucleic acid denaturation reaction of a primer probe (forward type, riverse type) with a target nucleic acid are defined as one cycle, and by repeating this cycle, nucleic acid amplification is performed.
  • This is a method for real-time monitoring (measurement) of changes in the optical character of a PCR reaction system in a state in which the amplified nucleic acid is denatured and in a state of a primer-nucleic acid complex at at least one measurement wavelength. Then, it is a method of determining the concentration of the target nucleic acid before amplification of the curve force of the change rate of the fluorescence intensity obtained by measuring at least one measurement wavelength.
  • the purpose of the PCR reaction is achieved by performing the reaction up to a cycle in which the nucleic acid is amplified exponentially and then examined. Known reaction conditions may be used. Specific examples are shown in the examples. In the above-mentioned PCR method, the actual measurement is carried out using a measurement 'data analyzer (which naturally includes a device equipped with an electronic recording medium recording the procedures of those methods).
  • the actual measurement of the real-time quantitative PCR method used in the present invention and the method of analyzing data obtained by the real-time quantitative PCR method may be performed by a known method.
  • the method of KURATA et al. KURATA et al., Ucleic Acids
  • the real-time quantitative PCR method currently consists of a reaction device that performs PCR, a device that detects changes in the optical character of a fluorescent substance at at least one measurement wavelength, and a user interface, that is, a program for each step of the data analysis method. And the record that recorded it A computer-readable recording medium ( ⁇ 1 J: Sequence Detection Software System) and a computer configured to control them and analyze data. These devices are measured in real time using at least one measurement wavelength. I have. Therefore, the measurement of the present invention is also performed by such an apparatus.
  • the device used in the present invention may be any device that can monitor PCR in real time using at least one measurement wavelength.
  • ABI PRISM TM 7700 base distribution system detection system (Sequence detection system SDS 7700) (Perkin 'Elmer 1 to Applied' Bio-systems, Inc. (Per kin Elmer Applied Biosytems, Inc., USA)
  • light cycler one TM system (Roche die ⁇ Diagnostics sticks stock company, Germany ) Etc. can be mentioned as particularly suitable.
  • the above-mentioned PCR reaction apparatus is an apparatus that repeatedly performs a thermal denaturation reaction, an annealing reaction, and a nucleic acid extension reaction of a target nucleic acid (for example, it can be repeatedly performed at a temperature of 95 ° C, 60 ° C, or 72 ° C).
  • the detection system is equipped with an argon laser for fluorescence excitation, a spectrograph, a CCD camera, and at least one fluorescence measurement channel.
  • a computer-readable recording medium in which each procedure of the data analysis method is programmed and recorded is used by being installed in a computer, controlling the above system via the computer, and outputting from the detection system. It records a program that analyzes and processes the data obtained.
  • the data analysis program recorded on the computer-readable recording medium is a process for measuring the fluorescence intensity at each cycle, and the measured fluorescence intensity is displayed on a computer display as a function of the cycle, that is, as an amplification plot of PCR.
  • the process shown above the process of calculating the PCR cycle number (threshold cycle number: Ct) at which the fluorescence intensity starts to be detected, the process of creating a calibration curve for determining the copy number of the sample nucleic acid from the Ct value, the data of each of the above processes, The process of printing the plot values, the color!
  • the first characteristic is that the nucleic acid to be analyzed in each cycle corresponds to the nucleic acid obtained in the real-time quantitative PCR method, and the amplified nucleic acid in each cycle is compared with a method of analyzing data obtained at each measurement wavelength
  • the primer probe is hybridized to the primer probe (which specifically hybridizes)
  • the fluorescence intensity value of the fluorescent substance obtained by labeling the primer probe is determined by the hybridization between the primer probe and the nucleic acid in each cycle (ie, , A primer-nucleic acid complex) is dissociated, and is an arithmetic processing step of correcting with the fluorescence intensity value of the fluorescent substance, that is, a correction arithmetic processing step.
  • the fluorescence intensity value of the fluorescent substance that has labeled the primer probe when the nucleic acid that has been amplified is hybridized to the primer probe corresponding to (specifically hybridizes to) the nucleic acid” For example, 40 to 85 ° C, preferably 50 to 80 in each cycle of PCR.
  • 0 means a reaction system in which the reaction is completed. The actual temperature depends on the length of the amplified nucleic acid.
  • the “fluorescence intensity value of the fluorescent substance when the primer-nucleic acid complex is dissociated” refers to a reaction system for heat denaturation of nucleic acid in each cycle of PCR, specifically, a reaction temperature of 90 to: ° C, preferably 94-96 ° C, and the fluorescence intensity value of the reaction system when measured at the measurement wavelength related to the fluorescent substance of the primer probe in the reaction system in which the reaction is completed (hereinafter, referred to as The same is true.)
  • any process can be used as long as it meets the object of the present invention, but specifically, the following [Equation 1] or the female formula 2]
  • a process including the following process can be exemplified.
  • the second feature is that, in each cycle, [Equation 1] or [Layer] substitutes the corrected calculation processing value by [Equation 2] into the following [Equation 3] or [Equation 4], and changes the fluorescence between each sample.
  • This is a data analysis method that calculates the amount (fluorescence change rate or fluorescence change rate) and compares them.
  • f a Force calculated by (Equation 1) or (Equation 2), force of any number of cycles before change in f n is observed Normally, for example, 10 to 40 cycles Preferably, 15 to 30 cycles, more preferably 20 to 30 cycles are employed. ]
  • the third feature is
  • A, b arbitrary numerical values, preferably integer values, more preferably natural numbers.
  • F n Fluorescence change amount in n cycles calculated by (Equation 3) or (Equation 4) (Fluorescence change Conversion ratio or fluorescence conversion ratio)].
  • reaction system for measuring the standard nucleic acid and the reaction system for measuring the target nucleic acid may be used together or separately.
  • the data analysis method is particularly effective when the real-time quantitative PCR method is used to measure the amount of decrease in the emission of a fluorescent substance.
  • a fourth feature is a method for analyzing the temperature dissociation curve (melting curve) of the amplified nucleic acid of the present invention, and a method for analyzing data obtained by a method for obtaining the Tm value of the nucleic acid by performing the PCR method of the present invention. It is.
  • the temperature is gradually increased (for example, from 50 ° C to 95 ° C) until the nucleic acid is completely denatured at a low temperature.
  • Measuring the fluorescence intensity of at least one measurement wavelength at a short time interval e.g., an interval corresponding to a temperature rise of 0.2 ° C to 0.5 ° C
  • the process of displaying the measurement result of wavelength on the display as a function of time that is, the process of displaying the melting curve of nucleic acid, the differentiation of this melting curve and the differential value (one dFZdT, F: fluorescence intensity, T: time ),
  • the process of displaying the value on the display as a derivative, and the process of finding the inflection point of the derivative force is provided.
  • the fluorescence intensity increases with each increase in temperature.
  • a process of performing a calculation process of dividing the fluorescence intensity value at the time of the nucleic acid extension reaction in each cycle, preferably at the end of the PCR reaction by using the fluorescence intensity value at the time of the thermal denaturation reaction More favorable results are obtained.
  • the temperature dissociation curve of the amplified nucleic acid of the first invention can be measured.
  • the base sequence is designed based on the data of the result of the partial base sequence decoding, and a currently known method is used. Can be prepared.
  • “Anchored PCR method” refers to the use of an antisense primer probe and / or a sense primer probe and a sense adapter primer probe and / or an antisense adapter primer probe as shown in FIG. This is a PCR method for nucleic acid amplification (Experimental Medicine, Vol. 15, No. 7, pp. 52-59, 1997).
  • linker 1 and linker 1 2 refers to an oligonucleotide pair having a region where two oligonucleotide chains are hybridized and each other has a hybridized region and a non-hybridized region. .
  • linker 11 one is referred to as linker 11 and the other as linker 12.
  • Anchor Used in the PCR method also called anchor (see Fig. 1).
  • the base sequence may be arbitrary.
  • an “antisense primer probe” is a single type of a single-stranded oligonucleotide probe.
  • a nucleic acid for example, in the case of DNA
  • a sense strand a nucleic acid (for example, in the case of DNA) base sequence having genetic information
  • an antisense strand is a primer probe that contains at least a base sequence that hybridizes to the antisense strand (ie, contains a part or all of the base sequence of the sense strand) (see FIG. 1).
  • the meaning of “at least” means that the signal may be hybridized over a part of the linker region in addition to the antisense strand.
  • the “sense primer probe” refers to a primer probe that hybridizes to the sense strand in the opposite direction to the antisense primer probe (see FIG. 1).
  • sense adapter primer probe or “antisense adapter primer probe” means that the nucleotide sequence of the probe is at least the nucleotide sequence of the linker, especially the non-acceptable region (where linker 11 and linker 12 It contains a base sequence that does not hybridize (see Figure 1).
  • bases that hybridize to the base sequence portion of the nucleic acid contained in the sample May contain an array.
  • An antisense primer probe or a sense primer probe hybridizes to an extended nucleic acid that has been extended and synthesized, and becomes a primer for a PCR reaction.
  • the meaning of “at least” means that the base sequence may have a base sequence region that extends beyond the non-complementary region.
  • the sense adapter primer probe contains a non-complementary nucleotide sequence of the linker on the sense strand of the target nucleic acid.
  • the antisense adapter primer probe contains the non-complementary nucleotide sequence of the linker on the antisense strand.
  • the relationship between the antisense or sense primer probe and the sense or antisense adapter primer profile is that one is a forward primer and the other is a reverse primer.
  • the sense adapter primer probe and the antisense adapter primer probe may be common (identical). The ones shown in Figure 1 are common (identical).
  • a nucleic acid obtaining method characterized by at least the following procedures (see Fig. 1): 1) At least one kind of restriction enzyme treatment is performed on a sample containing at least one kind of nucleic acid. .
  • the restriction enzyme treatment may be performed in the same manner as in the method described in the section of polymorphism analysis of the first invention.
  • the reaction for example, in the case of Accl, it is preferable to inactivate the restriction enzyme by heating at 65 ° C for 30 minutes. Care must be taken because the inactivation temperature and time vary depending on the restriction enzyme.
  • the treated product is, for example, spin column (MicroconPCR) (Millipore
  • linker 11 and linker 12 At least one pair of linkers (hereinafter, referred to as linker 11 and linker 12) is added to the restriction enzyme-treated product of (1), and a ligation reaction is performed.
  • the ligation reaction of the present invention can be carried out by a conventional known method such as blunt-end ligation and TA binding reaction (Experimental Medicine, pp. 12-19, Vol. 15, No. 7, 1997, sheep It is not particularly limited as long as it is performed at an earthworks company.
  • the ligation reaction format depends on, for example, the structure of the terminal end of the nucleic acid fragment generated by the restriction enzyme treatment. If the 5 'end of the nucleic acid fragment is phosphorylated, there is no linker Processing is fine.
  • the 5 'end of the linker may be phosphorylated with a kit reagent, and the nucleic acid fragment may be treated with phosphatase.
  • kit reagents Experimental Medicine, pp. 12-19, Vol. 15, No. 7, 1997, Yodosha).
  • Hirakatadaira's Blu-Daiichi Blunting kit (TAKARA BIO INC.), Ligation reaction DNA Ligation Kit Ver. 2 (TAKARA BIO INC.), 5, phosphorylation
  • MEGALABEL TM (TAKARA BIO INC.) And the like.
  • T-Vector Kits Novagen, TA Cloning Kits (Invitrogen), PGEM-T Vector System (Promega), etc. Specific procedures, reaction conditions, etc. are attached to the kit reagents. If you follow the protocol.
  • the reaction may or may not be purified.
  • purification it is convenient to use the above-mentioned spin column.
  • the purified product is preferably dried and dissolved in a small amount of sterile water to increase the concentration of the ligated nucleic acid. Transfer this DNA to the next anchor PC
  • At least one antisense primer probe is added to A, and a nucleic acid extension reaction is performed.
  • a nucleic acid extension reaction is performed by adding at least one kind of anti-adapter primer probe to A or the reaction product of (a).
  • Reaction conditions may be the same as the above PCR reaction conditions.
  • the concentration of the antisense primer / probe added is 0.0 :! ⁇ S imol / L, preferably 0.1-0.5 tmolZL, and the concentration of the adapter probe added is 0.101-2 molZL, preferably 0.1-0.5 ⁇ mol / L. is there.
  • the nucleic acid extension reaction time is:! To 1800 sec, preferably 10 to 400 sec.
  • polymerases such as DNA and RNA, those similar to those described in the PCR can be used.
  • the working concentration is 0.01-:! OuZ reaction solution (20 ⁇ l), preferably 0 :! 33 u / reaction solution (20 ⁇ l).
  • Reaction temperature The degree may be the same as the PGR reaction described above. For example, it is 40 to 85 ° C, preferably 50 to 80 ° C.
  • Reaction temperature The degree may be the same as the PGR reaction described above. For example, it is 40 to 85 ° C, preferably 50 to 80 ° C.
  • At least one kind of sense primer probe is added to B, and a nucleic acid extension reaction is performed.
  • At least one kind of sense adapter primer probe is added to B or the reaction product of the above (c) to perform a nucleic acid extension reaction.
  • Reaction conditions may be the same as described above.
  • the antisense primer probe and the sense primer probe it is preferable to use a probe labeled with at least one of the aforementioned violet substances. This is because the reaction product can be confirmed by performing a dissociation curve analysis of the reaction product. Then, the labeling position of the fluorescent substance is preferably a base on the 5 ′ side of the oligonucleotide. In that case, it is preferable that the amino group is C amino group or OH group. Among these, it is particularly preferable to use the above-mentioned Q probe type.
  • the nucleic acid extension reaction is performed using a plurality of kinds of fluorescent substance-labeled antisense primer probes and a plurality of kinds of anti-adapter primer probes.
  • the nucleic acid extension reaction may be performed using a plurality of types of fluorescent substance-labeled sense primer probes and a plurality of types of sense adapter primer probes.
  • the plurality of types of sense primer probes and antisense primer probes each have a different base sequence and are at least one type labeled with a fluorescent substance giving a different fluorescent emission color.
  • a plurality of at least one antisense primer probe and at least one Z or at least one sense primer probe labeled with at least one fluorescent substance, and the plurality of fluorescent substance labeled probes are different from each other.
  • a method for obtaining a nucleic acid using at least one fluorescent substance-labeled antisense primer probe and a Z or at least one sense primer probe, which has a base sequence and is labeled with a fluorescent substance giving a different fluorescent emission color. is there.
  • a plurality of types of useful nucleic acids (target genes) can be obtained.
  • the final reaction product of step 3), which proceeds to step 4) may be purified by various electrophoresis methods or various HPLC methods. Further, the object of the present invention can be sufficiently achieved even with the final reaction solution itself.
  • the PCR product of step 4) of the second invention may be subsequently separated by electrophoresis as described above. Then, the useful nucleic acid (target gene) is separated from the linker with a restriction enzyme, whereby the useful nucleic acid can be separated. It is preferred that the linker be cut off with a restriction enzyme before electrophoresis before separation by electrophoresis.
  • the size of the target base sequence portion in the target nucleic acid that is, the size of the target nucleic acid (target gene) is determined and obtained by the series of procedures of the second invention.
  • the expression amount and Z or expression product of the nucleic acid (gene) separated as described above are examined in the same manner as in the first invention.
  • the sample containing at least one nucleic acid of the second invention is at least one of the following:
  • the final product according to the first invention that is, the final reaction solution (product), or a product that is separated and purified (for example, separated and purified by various electrophoresis methods and various HPLC methods). That is, a nucleic acid identified as a new or the same functional nucleic acid species in the first invention or a nucleic acid containing the same.
  • the invention that has been treated with the restriction enzyme of the second invention is the invention that uses the restriction enzyme treatment for polymorphism analysis in the first invention.
  • the invention uses the information obtained in the first invention.
  • the novel useful nucleic acid thus obtained is preferably ligated to a vector by blunt-end cloning, TA cloning, cloning using a restriction enzyme, or the like to prepare a clone library. It is also preferable to use a novel useful nucleic acid linked to a vector to transform an appropriate expression cell (for example, Escherichia coli, yeast, etc.) and to test the actual expression. This is because even a nucleic acid recognized as a single nucleic acid by the electrophoresis method is not necessarily substantially single. Thus, the separated and obtained nucleic acid finally becomes a new useful nucleic acid.
  • the useful nucleic acid (target gene) present in one system in nature can be simply and quickly detected, and the concentration thereof can be determined.
  • Stool Quickly isolates a new useful nucleic acid (target gene). From the data of this examination result, the entire base sequence of the novel useful nucleic acid (gene) is determined, and it is clear that the nucleic acid is a novel useful nucleic acid (gene). Then, a novel useful gene is obtained. Since the data thus obtained is recognized as any polymorphic nucleic acid, it is very useful for modifying the genetically engineered base sequence of the next gene.
  • the fourth invention is a fluorescent substance-labeled primer probe. That is, it is a primer probe that can simply and quickly detect and measure the useful nucleic acid (target gene) existing in one system in nature, know its concentration, and separate the useful nucleic acid (target gene).
  • 3′-terminal ribose or deoxylipose 3′-OH group power Oligonucleotide containing S-free and complementary base sequence that hybridizes to the complementary base sequence formed and hybridizes to the complementary base sequence to form an optical character Is a phosphor-labeled primer having the property of changing. And it is a fluorescent substance labeling primer comprising at least one of the following.
  • the 5 'end is C (cytosine).
  • C may or may not hybridize to the complementary base sequence of the target nucleic acid. Good.
  • the base sequence does not hybridize with the complementary base sequence of the target nucleic acid or hybridizes with the complementary base sequence of the target nucleic acid between the complementary base sequence that hybridizes with C and does not hybridize with the base sequence of the target nucleic acid.
  • Arbitrary base sequence intervenes.
  • the intervening base sequence has 1 to 20 bases.
  • a fluorescent substance-labeled primer comprising at least one of the following.
  • the terminal is C (cytosine).
  • the intervening base sequence has the number of bases
  • any base sequence portion that does not hybridize to the target nucleic acid may have a hairpin structure.
  • nucleotide sequences of the consensus primer probes, linker-1, linker-1, target gene-specific antisense primer probe, target gene-specific sense primer probe, and adapter primer probe used in this example are as follows.
  • the base sequence is 5 'on the right and 3 on the left.
  • Consensus primer probe 1 forward type (consensus system IJ underlined, 5, terminal phosphate group is labeled with BODIPY FLC6):
  • GGTTACCTTGTTACGACTT Consensus primer probe 1 and consensus primer probe 2 are primers targeting a highly conserved region of the eukaryotic 16S rRNA gene, and are capable of PCR-amplifying all the eukaryotic 16S rRNA gene.
  • Linker 1 (the underlined portion does not hybridize to one of the oligonucleotides, sequence):
  • Linker 2 (a sequence in which the underlined portion does not hybridize to one of the oligonucleotides):
  • Antisense primer probe that specifically hybridizes to the target gene (the underline hybridizes to the nucleotide sequence of the antisense strand of the nucleic acid contained in the sample, and the phosphate group at the 5 'end is labeled with BODIPY FLC6 ):
  • a sense primer probe that specifically hybridizes to the target gene (the underlined portion is hybridized to the base sequence of the sense strand of the nucleic acid contained in the sample, and is labeled with the 5'-terminal phosphate group S BODIPY FLC6). ):
  • the sense primer probe and antisense primer probe are primers specific to the 16S rRNA gene of Escherichia coli used as model target genes.
  • Adapter primer probe (The underlined part will hybridize to the nucleic acid synthesized during the gene amplification process.
  • the phosphate group at the 5 'end is labeled with BODIPY FLC6.):
  • the adapter primer probe has the same sequence as the non-extrapolative region of the linker (the region where the linkers 1 and 2 have hybridized to each other). Therefore, it hybridizes to the base sequence of the amplified nucleic acid extended from the sense primer probe or antisense primer probe, and becomes a primer for the subsequent PCR reaction.
  • Figure 1 shows the state when the linker has been ligated to the restriction enzyme fragment site containing the target gene (16S rRNA gene of Escherichia coli). Shown.
  • the model target gene (Escherichia coli 16S rRNA gene) in the type III sample was quantified, and the ability to identify a sample containing a large amount of the target gene (Escherichia coli 16S rRNA gene) was verified.
  • samples were prepared by adding Escherichia coli genomic DNA at varying concentrations to DNA samples extracted from activated sludge, and nucleic acid amplification and quantification of the samples were performed by a real-time quantitative PCR method.
  • a gas test was performed which enabled identification of a sample containing a large amount of the target gene (Escherichia coli 16S rRNA gene).
  • Activated sludge which is known to contain almost no Escherichia coli, collected from food wastewater treatment equipment was used as a sample.
  • Type I DNA containing the model target gene Esscherichia coli 16S rRNA gene
  • Escherichia coli genomic DNA was Escherichia coli genomic DNA (Takara Shuzo).
  • the nucleic acid was extracted from the activated sludge using a BiolOl kit (for soil), and further purified using a Wizard DNA Clean-Up system. Next, the absorbance at 260 nm / 280 nm, and the DNA concentration were measured. The extracted DNA was a 120: ⁇ / 1-extract. And the extracted DN A known concentration of E. coli genomic DNA (10 5, 10 s, 10 7 copies E. coli genomic DNA equivalent) added and the ⁇ sample.
  • QP-PCR method As a real-time quantitative PCR method, a method (hereinafter, referred to as QP-PCR method) using Quenching primers (consensus probes 1 and 2 composed of a Q probe) was applied.
  • Denaturation reaction 95 ° C, 15 sec; Annealing: 53 ° C, 5 sec; extension and detection: 72 ° C, SOsec; Measurement device: Light cycler system (manufactured by Roche) (hereinafter referred to as “light cycler” for convenience); Fluorescence detection channel used: Channel 1 (Ex, 470 nm; Em,
  • Reaction solution total volume 20 ⁇ 1; rTaq polymerase (Takara Shuzo), 0.5 U reaction (20 ⁇ l); XlObuffer attached to rTaq polymerase, 2.0 ⁇ l / reaction (20 ⁇ l); dNTPs final concentration, each 2 mM; Mg ++ ion concentration, final concentration 2 mM; bovine serum albumin (BSA), final concentration 0.25 mg / ml; activated sludge extracted DNA, ⁇ . ⁇ 1 / reaction (20 ⁇ 1); E. coli genomic DNA, ⁇ . ⁇ 1 reaction (20 ⁇ l); final concentration of each primer probe (consensus primers 1 and 2), 100 nM; Taq start solution (Clontech, 0.4 ⁇ 1 / reaction (201)).
  • Quantification of all 16S rRNA genes contained in the samples was performed by QP-PCR.
  • Table 1 shows the results of quantification of all 16S rRNA genes in the sample by QP-PCR.
  • a dissociation curve of the obtained amplification product was prepared.
  • the instrument used a light cycler system, and the temperature was changed from 0.1 to 60 ° C at 0.1 ° C / sec, during which the fluorescence was continuously measured. Since the product obtained by the QP-PCR method has BODIPY FL modification at the terminal C base, the amount of fluorescence increases as the product dissociates and becomes single-stranded. Therefore, it was possible to easily obtain a dissociation curve of the amplification product.
  • FIG. 3 shows a dissociation peak obtained by differentiating the dissociation curve of FIG.
  • the dissociation peak is obtained by differentiating the dissociation curve. Therefore, the approximate Tm value can be obtained from the position of this peak apex.
  • the dissociation peak was detected at around 85 ° C.
  • An amplification product having the same fragment length as the target amplification product was confirmed by electrophoresis (Data not shown), and it was confirmed that the amplification product having a dissociation peak near 85 ° C was a specific product Was done.
  • the dissociation peak of the product obtained in Sample No. 1 was around 75 ° C. From this, it was confirmed that the product obtained in Sample No. 1 was a non-specific amplification product. This non-specific amplification product was presumed to be a primer dimer with its Tm value.
  • the dissociation curve of the obtained product can be easily prepared, it is possible to quickly determine whether the amplification product is specific or not. is there.
  • composition ratio of each gene is determined from the peak height ratio, and this composition ratio is multiplied by the total 16S rRNA gene amount quantified in the QP-PCR method step to obtain the copy number of each gene (polymorphism). Was calculated.
  • Example 1 sample No. 5 capable of amplifying the target gene to be a priority species in terms of concentration, activated sludge extracted DNA (60 ng Z At 1) and 0.24 pg Z 1 E. coli genomic DNA (about 100 An attempt was made to screen a target nucleic acid (Escherichia coli 16S rRNA gene) from a sample (sample No. 8) obtained by mixing equal amounts of E. coli. If an unknown sample such as a natural environment sample is the target sample, cloning and sequencing are performed on the amplified products of all identically functional genes obtained by the QP-PCR method, and a new gene (polymorphism) sequence is determined. Then, in order to perform the anchor PCR method, it is necessary to prepare an antisense primer probe and a sense primer probe specific to a novel gene.
  • activated sludge extracted DNA 60 ng Z At 1
  • 0.24 pg Z 1 E. coli genomic DNA about 100
  • the Escherichia coli 16S rRNA gene was assumed to be an unknown gene (target gene), and the study was carried out. Therefore, the steps of closing and sequencing were omitted.
  • the antisense primer probe and the sense primer probe specific to Escherichia coli were designed based on Escherichia coli 16S rRNA gene sequence information known in advance.
  • the restriction enzyme fragment was bound to the linker by the following method. First, add Linker 1 and Linker 1 to 1.5 ⁇ M and 1.M, respectively, and bind them beforehand.After that, add all of the sample No. 5 and Sample No. 8 treated restriction enzyme treated sump noles. Was. 1.5 units of 4 DNA ligase was added to this solution, and a ligation reaction was performed at 16 ° C for 24 hours. The ligated sample was purified by a spin column (MicroconPCR). The purified DNA solution was dried and dissolved with 101 of sterile water. This DNA solution was used as type I DNA for anchor QP-PCR performed in the following steps.
  • the reactant was split into two, one as A and the other as B.
  • the nucleotide sequence of the product obtained in 7) was decoded by a direct sequence using the dye terminator method (Experimental Medicine, Volume 15, pages 29 to 35, 1997, Youtosha).
  • the sequence primer is Consensus Primer Probe 2
  • Sequencer used ABI PRISM TM 310.
  • the target gene was E. coli 16S rRNA gene and 100% The base sequences matched. From the above results, it was suggested that the target gene could be easily and rapidly obtained by PCR reaction between the products obtained by the two anchor QP-PCR and the anchor QP-PCR.
  • the method of the present invention requires that (1) amplification and quantification of all identically functional nucleic acid (gene) species can be performed. (2) It was suggested that simultaneous quantification of polymorphism of each nucleic acid (gene) constituting the same functional nucleic acid (gene) was possible.
  • polymorphism analysis T-RFLP
  • T-RFLP polymorphism analysis
  • novel nucleic acid (gene) polymorphisms obtained in the process of real-time quantitative PCR, the partial sequence of the novel nucleic acid (gene) polymorphism is decoded, and this information is obtained. Based on this, it is possible to create primers specific to polymorphisms of novel nucleic acids (genes).
  • target nucleic acids (genes) can be directly and selectively separated from the specified sample by anchor QP-PCR using a specific primer.
  • target nucleic acids (genes) can be directly and selectively separated from the specified sample by anchor QP-PCR using a specific primer.
  • it was not possible to separate target nucleic acids (genes) from samples with low abundance of target nucleic acids (genes) it was important to identify Sex was suggested.

Landscapes

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

Abstract

Cette invention concerne une méthode permettant de détecter et de mesurer rapidement, facilement et avec une sensibilité élevée plusieurs acides nucléiques de même fonction parmi la totalité des acides nucléiques présents à l'état naturel dans un système, puis d'obtenir sélectivement, rapidement et facilement un acide nucléique nouveau et utile. Cette méthode consiste à amplifier des acides nucléiques présents dans le système au moyen d'une séquence de base consensuelle pour une variété d'acides nucléiques connus dotés de la même fonction ; et à rechercher un acide nucléique nouveau ou ayant la même fonction que les acides nucléiques ainsi amplifiés, ou bien à obtenir à partir desdits acides un acide nucléiques nouveau et utile (dont un gène).
PCT/JP2003/008311 2002-07-01 2003-06-30 Methode d'obtention d'un acide nucleique ou d'un gene Ceased WO2004003199A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2002192562 2002-07-01
JP2002-192562 2002-07-01

Publications (1)

Publication Number Publication Date
WO2004003199A1 true WO2004003199A1 (fr) 2004-01-08

Family

ID=29996978

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2003/008311 Ceased WO2004003199A1 (fr) 2002-07-01 2003-06-30 Methode d'obtention d'un acide nucleique ou d'un gene

Country Status (1)

Country Link
WO (1) WO2004003199A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988006634A1 (fr) * 1987-02-26 1988-09-07 The University Of Sydney Procede de detection de papillomavirus humains carcinogenes
JP2001286300A (ja) * 1999-04-20 2001-10-16 Japan Bioindustry Association 核酸の測定方法、それに用いる核酸プローブ及びその方法によって得られるデータを解析する方法
WO2002001412A1 (fr) * 2000-06-28 2002-01-03 Sanyo Electric Co.,Ltd. Systeme d'analyse microbienne, methode correspondante et base de donnees en rapport
WO2002046393A1 (fr) * 2000-12-07 2002-06-13 Toyo Boseki Kabushiki Kaisha Procede d'identification de polymorphisme nucleotidique

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988006634A1 (fr) * 1987-02-26 1988-09-07 The University Of Sydney Procede de detection de papillomavirus humains carcinogenes
JP2001286300A (ja) * 1999-04-20 2001-10-16 Japan Bioindustry Association 核酸の測定方法、それに用いる核酸プローブ及びその方法によって得られるデータを解析する方法
WO2002001412A1 (fr) * 2000-06-28 2002-01-03 Sanyo Electric Co.,Ltd. Systeme d'analyse microbienne, methode correspondante et base de donnees en rapport
WO2002046393A1 (fr) * 2000-12-07 2002-06-13 Toyo Boseki Kabushiki Kaisha Procede d'identification de polymorphisme nucleotidique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KURATA S. ET AL.: "Fluorescent quenching-based quantitative detection of specific DNA/RNA using a BODIPY(R) FL-labeled probe or primer", NUCLEIC ACIDS RES., vol. 29, no. 6E34, 2001, pages 1 - 5, XP002953169 *

Similar Documents

Publication Publication Date Title
US9938570B2 (en) Methods and compositions for universal detection of nucleic acids
CN102348811A (zh) 单细胞核酸分析
CA2400601A1 (fr) Quantification en temps reel de genes avec etalons internes
EP2722388B1 (fr) Sonde acide nucléique pour le dosage d'acides nucléiques
JP3844996B2 (ja) 反復性ｐｃｒ産物の融解曲線解析方法
KR20110036646A (ko) 변이의 검출 방법 및 그것에 이용하는 키트
CA2318371A1 (fr) Procede de detection de sequences nucleotidiques
EP1390542A2 (fr) Systeme de detection d'acides nucleiques
AU2002311414A1 (en) Nucleic acid detection method
EP2971147A1 (fr) Détection d'allèles multiplex
JP5207355B2 (ja) 標的ヌクレオチド配列を有する核酸の検出方法、プローブセット、及び核酸の識別方法
Seifi et al. Overview of real-time PCR principles
US20130288246A1 (en) Compositions, methods, and kits for analyzing dna methylation
JP4190562B2 (ja) 遺伝子配列検査法
WO2007044057A2 (fr) Procedes de quantification de precurseurs de micro-arn
JP2009077736A (ja) 核酸の新規測定方法
KR20080073321A (ko) 핵산 혼성화에서 Ｃｏｔ-1 ＤＮＡ 왜곡의 완화
JP6205216B2 (ja) 変異検出用プローブ、変異検出方法、薬効判定方法及び変異検出用キット
Ünsal et al. A novel method of multiplex SNP genotyping assay through variable fragment length allele-specific polymerase chain reaction: Multiplex VFLASP-ARMS
WO2004003199A1 (fr) Methode d'obtention d'un acide nucleique ou d'un gene
KR101669089B1 (ko) Ｅｇｆｒ 엑손 19 다형 검출 시험용 올리고뉴클레오티드 및 그 용도
US20040096829A1 (en) Absolute quantitation of nucleic acids by RT-PCR
JP2004081214A (ja) 核酸若しくは遺伝子の新規取得方法
EP4350002A1 (fr) Detection of molecular analytes based on tailored probe competition
JP6389473B2 (ja) 高解像能融解の較正の改善

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase