WO1999058716A1 - Procede servant a quantifier une sequence specifique de genes et reactif de quantification - Google Patents

Procede servant a quantifier une sequence specifique de genes et reactif de quantification Download PDF

Info

Publication number
WO1999058716A1
WO1999058716A1 PCT/JP1999/002374 JP9902374W WO9958716A1 WO 1999058716 A1 WO1999058716 A1 WO 1999058716A1 JP 9902374 W JP9902374 W JP 9902374W WO 9958716 A1 WO9958716 A1 WO 9958716A1
Authority
WO
WIPO (PCT)
Prior art keywords
carrier
stranded dna
bound
gene sequence
dna probe
Prior art date
Application number
PCT/JP1999/002374
Other languages
English (en)
Japanese (ja)
Inventor
Haruma Kawaguchi
Keiji Fujimoto
Mamoru Hatakeyama
Kyoko Nakamura
Masanori Fukui
Original Assignee
Kyowa Medex 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 Kyowa Medex Co., Ltd. filed Critical Kyowa Medex Co., Ltd.
Priority to AU36289/99A priority Critical patent/AU3628999A/en
Publication of WO1999058716A1 publication Critical patent/WO1999058716A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

Definitions

  • the present invention relates to a simple detection or standard method for early diagnosis of a mutation or the like in an awake gene and a detection or fibril reagent used therefor, and in particular, to a detection or ⁇ method in which a specific reaction is suppressed, And a reagent for detection or quantification used for Background art
  • PCR Polymerase Chain Reaction
  • a method for purifying a substance utilizing specific affinity for example, a method for purifying a protein specifically adsorbed to the probe DNA using a DNA probe bound to a carrier (Japanese Patent Application Laid-Open No. 3-61493) Is known. In addition, using a carrier that does not show nonspecific adsorption, using a DNA probe bound to the carrier, and concentrating or purifying DNA or RNA specifically binding to the probe. A method [Journal of Colloid and Interface Science (j. Colloid and Interface Sci.), 177, 245 (1996)] is known.
  • An object of the present invention is to provide a method for detecting or quantifying a single-stranded DNA fragment having a non-specific reaction and having a specific gene sequence using a hybridization method, and a method for detecting or quantifying the same.
  • the purpose is to provide a detection or quantification reagent to be used.
  • the method is useful for detecting or quantifying a point mutation or the like generated in connection with various diseases such as cancer.
  • a carrier-bound DNA probe in which a first single-stranded DNA probe having a sequence complementary to the gene sequence of the DNA fragment and a carrier are bound with or without a spacer; Detecting or quantifying a single-stranded DNA fragment having a specific gene sequence comprising hybridizing a DNA fragment in a sample with the carrier-bound DNA probe and detecting or quantifying the DNA fragment in the sample hybridized with the carrier-bound DNA probe.
  • the carrier is a polymer having an epoxy group on the surface, and after binding the carrier to the first DNA probe, treating the epoxy group of the carrier with an SH compound having a polar group.
  • a method for detecting or standardizing a single-stranded DNA fragment having a specific gene sequence which is characterized by using a carrier-bound DNA probe to be used (hereinafter sometimes referred to as the first method).
  • a carrier-bonded DNA probe in which a first single-stranded DNA probe having a sequence complementary to the gene sequence of a single-stranded DNA fragment having a carrier and a carrier are bound with or without a spacer;
  • the carrier is a polymer having an epoxy group on the surface, and after binding the carrier to the first DNA probe, the epoxy group of the carrier is converted to a polar group.
  • Carrier-bound DNA probe obtained by treatment with SH compound having And a method for detecting or standardizing a single-stranded DNA fragment having a gene sequence characterized by using a probe (hereinafter sometimes referred to as the second method).
  • a single-stranded DNA fragment having a specific gene sequence to be detected or detected in the sample The carrier-bound DNA probe in which the first single-stranded DNA probe having a sequence complementary to the gene sequence of the first and the carrier is bound with or without a spacer and the DNA fragment in the sample After hybridization with an enzyme that cuts the single-stranded DNA fragment, and then detecting or quantifying the DNA fragment in the sample hybridized with the carrier-bound DNA probe.
  • the carrier is a polymer having an epoxy group on its surface, and after binding the carrier to the first single-stranded DNA probe,
  • a method for detecting or quantifying a single-stranded DNA fragment having a specific gene sequence, comprising using a carrier-bound DNA probe obtained by treating a group with an SH compound having a polar group (hereinafter referred to as a third method) Sometimes) power is provided.
  • a first single-stranded DNA probe having a sequence complementary to a specific gene sequence of a single-stranded DNA fragment having a specific gene sequence to be detected or quantified in a sample, and an epoxy group The present invention provides a carrier-bound DNA probe obtained by binding a carrier having the formula (I) with or without a spacer and treating the epoxy group of the carrier with an SH compound having a polar group.
  • a sequence complementary to a partial sequence other than the gene sequence of the single-stranded DNA fragment having the specific gene sequence to be detected or quantified in the carrier-bound DNA probe and the sample is provided.
  • a test consisting of a labeled binding DNA probe in which a second single-stranded DNA probe having a labeled compound is bound to a labeled compound.
  • a detection or detection reagent kit is provided.
  • a first single-stranded DNA probe having a sequence complementary to the characteristic sequence of a single-stranded DNA fragment having a gene sequence to be detected or quantified in a sample is provided.
  • the epoxy group of the carrier is treated with an SH compound having a polar group.
  • a reagent having a gene sequence comprising a reagent containing the carrier-bound labeled DNA probe, a reagent containing an enzyme that cleaves single-stranded DNA, and a reagent for detecting or quantifying a labeled compound.
  • Reagent kits for detection or quantification of strand DNA fragments are provided.
  • the first method having a sequence complementary to the characteristic sequence of a single-stranded DNA fragment having a specific gene sequence to be detected or quantified in a sample.
  • the single-stranded DNA probe is bonded to a carrier having an epoxy group with or without a spacer, and then the carrier-bonded DNA obtained by treating the epoxy group of the carrier with an SH compound having a polar group Use a probe.
  • the first single-stranded DA probe having a sequence complementary to the gene sequence of a single-stranded DNA fragment having a specific gene sequence to be detected or quantified in a sample is provided.
  • the epoxy group of the carrier is treated with an SH compound having a polar group. It is preferable to use a carrier-bound labeled DNA probe obtained by the above method.
  • the shape of the carrier is not particularly limited, and examples thereof include a plate-shaped carrier and a particulate carrier.
  • the particulate carrier is preferable because it can bind a large amount of DNA per unit volume.
  • the particle size of the particulate carrier is preferably from 0.01 to: L 00 / m, particularly preferably from 0.05 to 20 / z m.
  • the carrier is a synthetic polymer having an epoxy group on the surface.
  • examples of a monomer forming the polymer include an epoxy group-containing glycidyl (meth) acrylate.
  • the synthetic polymer conjugate may form at least a surface layer of a plate or a particle, and the inside may be a hydrophobic polymer such as polystyrene or polyvinyl. It is particularly preferred that the surface of the plate or particle is prepared to be rich in epoxy groups.
  • Such particles include For example, particles having a polystyrene / polyglycidyl methacrylate core.
  • the polymer compound can be synthesized by a known method.
  • a suspension polymerization method or an emulsion polymerization method ⁇ is used.
  • polyacrylamide and its limited hydrolyzate, polyacrylic acid, hydroxypropylcellulose, ethylcellulose, methylcellulose, polyvinyl alcohol, and polyacrylamide are used as a dispersion stabilizer in the reaction.
  • a water-soluble polymer such as vinyl acetate can be used.
  • polymerization initiator use azo-based initiators such as azobisisobutyronitrile and 2,2′-azobis (2-aminobronone) dihydrochloride, and peroxides such as benzoyl peroxide. Can be.
  • the polymerization initiator in the emulsion polymerization is not particularly limited as long as it is used in ordinary emulsion polymerization.
  • a batch system, a semi-batch system, a continuous system or the like is used as the polymerization method in the emulsion polymerization method.
  • emulsion polymerization method it is preferable to use a so-called emulsion polymerization method comprising water, a monomer, and a polymerization initiator in order to make the particle surface clean and to prevent the adsorbed substance from being brought in.
  • a two-stage soap free polymerization method is preferred.
  • Examples of the SH compound include a hydrocarbon having an SH group having 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms.
  • Examples of the polar group include hydroxy, carboxy and the like.
  • Polar groups can be present in the SH compound:!-3.
  • Examples of the SH compound having a polar group include 2-mercaptoethanol, 2-mercaptoacetic acid, 3-mercaptopropionic acid, 3-mercapto-1,1,2-propanediol, and 3-mercapto-1-propanol.
  • These SH compounds having polar groups can be used directly for the purpose of opening the epoxy group of the carrier, but they can also be used by dissolving them in an aqueous medium such as water or a buffer solution.
  • Examples of the buffer include a phosphate buffer, an acetate buffer, a Tris buffer and the like.
  • the spacer is a first single-stranded DNA having a sequence complementary to a specific gene sequence of a single-stranded DNA fragment having a gene sequence to be detected or quantified in a carrier and a sample. It binds the probe.
  • Examples of the spacer include a single-stranded DNA chain and a single-stranded RNA chain.
  • the base sequence of the chain may be any, but it has an amino group at the base, adenine (A), guanine (G), Those having cytosine (C) at the terminal are preferred.
  • the length of the single-stranded DNA chain and the single-stranded RNA chain is preferably 5 to 4 Omer, and more preferably 10 to 20 mer.
  • the length of the DNA fragment of the first single-stranded DNA probe having a sequence complementary to the gene sequence is as follows: 0 to 50 bases, more preferably 10 to 30 bases, and particularly preferably 15 to 25 bases.
  • genes involved in various traits include PS1 (priserinin1) gene, PS2 (priserinin2) gene, APP (beta-amyloid breaker-protein) gene, lipoprotein gene, and HLA (human leukocyte antigen).
  • PS1 priserinin1 gene
  • PS2 priserinin2 gene
  • APP beta-amyloid breaker-protein gene
  • lipoprotein gene lipoprotein gene
  • HLA human leukocyte antigen
  • Gene hepatitis C virus gene
  • hepatitis B virus gene hepatitis B virus gene
  • hMSH2 gene hMSH2 gene and the like.
  • genes involved in the disease include the K_ras gene and the p53 gene. Mutations in the K-ras gene are described in Japanese Journal of Obs. Kyansa-Research (jpn. J.
  • Cancer Res. 84, 961 (1993), 85, 147 (1994), 85 , 1240 (1994), 85, 1005 (1994), 86, 1150 (1995), 86, 737 (1995), 87, 466 (1996), 87, 1 056 (1996) and 87, 793 (1996).
  • mutations in the p53 gene see Japanese Journal of Cancer Research (Jpn. J. Cancer Res.), 85, 1087 (1994), and 85, 1247 (19). 94), 86, 1143 (1995), 86, 57 (1 995), 86, 174 (1995), 86, 730 (1995), and 87, 930 (1996).
  • the hMSH2 gene is described, for example, in Japanese's Journal of Cancer, Jpn. J. Cancer Res., 87, 279 (1996).
  • a DNA fragment complementary to a specific gene sequence containing the nucleotide sequence described in these documents is suitably used as a probe DNA sequence.
  • the sequence of the first single-stranded DNA probe may be designed based on these known sequences.
  • a gene sequence to be detected or quantified particularly suitably applicable in the present invention is a sequence known to have an internal point mutation of a known sequence.
  • the position in the first single-stranded DNA probe having a base sequence complementary to the mutated base is preferably at least three bases at both ends of the DNA probe, and the DN It is particularly preferable to be within 2 bases from the center of the A probe.
  • the first single-stranded DNA probe having a sequence complementary to the gene sequence is used for DNA synthesis of DNA.
  • the reaction can be synthesized using a DNA synthesizer or the like, which is automated by a solid phase method using a carrier such as Siri force.
  • a carrier such as Siri force.
  • As the reaction substrate use is made of a nucleotide derivative in which the base amino group and ribose 5, -OH are protected with a protecting group, and the 3'-OH of ribose is conjugated to diisopropylphosphoramidite.
  • Examples of the protecting group for the amino group of the base include a benzoinole group and an isobutyl group, and examples of the protecting group for the 5′-OH of ribose include a dimethoxytrityl group.
  • a starting material is a column in which a nucleotide of any of adenine, thymidine (T), cytosine, and guanine, whose amino group and 5'-OH group are protected, is fixed via 3, -OH, After removal of the trityl group with an acid (formation of 5′-OH), the above nucleotide derivative having a phosphoramidite group at the 3 ′ end is added under a suitable condensing agent such as tetrazole.
  • both bonds are converted into stable phosphotriester bonds with iodine and water.
  • This cycle of the detrichidani and condensation reaction is repeated, and finally the ammonia treatment To cleave from the support and the support.
  • the desired first single-stranded DNA probe can be synthesized.
  • the spacer is a single-stranded DNA
  • its sequence may be synthesized continuously.
  • a first single-stranded DNA probe having a DNA spacer can be synthesized.
  • Identification of a single-stranded DNA fragment having a gene sequence to be detected or quantified in a sample First single-stranded DNA probe having a sequence complementary to the gene sequence and a substance that is unlikely to adsorb DNA
  • a method for preparing a carrier-bound DNA probe bound to a carrier consisting of or without a spacer will be described.
  • the binding method of the carrier, the spacer, and the first single-stranded DNA probe can be determined.
  • the first single-stranded DNA probe or the base at one end of the spacer is used to bind an amino group to an epoxy group of a carrier.
  • the binding between the DNA probe and the carrier is performed, for example, as follows.
  • a first single-stranded DNA probe having a nucleotide sequence and a sequence complementary to the probe are provided. Synthesize single-stranded DNA.
  • both are hybridized to form a double-stranded form by protecting the amino group of the base of the gene sequence to be detected or quantified, and the amino group of the base in the free spacer and the epoxy group of the carrier.
  • dissociate the single-stranded DNA that has a sequence complementary to the first single-stranded DA probe and no spacer sequence to prepare the target carrier-bound DNA probe can do.
  • Hybridization is performed with an aqueous solution containing formamide, salt, protein, stabilizer, buffer and the like as necessary.
  • this solution is referred to as a hybridization solution.
  • the formamide concentration is 0-60%, preferably 10-50%, more preferably 20-30%.
  • the salt include inorganic salts such as sodium salt sodium and potassium salt sodium and organic acid salts such as sodium citrate and sodium oxalate.
  • the salt concentration is 0 to 2.0 M, preferably 0.15 to 1 M. 0M. Serum albumin as a protein And the like.
  • the stabilizer include ficoll.
  • the buffer include a phosphate buffer and the like, and the concentration is preferably 1 to 10 OmM.
  • the above-mentioned two types of DNA hybridization are prepared by adding the synthesized single-stranded DNA to the hybridization solution at an equimolar concentration, and adding 1 to 6 at 60 to 90 ° C. After heating for 0 minutes, it can be performed by gradually cooling from 0 to 40 over 1 to 24 hours. Thus, a partially double-stranded DNA having a spacer of a single-stranded polynucleotide is prepared.
  • the carrier particles having an epoxy group are washed with 1 to 100 mM phosphate buffer, and after equilibration of the particles, the carrier is mixed with the carrier in the same solution as the buffer used for washing.
  • the method is performed by mixing the partial double-stranded DNAs having the spacer of the single-stranded DNA prepared by the method described in the above, and keeping the mixture at 20 to 50 ° C for 5 to 50 hours. Unreacted DNA is removed by washing with an aqueous solution containing 1 to 3 M of a salt such as sodium chloride.
  • a carrier-bound single-stranded DNA can be prepared. Washing is performed several times using a hybridization solution at 70 to 90 ° C and centrifugation at several thousand to several times.
  • the carrier is added to the polar group-containing SH compound, preferably a polar group-containing SH compound dissolved in an aqueous medium to have a concentration of 1 to 3 M, more preferably 1.5 to 2.5 M.
  • the temperature is maintained at 10 to 60 ° C., preferably at room temperature, for 15 to 50 hours to open the unreacted epoxy groups present on the carrier.
  • a biological test sample used for detection or quantification can be prepared from various organs, tissues, blood, etc. by a known DNA extraction method.
  • the isolated DNA is cleaved with a specific restriction enzyme and contains a specific gene sequence to be detected or quantified, 10 to 20 Omer, preferably 20 to:!
  • a specific restriction enzyme for example, when DNA extracted from cells is treated with the restriction enzymes Dde 1 and Hi ⁇ ⁇ I, K
  • a DNA fragment of 70 bases length including codon 12 of _ras can be prepared. In the present invention, it is only necessary to fragment DNA so as to have a specific gene sequence to be detected or quantified.
  • the target DNA to be labeled with the labeled conjugate may be a DNA fragment in a sample or a single-stranded DNA fragment having a gene sequence to be detected or 3 ⁇ 4fid in a sample, depending on the detection or 3 ⁇ 4fi method.
  • a single-stranded DNA fragment in the second single-stranded DNA probe having a sequence complementary to a partial sequence other than the gene sequence, a single-stranded DNA fragment having a specific gene sequence to be detected or quantified in a sample The first single-stranded DNA probe, which has a sequence complementary to the gene sequence of the single-stranded DNA fragment, and a carrier made of a substance that is difficult to adsorb DNA are bound with or without a spacer Any of the single-stranded DNA fragments in the carrier-bound DNA probe.
  • the labeled DNA is simply referred to as a labeled DNA probe.
  • the first single-stranded DNA probe having a sequence complementary to the specific gene sequence of the single-stranded DNA fragment having the specific gene sequence to be detected or quantified in the sample and a substance that does not easily adsorb DNA is labeled with a carrier-bound DNA. It is called a probe.
  • any labeled conjugate can be used as long as it can be detected or quantified directly or indirectly.
  • a heat-stable antigen biotin
  • the labeling compounds for the label-bound DNA probe and the carrier-bound labeled DNA probe include thermally stable labeling compounds such as radioisotopes, coloring reagents, fluorescent reagents, and luminescent reagents, and particularly preferred is biotin.
  • the method for binding DNA and biotin is described.
  • Pyotinylation of DNA Can be enzymatically linked using a biotin-linked deoxyribonucleoside triphosphate, for example, by 3, end-labeling, bio-bridge, nick translation, etc. it can.
  • biotin-linked deoxyribonucleoside triphosphates include Bio-11-dUTP, Bio-16-dUTP, and Bio-11-dCTP, and are commercially available from Enzo.
  • the DNA is a synthetic DNA
  • the 5′-end biotin-labeled DNA can be synthesized by an automatic synthesizer based on the cyanoethyl phosphoramidite method.
  • the biotinylated DNA can be purified, for example, by gel filtration of a spin column using Sephadex G-50.
  • the detection or quantification reagent comprises a labeled enzyme-conjugated avidin and the labeled enzyme activity detection or quantification reagent.
  • the labeling enzyme include peroxidase and alkaline phosphatase.
  • reagents for detecting or quantifying the enzyme activity include, for example, hydrogen peroxide and 3,3 ′ diaminobenzidine, o-dianicidine, 4-methoxy-11-naphthol, 2, 2, 1-Azinobis (3-ethylbenzothiazoline) — 6-sulfonic acid (ABTS), 10-N-methylcarbamoyl-3,7-dimethylamino-1H-phenothiazine (MCDP), 10-N-carboxymethylcarbamoyl-3, 7-Dimethylamino-10H-phenothiazine (CCAP), N- (carboxymethylaminocarbonyl) 1,4,4,1-bis (dimethylamino) diphenylamine sodium (DA-64), 4,4,1-bis (dimethylamino) Coloring reagents such as diphenylamine or bis [3-bis (4-chlorophenyl) methyl-4-dimethyl-1-a
  • a detection or quantification reagent composed of a color-forming reagent of a 11-naphthol derivative such as 4-methoxy-11-naphthol, a lanthanide fluorescent reagent such as palladium europium, and a luminescent reagent such as acridinium is exemplified.
  • Examples of the detection or quantification reagent when the labeling enzyme is alkaline phosphatase include a detection or quantification reagent comprising a luminescent reagent of a phosphate ester such as para-nitrophenyl phosphate or an adamantyl oxetane derivative.
  • a detecting or quantifying reagent comprising NADP, INT-biolet, NADH, ethanol, diaphorase and alcohol dehydrogenase is preferable.
  • Buffers, other substrates, other enzymes, surfactants, enzyme stabilizers, and the like may be added to these enzyme activity detection or quantification reagents as necessary.
  • the enzyme activity detection or quantification reagent may be prepared as a kit comprising two or more reagents as necessary.
  • Examples of enzymes that cleave single-stranded DNA include nucleases such as S1 nuclease and mangbean nuclease;
  • the first single-stranded DNA probe having a sequence complementary to the specific sequence of the single-stranded DNA fragment having the specific sequence to be detected or quantified, and a carrier that does not easily adsorb DNA are used.
  • the carrier-bound DNA probe bound with or without the probe and the single-stranded DNA fragment having the specific gene sequence to be detected or quantified in the sample Characteristics of a single-stranded DNA fragment having a gene sequence to be detected or quantified, consisting of a second single-stranded DNA probe having a complementary sequence and a labeled DNA probe to which a labeling compound is bound ⁇ ⁇ Array and complementary distribution
  • the first single-stranded DNA probe having a column and the labeled binding DNA probe are bonded to a labeled binding DNA probe and a carrier that does not easily adsorb the DNA are bound with or without a spacer, resulting in carrier binding. It is preferable that, for example, the above-mentioned hybridization solution or the like is added to the reagent compris
  • Hybridization is performed under stringent conditions using the above-mentioned hybridization solution.
  • a sample containing the carrier-bound DNA probe of the present invention and a DNA fragment is added to the hybridization solution, and the mixture is placed at 70 to 90 ° C., preferably 80 to 85 ° C. for 1 to 20 minutes, preferably After heating for 5 to 15 minutes, the mixture is gradually cooled to 0.5 to 24 hours, preferably 1 to 6 hours, to 20 to 50 ° C, preferably 20 to 30 ° C. Do.
  • Hybridization is performed by adding a sample containing the carrier-bound DNA probe of the present invention and a DNA fragment to the hybridization solution at room temperature for 0.5 to 24 hours, preferably 1 to 6 hours. Can also. Thereafter, it is preferable to wash with the above-mentioned washing solution at an intermediate temperature between the melting temperatures of the completely complementary double strand and the mismatched duplex, usually 45 to 65 ° C, preferably 50 to 60 ° C. .
  • the DNA fragments in the unhybridized sample are removed by centrifugation if the particles are used as the carrier, or by washing when the plate is used as the carrier.
  • Detection or quantification of the DNA fragment hybridized to the carrier-bound DNA probe can be performed by a method of detecting or quantifying the DNA in a hybridized state or after isolation from the carrier probe.
  • the detection or quantification of the hybridized DNA fragment is preferably performed using a labeled compound.
  • the detection or quantification method in the case of labeling all sample DNA is described. According to the method described above, all the DNA fragments in the sample are labeled. Next, the carrier-bound DNA probe of the present invention and the labeled DNA are hybridized. The hybridization conditions are the same as the strict hybridization conditions described above. Hybrida Detection or quantification of the hybridized DNA fragment is carried out by removing the non-hybridized DNA fragment and then detecting or quantifying the label of the labeled DNA sample hybridized to the carrier-bound DNA probe.
  • sample DNA ⁇
  • the carrier-bound DNA probe and the labeled DNA probe of the present invention are hybridized under the above hybridization conditions.
  • the conditions for hybridization are the same as the strict hybridization conditions described above.
  • a sample DNA fragment and a label-bound DNA probe which are not hybridized to the carrier-bound DNA probe of the present invention are subjected to centrifugation if the particles are used as the carrier, or to washing if the plate is used as the carrier. Is removed.
  • the detection or quantification of the hybridized DNA fragment is carried out by detecting or quantifying the label of the labeled DNA probe further hybridized to the sample DNA fragment hybridized to the carrier-bound DNA probe.
  • the sample DNA fragment and the carrier-bound labeled DNA probe of the present invention are hybridized.
  • the conditions for the hybridization are not particularly limited, and the conditions may be such that a DNA fragment having a single-base mismatch hybridizes if completely complementary DNA fragments hybridize.
  • an enzyme such as S1 nuclease that cleaves single-stranded DNA is allowed to act to cleave the labeled DNA probe of the present invention that has not formed double-stranded DNA. DNA fragments that are not completely complementary hybridize to the probe DNA, and the partially single-stranded probe DNA is cleaved by the enzyme reaction.
  • Detection or quantification of a completely hybridized DNA fragment is performed by detecting or quantifying the label of the carrier-bound labeled DNA probe.
  • this carrier-bound DNA-labeled probe it is preferable not to use a spacer that binds the DNA probe and the carrier, or to use a probe other than a DNA chain. In this method, even if a sample DNA fragment that binds with a mismatch is generated, this is eliminated. This is particularly preferable in that only a sample DNA having a sequence completely complementary to the probe DNA can be detected.
  • the concentration of DA to be detected or quantified in a sample can be detected or quantified by preparing a calibration curve using a known amount of the sample DNA in advance.
  • the piotin can be detected or quantified by, for example, binding a labeled enzyme-conjugated avidin to the biotin, and then detecting or quantifying the enzyme activity of the labeled enzyme-conjugated avidin bound to the biotin.
  • Binding of biotin and labeled enzyme-conjugated avidin is performed, for example, by allowing the mixture to stand at room temperature for 0.5 to 2 hours in a solution containing a surfactant, salts, buffers, etc. Is removed by centrifugation or washing.
  • the activity of the enzyme bound to avidin can be determined by a conventional method using the aforementioned enzyme activity detection or quantification reagent.
  • the biotin-binding labeling enzyme is alcohol phosphatase
  • a method using a sensitive system [(Ann. Biol, clin), 47, 527 (1989)] using alcohol dehydrogenase and diaphorase is preferable.
  • Detection or quantification is performed by reacting alcohol dehydrogenase with NAD and ethanol produced by alkaline phosphatase to produce NADH, and then using the produced NADH and diaphorase as a substrate for the diaphorase such as INT-biolet. It can be carried out by colorimetrically producing a dye, for example, formazan at 492 nm.
  • the reaction solution was centrifuged (30,000 g, 10 minutes), and the precipitate was washed with distilled water three times and purified to obtain carrier particles.
  • Particles created by this method are abbreviated as SG particles below.
  • the prepared particles were monodisperse and had a particle size of about 0.2 ⁇ m.
  • the particles were placed in 0.1 M phosphate buffer (pH 8.0) containing 2 M mercaptoethanol at room temperature for 24 hours. Reacted. After completion of the reaction, the mixture was centrifuged and washed three times with distilled water, and further subjected to dialysis treatment for 2 minutes to completely remove unreacted mercaptoethanol, thereby producing carrier particles treated with mercaptoethanol.
  • 0.1 M phosphate buffer pH 8.0
  • tris-treated carrier particles were produced in the same manner as described above, except that 2 M mercaptoethanol was replaced with 1 OmM Tris buffer “Tris (hydroxymethyl) aminomethane, (pH 8.0)”.
  • each of the carriers produced above and avidin-labeled peroxidase (manufactured by Behringa-Heim) were combined with 25% formamide, 0.75 M sodium chloride, 0.075 M sodium citrate, The solution was added to a hybridization solution consisting of 1% bovine serum albumin, 1% polyvinylpyrrolidone, and 1% phycol, and incubated at 25 ° C for 30 minutes. Thereafter, the mixture was treated at 55 ° C. for 1 hour, and the carrier was washed with the hybridization solution by centrifugation under the temperature conditions.
  • the carrier was recovered, and the surface activity of 1Z50 was neutralized (neutral washing solution containing nonionic surfactant, manufactured by Kyowa Medex Co., Ltd.). From 1M sodium salt, 1OmM phosphate buffer (pH 8.0) The solution was dispersed in 10 ⁇ l of the resulting solution, 100 ⁇ M MCDP (manufactured by Kyowa Medex) and 1 M hydrogen peroxide were added, and the mixture was allowed to react at 37 ° C. for 30 minutes. Measured at 2 O nm. The concentration of avidin-labeled peroxidase is
  • TRIS Tris (hydroxymethyl) aminoaminomethane
  • Test Example 1 mercaptoethanol-treated carrier or Tris-treated carrier
  • the labeled DNA prepared in Reference Example 2 were combined with 25% formamide and 0.75 M sodium chloride, respectively. It was added to a hybridization solution consisting of 0.75 M sodium citrate, 1% bovine serum albumin, 1% polyvinylpyrrolidone, and 1% ficoll, and reacted at 25 ° C. for 30 minutes. Thereafter, the mixture was treated at 55 ° C. for 1 hour, and the carrier was washed with the hybridization solution by centrifugation under the above temperature conditions.
  • the carrier was recovered and consisted of 1,500 surfactants (neutral washing solution containing nonionic surfactant, manufactured by Kyowa Medex Co., Ltd.) 1M sodium chloride, 1 OmM phosphate buffer ( ⁇ 8.0) Disperse the solution in 100 ⁇ , add 100 / ⁇ MCDP (manufactured by Kyowa Medex) and 1 ⁇ hydrogen peroxide, and react at 37 ° C for 30 minutes. The change was measured at 62 nm.
  • surfactants neutral washing solution containing nonionic surfactant, manufactured by Kyowa Medex Co., Ltd.
  • 1M sodium chloride 1 OmM phosphate buffer ( ⁇ 8.0)
  • Disperse the solution in 100 ⁇ add 100 / ⁇ MCDP (manufactured by Kyowa Medex) and 1 ⁇ hydrogen peroxide, and react at 37 ° C for 30 minutes. The change was measured at 62 nm.
  • TRIS Tris (hydroxymethyl) aminoaminomethane
  • the reaction mixture was centrifuged (30,000 g, 10 minutes), and the precipitate was washed three times with distilled water and purified to obtain carrier particles.
  • Particles created by this method are abbreviated as SG particles below.
  • the prepared particles were monodisperse and had a particle size of about 0.2 ⁇ m.
  • the SG particles prepared in (2) above were washed with 1 OmM phosphate buffer (pH 8.0) by centrifugation to equilibrate the particles.
  • SG particles obtained by equilibrating 8 // g and 30 mg of the double-stranded DNA prepared in (1) above were reacted at 37 ° C for 24 hours in a 4001 phosphate buffer solution and fixed. Unbound DNA was removed by washing three times with a 2.5 M sodium salt solution by centrifugation.
  • the double-stranded DNA immobilized on 2 mg of the SG particles obtained in (3) above was mixed with 25% formamide, 0.75 M sodium chloride, 0.075 M sodium citrate, 1% cow It was added to 400 ml of a hybridization solution consisting of serum albumin, 1% polyvinylpyrrolidone, and 1% ficoll, treated at 80 ° C for 5 minutes, and washed 5 times at this temperature condition. 5, terminal and S of base sequence. A carrier-bound probe was obtained.
  • the DNA-bonded particles were reacted in a 0.1 M phosphate buffer (pH 8.0) containing 2 M mercaptoethanol at room temperature for 24 hours in order to open the epoxy groups remaining on the surface of the SG particles.
  • the mixture was centrifuged and washed three times with distilled water, and further subjected to dialysis treatment to completely remove unreacted mercaptoethanol, thereby producing a carrier-bound DNA probe treated with mercaptoethanol.
  • a probe in which the 5 'end of the base sequence of SEQ ID NO: 2 and the SG carrier were bound was obtained.
  • the DNA-bound particles were placed in 1 OmM Tris buffer (pH 8.0). The reaction was performed at room temperature for 24 hours. After completion of the reaction, the mixture was centrifuged and washed three times with distilled water to produce a carrier-bound DNA probe treated with trisaminomethane.
  • a K-ras mutation detection kit having the following composition was prepared.
  • Example 2 having the DNA of SEQ ID NO: 2) 2 Omg / m1 and the biotin-labeled DNA probe prepared in Reference Example 1 (DN of SEQ ID NO: 3)
  • a K-ras mutation detection kit having the following composition was prepared.
  • Example 3 To a 2 ml microtube, 120 ⁇ l of the first reagent prepared in Example 2 or Comparative Example 2 and 120 ⁇ 1 prepared in Reference Example 2 were respectively added, and the mixture was treated at 55 ° C for 1 hour. 2. After c wash was washed with 2 times centrifugation at 5 M Shioi ⁇ solution of sodium 400 ⁇ 1, at room temperature was added the second reagent 20 mu 1 prepared in Example 2 or Comparative Example 2, respectively centrifuge tube 1 Let stand for hours.
  • TRIS Tris (hydroxymethyl) aminoaminomethane
  • a carrier-bound DNA probe in which the 5 'end of the nucleotide sequence of SEQ ID NO: 2 and the SG carrier were bound was prepared. Then, 15 g of the carrier-bound DNA probe (15 g as DNA) and 15 g of biotinylated dUTP (manufactured by Enzo) were transferred to the terminal reaction buffer in the standard reaction buffer using the 3 'end labeling kit of the same. The reaction was carried out in the presence of a transferase, and a biotin was bound to the 3'-end. Unreacted biotinylated d UTP is washed by centrifugation, and the carrier-bound labeled DNA probe is Manufactured.

Landscapes

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

Abstract

Procédé servant à détecter ou à quantifier un fragment d'ADN monocaténaire possédant une séquence spécifique de gènes et consistant à hybrider ce fragment d'ADN à un spécimen comportant une sonde d'ADN liée à un support et constituée par une sonde d'ADN monocaténaire possédant une séquence complémentaire à la séquence spécifique de gènes du fragment d'ADN monocaténaire contenant la séquence spécifique de gènes à détecter ou à quantifier dans le spécimen et un support lié à ladite sonde éventuellement par l'intermédiaire d'un espaceur; à détecter ou à quantifier le fragment d'ADN ayant été hybridé à la sonde d'ADN liée au support du spécimen. Ce procédé est caractérisé par le fait que, dans la sonde d'ADN liée à un support et mise en application dans ce procédé, le support est un polymère dont la surface possède un groupe époxy et qu'après liaison du support à la sonde d'ADN, on traite le groupe époxy du support avec un composé de SH possédant un groupe polaire. Réactif utilisé dans ce procédé.
PCT/JP1999/002374 1998-05-08 1999-05-07 Procede servant a quantifier une sequence specifique de genes et reactif de quantification WO1999058716A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU36289/99A AU3628999A (en) 1998-05-08 1999-05-07 Method for quantitating specific gene sequence and quantitating reagent

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP10/125858 1998-05-08
JP12585898 1998-05-08

Publications (1)

Publication Number Publication Date
WO1999058716A1 true WO1999058716A1 (fr) 1999-11-18

Family

ID=14920694

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1999/002374 WO1999058716A1 (fr) 1998-05-08 1999-05-07 Procede servant a quantifier une sequence specifique de genes et reactif de quantification

Country Status (2)

Country Link
AU (1) AU3628999A (fr)
WO (1) WO1999058716A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001067129A3 (fr) * 2000-03-09 2002-06-06 Surmodics Inc Surfaces en polymeres epoxydes
WO2004061100A1 (fr) * 2002-12-10 2004-07-22 Olympus Corporation Procede d'analyse d'une mutation de l'acide nucleique et procede d'analyse de l'expression de genes
US7309593B2 (en) 2003-10-01 2007-12-18 Surmodics, Inc. Attachment of molecules to surfaces
US7691787B2 (en) 1997-09-30 2010-04-06 Surmodics, Inc. Target molecule attachment to surfaces
US9163280B2 (en) 2001-06-30 2015-10-20 Enzo Life Sciences, Inc. Process for detecting or quantifying nucleic acids in a library

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10179179A (ja) * 1996-11-08 1998-07-07 Kyowa Medex Co Ltd 特定遺伝子配列の定量方法及び定量試薬

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10179179A (ja) * 1996-11-08 1998-07-07 Kyowa Medex Co Ltd 特定遺伝子配列の定量方法及び定量試薬

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF JAPANESE BIOCHEMICAL SOCIETY, XX, XX, 1 January 1991 (1991-01-01), XX, pages 169 - 180, XP002921822 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7300756B2 (en) 1997-09-30 2007-11-27 Surmodics, Inc. Epoxide polymer surfaces
US7691787B2 (en) 1997-09-30 2010-04-06 Surmodics, Inc. Target molecule attachment to surfaces
US6762019B2 (en) 1997-09-30 2004-07-13 Surmodics, Inc. Epoxide polymer surfaces
JP5005869B2 (ja) * 2000-03-09 2012-08-22 サーモディクス，インコーポレイティド エポキシドポリマーの表面
JP2003526791A (ja) * 2000-03-09 2003-09-09 サーモディックス，インコーポレイティド エポキシドポリマーの表面
WO2001067129A3 (fr) * 2000-03-09 2002-06-06 Surmodics Inc Surfaces en polymeres epoxydes
US9163280B2 (en) 2001-06-30 2015-10-20 Enzo Life Sciences, Inc. Process for detecting or quantifying nucleic acids in a library
US9650666B2 (en) 2001-06-30 2017-05-16 Enzo Biochem, Inc. Processes for detecting or quantifying nucleic acids using an array of fixed or immobilized nucleic acids
US9777406B2 (en) 2001-06-30 2017-10-03 Enzo Biochem, Inc. Process for detecting or quantifying nucleic acids in a library
WO2004061100A1 (fr) * 2002-12-10 2004-07-22 Olympus Corporation Procede d'analyse d'une mutation de l'acide nucleique et procede d'analyse de l'expression de genes
US7309593B2 (en) 2003-10-01 2007-12-18 Surmodics, Inc. Attachment of molecules to surfaces
US7829317B2 (en) 2003-10-01 2010-11-09 Surmodics, Inc. Attachment of molecules to surfaces
US8129159B2 (en) 2003-10-01 2012-03-06 Surmodics, Inc. Attachment of molecules to surfaces

Also Published As

Publication number Publication date
AU3628999A (en) 1999-11-29

Similar Documents

Publication Publication Date Title
JP4216333B2 (ja) オリゴヌクレオチドの化学結合による核酸検出及び増幅
JP4527789B2 (ja) 増加した標的特異的ｔｍを持つ修飾オリゴヌクレオチドを用いた核酸配列の検出および増幅のための方法
JP4634608B2 (ja) 核酸増幅産物を得るために異なるプライマー濃度を用いるための方法
JP2788786B2 (ja) 増幅生成物の迅速アッセイ
JP3021036B2 (ja) 核酸配列又はその中の変化の検出
US9593375B2 (en) Nucleic acid analysis using emulsion PCR
JP4499987B2 (ja) 非−標準塩基を使用する固体支持体アッセイ系及び方法
US11293053B2 (en) Bifunctional oligonucleotide probe for universal real time multianalyte detection
US20030148284A1 (en) Solid phase detection of nucleic acid molecules
US6482592B1 (en) Methods and kits for isolating primer extension products using modular oligonucleotides
JPH10506267A (ja) 核酸の配列または遺伝的変化のハイスループットスクリーニング法
US6054266A (en) Nucleic acid detection with separation
JP2008000143A (ja) オリゴヌクレオチド修飾粒子をベースとするバイオバーコード
JP2005507674A (ja) 迅速乾燥アッセイ法の形で核酸を検出する方法
JP2007506404A (ja) 核酸分子を検出するための迅速な方法
JP2007506402A (ja) Ｄｎａチップに基づく遺伝子型決定
JP2009261406A (ja) β２アドレナリン多型検出
WO2005001113A2 (fr) Procedes de detection de variations d'acides nucleiques
JP3384806B2 (ja) Ｈｌａ−ｄｒ型別判定の実施を可能にするプローブ系および該プローブを用いる型別判定法
JP2644419B2 (ja) 核酸の固定化
EP0843019A2 (fr) Méthode pour la détermination d'une séquence d'acide nucléique spécifique, et un réactif correspondant
WO2007032748A1 (fr) Procede de detection de la methylation de l'adn
WO1999058716A1 (fr) Procede servant a quantifier une sequence specifique de genes et reactif de quantification
US20030207289A1 (en) Detection of genetic sequences using a bipartite probe
IE910210A1 (en) Method and kits for detecting human leukocyte antigen dna

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BG BR CA CN CZ HU ID IL IN JP KR MX NO NZ PL RO SG SI SK UA US VN ZA

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
NENP Non-entry into the national phase

Ref country code: KR

122 Ep: pct application non-entry in european phase