WO1999024609A1 - Procede d'analyse de gene - Google Patents

Procede d'analyse de gene Download PDF

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Publication number
WO1999024609A1
WO1999024609A1 PCT/JP1998/004952 JP9804952W WO9924609A1 WO 1999024609 A1 WO1999024609 A1 WO 1999024609A1 JP 9804952 W JP9804952 W JP 9804952W WO 9924609 A1 WO9924609 A1 WO 9924609A1
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Prior art keywords
polynucleotide
binding substance
label
analysis method
analysis
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PCT/JP1998/004952
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English (en)
Japanese (ja)
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Kaoru Hirai
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Kyoto Daiichi Kagaku Co., Ltd.
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Publication of WO1999024609A1 publication Critical patent/WO1999024609A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention relates to a gene analysis method used for detecting and quantifying a gene in, for example, a clinical test or biochemical analysis.
  • the sample DNA is amplified by the polymerase chain reaction (PCR) method, the PCR product is subjected to electrophoresis, and this is subjected to the Southern blot method.
  • PCR polymerase chain reaction
  • the PCR method is a method in which the amplification can be repeated several million times only for the specific sequence of DNA.
  • an oligonucleotide complementary to a part of the base sequence of the target DNA is used as a primer, (1) dissociating the double-stranded DNA into a single strand, and (2) Annealing the single-stranded DNA and the primer, (3) DNA polymerase
  • the complementary strand is extended by the enzyme, and thereafter, the operations (1) to (3) are repeated to replicate the target DNA in large quantities. Therefore, this method is extremely useful when a small amount of DNA derived from blood, hair roots, or the like is used as a sample.
  • the PCR product (DNA sample) obtained by this PCR is treated with a restriction enzyme, and then subjected to electrophoresis, followed by the following Southern blotting method.
  • the Southern blot method is a method of transferring a DNA fragment fractionated in an electrophoresis gel by the electrophoresis to a nitrocellulose membrane or the like, and then detecting a target DNA on the nitrocellulose membrane or the like using a probe.
  • a probe a polynucleotide complementary to the target DNA is used and is usually labeled with a radioactive substance or the like.
  • Conventional gene analysis methods include, in addition to the Southern blot method, a Northern blot method in which ribonucleic acid (RNA) is immobilized and detected according to the type of the gene to be analyzed, and a dot blot hybridization method in addition to the above method.
  • RNA ribonucleic acid
  • a dot blot hybridization method in addition to the above method.
  • the conventional gene analysis method using the Southern blot method or the like is complicated, requires a long time, and requires safety and analysis. There was a problem with the reliability of the results.
  • an object of the present invention is to provide a gene analysis method which is easy to operate, can be analyzed in a short time, has high analysis accuracy, and is excellent in safety.o
  • the gene analysis method of the present invention is an analysis method for confirming the presence of a target gene in a sample and measuring the amount thereof, and comprises the following (A) to (G) It has a process of.
  • (B) a step of extending a polynucleotide chain complementary to the target gene in the sample using a primer complementary to the target gene and a detectable nucleotide triphosphate (NTP).
  • NTP detectable nucleotide triphosphate
  • (C) a step of dissociating the extended polynucleotide chain into a single strand.
  • (D) A step of repeating the steps (B) and (C) in this order to amplify the polynucleotide chain.
  • the detection principle of the gene analysis method of the present invention is different from the conventional method.
  • the detectable NTP since the detectable NTP is used, the extended polynucleotide chain itself can be detected.
  • this since this is used to form a polynucleotide complex using this and the immobilized single-stranded polynucleotide that is complementary to the NTP. , Obey The target gene can be detected simply and in a short time without the need for complicated operations in multiple steps as in the past. For this reason, variations in the analysis results can be prevented.
  • the analysis method of the present invention is a method that can be performed in a closed system, and therefore has high safety.
  • the detection sensitivity can be sufficiently increased as compared with the conventional analysis method. For this reason, for example, when the PCR method is applied to the present invention, the target DNA can be detected even if the number of PCR cycles is smaller than before, and the operation can also be simplified. Furthermore, in the gene analysis method of the present invention, there are two stages of selection steps: selection with a primer complementary to the target gene and selection with an immobilized single-stranded polynucleotide chain complementary to the target gene. Its analysis accuracy is extremely high. And, in the analysis method of the present invention, the use of primers and immobilized single-stranded polynucleotides relating to a specific infectious disease such as HIV enables a quick, easy and safe diagnosis of the infectious disease.
  • NTP used for elongation of the polynucleotide does not need to use all detectable NTPs, and general NTPs may be used in combination.
  • the detecting means is a means using a first binding substance and a second binding substance that specifically bind to each other, wherein the NTP comprises the first binding substance,
  • the second binding substance is labeled, the second binding substance is supplied to the polynucleotide complex to form a complex, and the second binding substance not involved in the complex formation is separated. After removal, it is preferably a means for detecting the label of the second binding substance.
  • the first binding substance and the second binding substance in the detection means are biotin and the second binding substance is avidin. This is because these two binding substances have high binding specificity and good operability.
  • examples of the combination of the first binding substance and the second binding substance include a combination of an antigen and an antibody.
  • the label of the second binding substance for example, at least one label selected from the group consisting of a radioactive label, a fluorescent label, a chromogenic label, and a specific wavelength light absorbing label can be used.
  • radioactive label examples include 14 C, 32 P, 35 s, and the like.
  • fluorescent label examples include fluorescein isothiocyanate
  • FITC FITC ⁇ tetramethylrhodamine isothiocyanate
  • the color label is a label that develops a color by a chemical reaction or the like, and includes, for example, indole and p-nitro-2-lide.
  • the specific wavelength light-absorbing label refers to, for example, a label showing absorption in a specific light wavelength region such as an ultraviolet region, and includes, for example, Rhodamine 8, Texas Red, Oregon Green and the like.
  • a light-absorbing label having a specific wavelength is preferred from the viewpoint of simplicity of handling and safety.
  • an enzyme that generates a detectable product by an enzymatic reaction is preferable.
  • an enzyme includes, for example, peroxidase, and its substrate is preferably o-phenylenediamine, a Trinder reagent, 41-aminoantipyrine and the like.
  • the polynucleotide chain is DNA, and NTP is deoxyribonucleotide. If it is dotriphosphate (dNTP), the amplification reaction is performed by PCR. If the polynucleotide chain is RNA and NTP is ribonucleotide triphosphate (rNTP), the amplification reaction is performed by RT- It is preferable to carry out by PCR.
  • dNTP dotriphosphate
  • rNTP ribonucleotide triphosphate
  • the carrier for immobilizing and supporting the immobilized single-stranded polynucleotide is surface-treated (blocked) so as not to react with the avidin.
  • a serum albumin, casein or the like can be used.
  • a serum serum albumin is preferable because the preparation is easy.
  • Carriers for immobilizing and supporting the immobilized single-stranded polynucleotide include, for example, plates, Beads, filter paper, nitrocellulose membrane, and the like can be used, but a plate is preferred because of simplicity in a series of operations.
  • the gene analysis method of the present invention can be performed, for example, using a gene analysis kit described below.
  • the analysis kit includes an immobilized single-stranded polynucleotide complementary to the target gene, a detectable NTP, and a primer complementary to the target gene. By using this analysis kit, the analysis method of the present invention can be performed easily and quickly.
  • a first binding substance and a second binding substance that specifically bind to each other wherein the NTP includes the first binding substance, and the second binding substance is a label. It is preferred that
  • the first binding substance and the second binding substance may be a combination of biotin and avidin, as described in the above analysis method. Matching is preferred.
  • the label of the second binding substance may be, for example, at least one selected from the group consisting of a radioactive label, a fluorescent label, a coloring label, and a specific wavelength light absorbing label. Only one sign can be used.
  • the label of the second binding substance is an enzyme, and the enzyme generates a detectable product by an enzymatic reaction, as in the analysis method described above.
  • an enzyme include peroxidase, and its base is preferably o-phenylenediamine, a Trinder reagent, 4-aminoantipyrine and the like.
  • the second binding substance is avidin
  • a surface treatment is performed so that the carrier for immobilizing and supporting the immobilized single-stranded polynucleotide does not react with the avidin. (Blocking) is preferred.
  • serum albumin is used for this.
  • the carrier for immobilizing and supporting the solidified single-stranded polynucleotide is preferably a plate, as in the above-described analysis method.
  • the gene analyzer is a gene analyzer for confirming the presence of a target gene in a sample and measuring the amount thereof, and has the following (A) to (G).
  • (D) means for amplifying a polynucleotide chain by repeating means (B) and means (C) in this order.
  • (E) A means for forming a polynucleotide complex using the immobilized single-stranded polynucleotide and the single-stranded extended polynucleotide.
  • (F) means for removing NTP not involved in the extension reaction.
  • (G) means for detecting the polynucleotide complex by a detection means.
  • FIGS. 1A to 1C are schematic diagrams illustrating an example of primer annealing in an embodiment of the analysis method of the present invention.
  • FIGS. 2A to 2D are schematic diagrams illustrating an example of a polynucleotide chain elongation reaction in one example of the present invention.
  • FIGS. 3A to 3D are schematic diagrams illustrating an example of detection of an elongated polynucleotide in one embodiment of the present invention.
  • FIG. 4 is a graph of absorbance measurement in another example of the present invention.
  • FIG. 5 is a graph of absorbance measurement in still another example of the present invention.
  • FIG. 6 is a graph of absorbance measurement in still another example of the present invention.
  • FIG. 7 is a graph of absorbance measurement according to still another embodiment of the present invention. Oo
  • FIG. 8 is a graph of absorbance measurement in still another example of the present invention.
  • FIG. 9 is a graph of absorbance measurement in still another example of the present invention.
  • FIG. 10 is a graph of absorbance measurement in still another example of the present invention.
  • FIG. 11 is a graph of absorbance measurement in still another example of the present invention.
  • FIGS. 1 to 3 show an example in which the target gene is DNA, and the first binding substance and the second binding substance are biotin and avidin, and the avidin is bound to peroxidase. It is.
  • the same parts are denoted by the same reference numerals.
  • the primer is annealed to the sample DNA. That is, as shown in FIG. 2A, a target double-stranded DNA 1 is prepared. Next, the double-stranded DNA 1 is dissociated into single-stranded DNAs la and lb, as shown in FIG. This dissociation is performed by a heat treatment, an alkali treatment, or the like, but a heat treatment that is simple in operation is preferable. Then, as shown in Figure (C), primers 2a and 2b complementary to the target DNA 1 are added thereto, and when cooled, the single-stranded DNA 1a, lb and primers 2a and 2b are added. And are combined respectively. If the target DNA is not present in the sample, the primers 2a and 2b do not bind to the DNA, and thus the following polynucleotide chain elongation does not occur. That is, Here, the first-stage selection is performed.
  • a polynucleotide chain elongation reaction is performed.
  • biotin is added to each of the complexes of single-stranded DNA A1a and primer 12a and single-stranded DNA A1a and primer 12b via a linker.
  • Add dNTP3 to which 4 is bound (piotinylated dNTP3).
  • the DNA polymerase (not shown) is allowed to act to generate a polynucleotide chain that is complementary to the target DNA by using piotinylated dNTP3. Extend.
  • PCR a large amount of DNA is replicated by repeating the above operations (A) to (C). Then, as shown in FIG. 2D, a single-stranded elongated polynucleotide having the target DNA in the form of ⁇ is obtained.
  • This extended polynucleotide has the same number of biotins as the number of incorporated nucleotide residues, and is used to detect extended polynucleotides, as described later. . Therefore, the number of PCR cycles can be reduced as compared with the conventional case.
  • the present invention it is not necessary to use all detectable NTPs for NTP used for polynucleotide extension, and for example, only dATP may be detectable. Even in this case, a significantly higher detection sensitivity than the conventional method can be obtained.
  • detection was performed using a polynucleotide complementary to the extended polynucleotide as a probe. Therefore, the detection sensitivity depends on the amount of the probe and the number of cycles of the extension reaction (ie, the number of the extended polynucleotides), and it was necessary to sufficiently repeat the PCR cycle.
  • the length of the linker that binds biotin and dNTP is preferably determined as appropriate according to the target gene, but is usually 3 to 30 atom linker, preferably 7 to 30 atom linker. ⁇ 14 atom linker One range.
  • a polynucleotide complex is formed using the extended polynucleotide and the immobilized single-stranded DNA, and then the extended polynucleotide is detected.
  • the immobilized single-stranded DNA is complementary to the target DNA.
  • an immobilized single-stranded DNA A5 in which single-stranded DNAs 1a and 1b complementary to a target DNA are immobilized on a carrier 6 is prepared.
  • a carrier 6 As described above, plates, beads, and the like can be used as the carrier.
  • a film such as a nylon membrane, glass such as quartz, nitrocellulose, or fiber can be used, and is not particularly limited.
  • the method for immobilizing the single-stranded DNA is not particularly limited, and a conventionally known method can be employed, and examples thereof include a method such as a heat treatment or an acid treatment.
  • the amount of immobilized single-stranded DN A in order to influence the detection sensitivity, each polynucleotide strand length of, depending on the sequence and GC content, etc., arbitrariness 0 preferred to determine the optimum amount
  • the extended polynucleotide obtained by the above-described operation is supplied to the immobilized single-stranded DNA 5, thereby forming a polynucleotide complex.
  • the dotted lines indicate hydrogen bonds.
  • dNTPs not involved in the extension reaction are removed (so-called B / F separation). This removal can be easily performed in a short time by washing. The washing can be performed by an ordinary method using a filter or the like.
  • a second-stage selection for detection of the target DNA is performed.
  • adivine 7 to which peroxidase 8 is bound is supplied to the complex.
  • the avidin 7 binds to the extended polynucleotide, biotin 4.
  • the avidin 7 not bound to biotin 4 is removed. This removal can be easily and quickly performed by washing or the like.
  • an enzyme reaction is carried out by adding the substrate 9 of the peroxidase 8 described above. If, for example, hydrogen peroxide and 0-phenylenediamine are used as the substrate, the product shows light absorption at a wavelength of 492 ⁇ m, and the absorbance is measured. This measurement is performed using an ordinary spectrophotometer or microplate reader. This absorbance increases in accordance with the number of piotinylated dNTPs incorporated into the extended polynucleotide, so that the detection sensitivity of the target DNA is extremely high.
  • the example described above is an example of the present invention. Therefore, for example, when the gene to be analyzed is RNA, the analysis of the target RNA can be performed quickly and easily by appropriately selecting a polynucleotide extension operation or the like in accordance with the gene.
  • the final detection was performed by measuring the absorbance of the enzyme reaction product.
  • radioactive labels there are other detection methods using radioactive labels and the like.
  • the polynucleotide is extended using NTP directly labeled with these labels to form a complex with the immobilized single-stranded polynucleotide, and A label or a fluorescent label is detected by a conventionally known method.
  • the gene analysis method of the present invention can be applied to diagnosis of infectious diseases.
  • infectious diseases include, for example, HIV infection, hepatitis C, HPV (human papilla-mavirus), HCMV (human cytomegalovirus), and the like.
  • HPV human papilla-mavirus
  • HCMV human cytomegalovirus
  • the analysis kit that can be used in the gene analysis method of the present invention includes an immobilized single-stranded polynucleotide complementary to the target gene, a detectable NTP, and a primer complementary to the target gene. Have.
  • the method of using the analysis kit is the same as the analysis method described above. By using this kit, it is not necessary to prepare a reagent or the like each time analysis is performed, and the gene analysis method of the present invention can be performed more quickly and easily. Then, in this kit, if a primer and an immobilized single-stranded polynucleotide relating to a specific infectious disease are used, it can be used as a diagnostic kit for the infectious disease.
  • the dCTP, dTTP, dGTP, dATP and TaqDNA polymerase used were those manufactured by Takara Shuzo.
  • Piotin-14-dATP and piotin-1 7-dATP (hereinafter referred to as B-14-dATP and B-7-dATP) were obtained from GIBC0.
  • Each number of the above-mentioned piotinylated ATP represents the length of a linker, and its unit is an atomic linker.
  • M13mpl8RFDNA manufactured by Takara Shuzo
  • M13mp18ssDNA and M13mpl8RFDNA manufactured by Takara Shuzo
  • M13 Primer M4 manufactured by Takara Shuzo
  • M13 Primer RV manufactured by Takara Shuzo
  • M13 Primer M4 manufactured by Takara Shuzo
  • M13mp18ss DNA which is a DNA complementary to the target gene, was heat-treated at 100 ° C. for 15 minutes and immediately cooled to 0 ° C. to prepare single-stranded DNA.
  • This single-stranded DNA was mixed with a 10 X SSC buffer solution mixed with 10 mM EDTA (pH 7.0) at a predetermined concentration (0 pmo ⁇ / 1, 0.00000069 pmol 1, 0.00021 pmol 1, 0.
  • the mixture was diluted with 00062 pmol 1, 0.0019 pmo 1 Z 1, 0.0056 pmo 10 .017 pmo 11) to prepare seven concentrations of single-stranded DNA solutions.
  • This solution was injected into a microplate well at 100 ⁇ 1 each. And incubate at 37 ° C for 2 hours After performing the washing and washing twice with the PBS_T buffer, PBS-1 buffer solution 1001 was added to each well, and the well was stored at 4 ° C.
  • Ml3mp18RFDNA was treated with restriction enzymes Av aII (Takara Shuzo) and Ec081I (Takara Shuzo), and subjected to agarose gel electrophoresis. Of the two bands obtained, a short Avail-Eco81I fragment was recovered by ethanol precipitation, and purified by a conventional method to obtain target DNA. This fragment becomes type I DNA in PCR.
  • Step 4 (72 ° C, 90 seconds): 30 cycles
  • Step 5 (72 ° C, 4 minutes): 1 cycle
  • the resulting biotinylated single-stranded DNA was diluted 10-fold with 6 X SSC buffer, 5 X Denhardt's solution, 50 mM PBS (pH 6.7) buffer and 25 gm 1 salmon sperm DNA.
  • the above-mentioned single-stranded DNA was added to each plate of the immobilized microplate in an amount of 100 1 and incubated at 55 ° C for 2 hours. Thereafter, the pellet was washed twice with PBS-T buffer.
  • microplate Ueru to 9 mM 0 of - phenylenediamine ⁇ Mi emissions OPD, manufactured by I CN Pharmaceutical Co.
  • 1 OmM H manufactured by Santoku Kagaku Co.
  • 0. 1M Kuen triphosphate 100 1 each of buffer (CP B, pH 5.0) was added and reacted at room temperature for 10 minutes . Thereafter, 3. 6 NH to 2 S 0 4 5 0 1 Dzu' added, off to stop the reaction ⁇ ⁇
  • the immobilized concentration means the concentration of immobilized single-stranded DNA.
  • Ml3mpl8 RFDNA which is a DNA complementary to the target gene, is treated with restriction enzymes with AVall and Bg1II, and subjected to electrophoresis.
  • the fragment was extracted and purified by a conventional method. After dissociating the DNA in the purified product into single-stranded DNA, five concentrations (0 pm 01 Z / z 1, 0.0 002 pmo 1/1, 0. ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ / ⁇ ⁇ 0.00 19 pmo 1/1, 0.005 po ⁇ / ⁇ ⁇ ), and these were prepared in the same manner as in Example 1. The operation was performed to prepare an immobilized single-stranded DNA.
  • a complex was formed in the same manner as in Example 1 except that the biotinylated single-stranded DNA prepared in Example 1 was supplied to the immobilized single-stranded DNA, and the absorbance was measured after the enzyme reaction. .
  • the results are shown in the graph of FIG.
  • a complex was formed in the same manner as in Example 1 except that the biotinylated single-stranded DNA prepared in Example 1 was supplied to the immobilized single-stranded DNA, and the absorbance was measured after the enzyme reaction. The results are shown in the graph of FIG.
  • the absorbance varied depending on the concentration of the immobilized single-stranded DNA, and the maximum absorbance was exhibited at a concentration of 0.74 pmo1.
  • M13 Primer RV which is a DNA complementary to the target gene, was dissociated into single-stranded DNA in the same manner as in Example 1, and the DNA was prepared at five different concentrations (O pmo ⁇ / nl ⁇ 0.03 pmol / 1, 0.08 pmo 1 / z K 0.2
  • a complex was formed in the same manner as in Example 1 except that the biotinylated single-stranded DNA prepared in Example 1 was supplied to the immobilized single-stranded DNA, and the absorbance was measured after the enzyme reaction. did. The results are shown in the graph of FIG.
  • Ml3mp18 RFDNA as target gene
  • M13PrimerM4 as DNA complementary to target gene to be immobilized
  • M13PrimerM4 and M13PrimerRV as primers for PCR The following operations were performed using each.
  • iDNA (Takara Shuzo) was replaced with Hindlll.
  • PCR was carried out in the presence of an ADNA-Hind III-treated product treated with a restriction enzyme by Takara Shuzo Co., Ltd. Then, PCR was performed by adding I DNA-Hindill-treated product to the target DNA; the result was used as a positive control (PC), and PCR-treated only with ⁇ -HindIII-treated product was used. Negative control (NC) was used.
  • PC-S and NC-S when B-dNTP was used for amplification by PCR in these PCs and NCs, and PC-N and NC when B-dNTP was not used, respectively. — N. This operation is the same in the following Examples 6 to 8.
  • the target gene (Ml 3mp) prepared by PCR in the same manner as in Example 1
  • the piotinylated single-stranded DNA of Avail (Eco 81 I fragment of 18RF DNA) was diluted 2700-fold and supplied to the immobilized single-stranded DNA to form a complex. After the enzyme reaction, the absorbance was measured. Was measured. The results are shown in the graph of FIG.
  • the absorbances of PC—S and PC—N are 0.760 and 0.151, respectively, which are about a five-fold difference. Since this PC-N does not use B-dNTP for PCR, this absorbance is not dependent on the enzymatic reaction, but is considered to be a value derived from the light absorption of the amplified DNA fragment itself. The same can be said for the measured values of NC-N.
  • the required measurement values are PC-S, which is the extended chain of the target gene using B-dNTP, and NC-S, which is the extended chain without the target gene. .
  • the difference ASIG (PC-NC) between these values is 0.651, and it can be said that the presence or absence of the target gene in this example can be clearly determined from the difference between these values.
  • HCMV-T synthetic DNA having recognition sequences for HCMVpF and HCMVpR
  • DNA complementary to the target gene to be immobilized primer HCMVpR
  • primer for PCR primer for PCR
  • HC which is a DNA complementary to the target gene, as in Example 1.
  • the MV pR was dissociated into single strands, the concentration was adjusted to be 0.003 pmo 1 Z ⁇ 1, and this was used to prepare immobilized single-stranded DNA.
  • the same operation as in Example 1 was performed unless otherwise indicated, except that the above-mentioned primers were used.
  • PCR was performed in the following cycle.
  • Step 1 (94 ° C, 30 seconds): 1 cycle Step 2 (94 ° C, 30 seconds)
  • Step 4 (72 ° C, 2 minutes): 30 cycles
  • Step 5 (72 ° C, 4 minutes): 1 cycle
  • the biotinylated single-stranded DNA of the target gene prepared by PCR was diluted 30-fold and supplied to the immobilized single-stranded DNA to form a complex. After the enzymatic reaction, the absorbance was measured. The results are shown in the graph of FIG. As shown in the graph of FIG. 9, the absorbances of PC—S, PC—N, NC—S, and NC—N were 1.161, 0.494, 0.000, and 0.078, respectively. From these results, it was confirmed that also in this example, the enzyme reaction of peroxidase was not performed unless the extended chain incorporating biotin bound to the immobilized single-stranded DNA.
  • the required measurement values are PC-S, which is the extended strand of the target gene using B-dNTP, and NC-I, which is the extended strand having no target gene. S.
  • the difference ASIG (PC-NC) between these values was 1.161. From the difference between these values, it can be said that in this example, the presence or absence of the gene derived from human cytomegalovirus could be clearly determined.
  • HPVT16 synthetic DNA having recognition sequences for HPVpF and HPVp16R
  • HPVbl6 DNA having recognition sequences for HP VT16
  • PCR DNA complementary to the target gene to be immobilized
  • HPVp F 25mo ⁇ / ⁇
  • HPVp16R 25mo1 // z1
  • Vb 16 is dissociated into single strands and its concentration reaches 0.003 pmol z / z 1. This was used to prepare immobilized single-stranded DNA. Hereinafter, unless otherwise indicated, the same operation as in Example 1 was performed.
  • PCR reaction solution was prepared with the composition shown below, and PCR was performed in the same cycle as in Example 6.
  • the piotinylated single-stranded DNA of the target gene prepared by PCR was diluted 20-fold and supplied to the immobilized single-stranded DNA to form a complex.After the enzymatic reaction, the absorbance was measured. . The results are shown in the graph of FIG.
  • the absorbances of PC—S, PC—N, NC—S, and NC—N were 1.711, 0.354, 0.023, and 0.000, respectively. From these results, it can be seen that in this example, as long as the extended strand incorporating the biotin did not bind to the immobilized single-stranded DNA, It was confirmed that the enzyme reaction of oxidase was not performed. Also, as the conditions of the actual measurement system, the required measurement values are PC-S, which is an extended chain of the target gene using B-dNTP, and NC-S, which is an extended chain having no target gene. . The difference between these values, SIG (PC-NC), was 1.688. From this difference, it can be said that in this example, the presence or absence of the gene derived from human papillomavirus could be clearly determined.
  • Control DNA synthetic DNA having recognition sequence for LAU-1 and LAC-3R
  • LAF-3R recognition sequence for control DNA
  • LAC-3R recognition sequence for control DNA
  • Example 2 LAF-3R, which is a DNA complementary to the target gene, was dissociated into single strands, and the concentration was adjusted to 0.0003 pmo ⁇ 1. Immobilized single-stranded DNA was prepared.
  • the same operation as in Example 1 was performed unless otherwise indicated, except for using the primer.
  • control DNA was designated as type I DNA, and PCR was performed in the following cycle.
  • Step 1 (94 ° C, 30 seconds): 1 cycle Step 2 (94 ° C, 30 seconds) —Step 3 (55 ° C, 1 minute)
  • Step 4 (72 ° C, 1 minute) 35 cycles
  • Step 5 (72 ° C, 4 minutes) 1 cycle
  • the piotinylated single-stranded DNA of the target gene prepared by PCR was diluted 20-fold and supplied to the immobilized single-stranded DNA to form a complex, and the absorbance was measured. The results are shown in the graph of FIG.
  • the absorbances of PC—S, PC—N, NC—S, and NC—N were 0.209, 0.000, 0.000, and 0.036, respectively. From these results, it was confirmed that also in this example, the enzyme reaction of peroxidase was not performed unless the extended chain incorporating the biotin bound to the immobilized single-stranded DNA.
  • the required measurement values are PC-S, which is an extended chain of the target gene using B-dNTP, and NC-S, which is an extended chain without the target gene. .
  • the difference ⁇ S IG (P C — N C) between these values was 0.209, and it can be said that from the difference between these values, it was possible to clearly determine the presence or absence of the gene derived from Hiochi in this example.
  • the analysis method of the present invention is a method that can easily and safely carry out analysis of a target gene in a short time, and the analysis results are extremely reliable and can be automated. . Therefore, if the analysis method of the present invention is applied to, for example, the field of clinical medicine such as diagnosis of infectious diseases, it can greatly contribute to efficiency of diagnosis. In particular, if the analysis method of the present invention is applied to the diagnosis of an infectious disease, such as HIV infection, which is difficult to detect in the early stage of infection by an immunological detection method, the effect is large.

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  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne un procédé permettant de détecter facilement un gène cible pendant une courte durée, avec une précision élevée, consistant (A) à préparer un polynucléotide (5) immobilisé à un seul brin, complémentaire du gène cible; (B) à allonger un brin de polynucléotide dans un échantillon complémentaire du gène cible, à l'aide de primaires (2a, 2b), eux-mêmes complémentaires du gène cible et d'un NTP détectable; (C) à dissocier le brin de polynucléotide allongé ainsi obtenu en brins simples; (D) à répéter les procédures des étapes (B) et (C) ci-dessus, de manière à amplifier ainsi les brins de polynucléotide; (E) à former des complexes de polynucléotide à l'aide du polynucléotide (5) immobilisé à un seul brin susmentionné avec les polynucléotides allongés, à un sel brin susmentionnés; à éliminer le NTP qui ne participe pas à la réaction d'élongation; et (G) à détecter les complexes de polynucléotides ainsi obtenus à l'aide d'un dispositif de détection.
PCT/JP1998/004952 1997-11-07 1998-10-30 Procede d'analyse de gene WO1999024609A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP9/306152 1997-11-07
JP9306152A JPH11137297A (ja) 1997-11-07 1997-11-07 遺伝子分析方法

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WO1999024609A1 true WO1999024609A1 (fr) 1999-05-20

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WO2002016528A1 (fr) * 2000-08-21 2002-02-28 National Institute Of Advanced Industrial Science And Technology Particules magnetiques et procede de fabrication correspondant

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002016528A1 (fr) * 2000-08-21 2002-02-28 National Institute Of Advanced Industrial Science And Technology Particules magnetiques et procede de fabrication correspondant
US7393698B2 (en) 2000-08-21 2008-07-01 National Institute Of Advanced Industrial Science And Technology Magnetic fine particles and process for producing the same

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