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Method of typing gene polymorphisms

A genetic polymorphism and gene technology, applied in the field of genetic polymorphism typing, can solve the problem that it cannot be used to detect trace target nucleic acids

Inactive Publication Date: 2005-09-21
TAKARA HOLDINGS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The above methods suffer from the following problems: cannot be used to detect trace amounts of target nucleic acids; must be performed under strict temperature / time conditions; annealing to target nucleic acids needs to be strictly controlled; and require enzymes with special properties required for detection

Method used

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Experimental program
Comparison scheme
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Embodiment approach

[0176] An embodiment of the method for typing genetic polymorphisms in multiple target nucleic acids according to the present invention is exemplified below: a method for typing genetic polymorphisms, wherein multiple target nucleic acids are detected in parallel, the method comprising:

[0177] (1) template nucleic acid, deoxyribonucleoside triphosphate, DNA polymerase with strand displacement activity, at least two nucleotides and RNase H are mixed to prepare a reaction mixture, wherein

[0178] (a) the 3' terminus of said nucleotides is modified so that extension from that terminus does not occur under the action of a DNA polymerase; and

[0179] (b) the nucleotides each have a nucleotide sequence that anneals to a specific region containing the specific nucleotide in the plurality of target nucleic acids; and

[0180] (2) incubating the reaction mixture for a period of time sufficient to generate reaction products to extend nucleic acids each containing a region of arbitra...

Embodiment 1

[0241] (1) Preparation of genomic DNA

[0242]After obtaining informed consent from 7 healthy human donors, 7 samples of 10ml blood were collected. Genomic DNA was prepared from 100 µl of each of the above bloods using Dr. GenTLE (for blood) (Takara Bio.). The concentration of the obtained genomic DNA solution is as follows:

[0243] Test sample 1: 182ng / μl

[0244] Test sample 2: 150ng / μl

[0245] Test sample 3: 156ng / μl

[0246] Test sample 4: 204ng / μl

[0247] Test sample 5: 105ng / μl

[0248] Test sample 6: 172ng / μl

[0249] Test sample 7: 253ng / μl

[0250] (2) Synthesis of primers and probes

[0251] Design oligonucleotide primers that can be used for ICAN reaction detection and contain 3 RNA residues at the 3' end, and based on the nucleotide sequence of the human glutathione-S-transferase M1 (GSTM1) gene (GenBank No. X51451) synthesis. Specifically, oligonucleotide primers GS-F (SEQ ID NO: 6) and GS-R (SEQ ID NO: 7) were synthesized. Oligonucleotide GS-F is a ...

Embodiment 2

[0263] (1) Allele-specific detection of human CYP2C19(636)

[0264] An assay for determining whether the allele at nucleotide 636 in human CYP2C19 is genetically homozygous or heterozygous (homozygous or heterozygous) was tested.

[0265] After obtaining informed consent, 200 μl whole blood was obtained from each healthy individual (samples 1-6) and processed using Dr.GenTLE TM (Takara Bio) Genomic DNA was prepared from the above whole blood. In a Thermal Cycler Personal (Takara Bio), a reaction mixture with a total volume of 5 μl containing 160 ng of prepared genomic DNA as a template, a sense primer for specific detection of 636G (SEQ ID NO : 14) or 636A (SEQ ID NO: 15) allele synthetic oligonucleotide and synthetic oligonucleotide (SEQ ID NO: 16) as an antisense primer: each 50 pmol, 1 μl 0.05% propylene imine aqueous solution , followed by annealing of the primers to the template at 53°C. 20 μl containing 0.625mM dNTP mixture, 40mM Hepes-KOH buffer (pH7.8), 125mM potass...

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Abstract

Accurate and highly reproducible means of detecting a base substitution mutation (for example, SNP), an insertion mutation or a deletion mutation with the use of a nucleic acid sample in a trace amount and a method of typing gene polymorphisms using the means.

Description

technical field [0001] The present invention relates to a method for detecting base substitution, insertion mutation or deletion mutation in a gene of interest and a kit suitable for genotyping method. Background technique [0002] It is known that the genetic codes contained in the genomes of individuals of the same species are different, and there are also differences in nucleotide sequences, which are called polymorphisms. Cases in which 1 to 10 nucleotides are deleted or inserted, cases in which a specific nucleotide sequence is duplicated, and the like are considered polymorphisms. A situation involving the substitution of a single nucleotide by another nucleotide is called a single nucleotide polymorphism (SNP). [0003] Conventional means for detecting SNPs are generally classified into hybridization-based means, primer extension-based means, and means utilizing the substrate specificity of enzymes. [0004] The presence of a base substitution is detected based on h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q2600/156C12Q1/6827C12Q1/6883C12Q2531/119C12Q2525/121C12Q2521/319C12Q1/6844
Inventor 小林英二榎龙嗣田边雅茂上田木绵奥田真治佐川裕章加藤郁之进
Owner TAKARA HOLDINGS
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