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Sequencing primer, kit and detection method for detecting CYP2C19 gene polymorphism

A CYP2C19, gene polymorphism technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of inflexible use, high cost, long time consumption, etc.

Inactive Publication Date: 2014-06-18
康熙雄 +7
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the shortcomings of DNA microarray chip method for detecting CYP2C19 gene polymorphism in the prior art, which takes a long time, costs high, and is inflexible to use, this application provides a sequencing primer for detecting CYP2C19 gene polymorphism by pyrosequencing method, Kit and detection method, the kit has short detection time, low detection cost, flexible use, and easy operation of the detection method

Method used

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  • Sequencing primer, kit and detection method for detecting CYP2C19 gene polymorphism
  • Sequencing primer, kit and detection method for detecting CYP2C19 gene polymorphism
  • Sequencing primer, kit and detection method for detecting CYP2C19 gene polymorphism

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preparation example Construction

[0152] (4) Preparation of single-stranded DNA template In the vacuum workstation, add 50ml of 70% (volume ratio) ethanol to position 1, add 40ml of Denaturation Buffer (denaturation buffer) to position 2, and add Washing Buffer (washing buffer) to position 3. ) 50ml, add 50ml of high-purity water to position 4, add 70ml of high-purity water to position 5; turn on the vacuum pump, clean the vacuum tool in position 5 for 15s, move to the PCR plate to absorb Beads15s, and place the amplification product in position 1 and position 2 in turn Position and No. 3 are cleaned for 5s, 5s, 10s.

[0153] (5) Primer hybridization Close the vacuum valve of the vacuum toolbox, put the single-stranded DNA template obtained in step (4) into the annealing buffer containing sequencing primers on the detection plate, shake fully to release the Beads, and hybridize at 80°C for 2 minutes, Let stand at room temperature for 5 minutes to cool down.

[0154] (6) Sequencing Add the product obtained in ...

Embodiment 1

[0165] The kit for detecting CYP2C19*17 gene polymorphism by pyrosequencing provided by the present invention comprises the following reagents:

[0166] Primers for detection of CYP2C19*17 gene polymorphism:

[0167] CYP2C19*17 upstream amplification primer: 5'-GTGATGGAGAAGGGAGAACTCTTA-3';

[0168] CYP2C19*17 downstream amplification primer: 5'-ATCGTGGCGCATTATCTCTTAC-3', the downstream amplification primer is labeled with biotin at the 5' end;

[0169] CYP2C19*17 sequencing primer: 5'-TTGTGTCTTCTGTTCTCAA-3'.

[0170] E.Z.N.A. Boold DNA Kit (Omega, Cat. no.: D3392-02);

[0171] 2XEasyTaq PCR SuperMix (Cat.no: AS111, Beijing TransGen Biotech Biotechnology Co., Ltd.);

[0172] PyroMark Gold Q24 Reagents (Cat. no.: 970802);

[0173] PyroMark Annealing Buffer (Cat. no: 979009);

[0174] PyroMark Bindng Buffer (Cat. no: 979006);

[0175] PyroMark Wash Buffer concentrate (Cat. no: 979008);

[0176] PyroMark Denaturation Solution (Cat. no: 979007);

[0177] Streptavidin Sephar...

Embodiment 2

[0179] The kit for detecting gene polymorphisms of CYP2C19*17 and CYP2C19*2 loci by pyrosequencing method provided by the present invention includes the following reagents:

[0180] Primers and other reagents for detecting CYP2C19*17 gene polymorphism described in Example 1. In addition, primers for detection of CYP2C19*2 gene polymorphisms are included:

[0181] CYP2C19*2 upstream amplification primer: 5'-ccagagcttggcatattgtatcta-3';

[0182] CYP2C19*2 downstream amplification primer: 5'-gtccatcgattcttggtgttct-3', the downstream amplification primer is labeled with biotin at the 5' end;

[0183] CYP2C19*2 sequencing primer: 5'-ccactatcattgattatttc-3'.

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Abstract

The invention relates to the technical field of gene polymorphism detection, and in particular relates to a sequencing primer, a kit and a detection method for detecting CYP2C19 gene polymorphism, which aim at overcoming the defects that a DNA microarray chip method for detecting CYP2C19 gene polymorphism is long in consumed time, relatively high in cost and inflexible in use in the prior art. The sequencing primer provided by the invention is the one for detecting CYP2C19*17 gene polymorphism, and the 3'-end base sequence of the sequencing primer is -TTGTGTCTTCTGTTCTCAA-3'. The kit provided by the invention is short in detection time, low in detection cost, flexible in use and very high in detection precision. The detection method provided by the invention is easy in operation.

Description

technical field [0001] The invention relates to the technical field of gene polymorphism detection, in particular to a sequencing primer, a kit and a detection method for detecting CYP2C19 gene polymorphism by pyrosequencing. Background technique [0002] Different individuals have great differences in the response, toxicity and therapeutic effect of drugs. There are many reasons for this individual difference. Among them, the gene polymorphism of drug metabolizing enzymes may lead to the drug efficacy and adverse reactions of many drugs in treatment. A cause of individual differences. With the continuous deepening of pharmacogenomics research, the study of the differences in individual allelic polymorphisms affecting individual responses to drugs has become a research hotspot at home and abroad. [0003] Cytochrome P450 (cytochrome P450, CYP450) enzyme system is an important drug-metabolizing enzyme in the human body, among which CYP2C19 is an important isoenzyme of the CY...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q2531/113C12Q2565/301
Inventor 康熙雄王谦张国军刘志忠马瑞敏吕虹杜亚梅方芳
Owner 康熙雄
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