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

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

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] The kit for detecting CYP2C19*2 gene polymorphism by pyrosequencing method provided by the present invention comprises the following reagents:

[0119] CYP2C19*2 upstream amplification primer: 5'-ccagagcttggcatattgtatcta-3';

[0120] CYP2C19*2 downstream amplification primer: 5'-gtccatcgattcttggtgttct-3', the downstream amplification primer is labeled with biotin at the 5' end;

[0121] CYP2C19*2 sequencing primer: 5'-ccactatcattgattatttc-3';

[0122] E.Z.N.A. Boold DNA Kit (Omega, Cat. no.: D3392-02);

[0123] 2XEasyTaq PCR SuperMix (Cat.no: AS111, Beijing TransGen Biotech Biotechnology Co., Ltd.);

[0124] PyroMark Gold Q24 Reagents (Cat. no.: 970802);

[0125] PyroMark Annealing Buffer (Cat. no: 979009);

[0126] PyroMark Bindng Buffer (Cat. no: 979006);

[0127] PyroMark Wash Buffer concentrate (Cat. no: 979008);

[0128] PyroMark Denaturation Solution (Cat. no: 979007);

[0129] Streptavidin Sepharose High Performance (Streptavidin Sepharose-HP, GEhealth Car...

Embodiment 2

[0131] The kit for detecting CYP2C19*2 and CYP2C19*3 gene polymorphisms by pyrosequencing provided by the present invention includes the following reagents:

[0132]CYP2C19*2 upstream amplification primer: 5'-ccagagcttggcatattgtatcta-3';

[0133] CYP2C19*2 downstream amplification primer: 5'-gtccatcgattcttggtgttct-3', the downstream amplification primer is labeled with biotin at the 5' end;

[0134] CYP2C19*2 sequencing primer: 5'-ccactatcattgattatttc-3';

[0135] The kit also includes:

[0136] CYP2C19*3 upstream amplification primer: 5'-ccctgtgatcccactttcat-3';

[0137] CYP2C19*3 downstream amplification primer: 5'-atgtacttcagggcttggtca-3', the downstream amplification primer is labeled with biotin at the 5' end;

[0138] CYP2C19*3 sequencing primer: 5'-ttgtaagcaccccct-3';

[0139] E.Z.N.A. Boold DNA Kit (Omega, Cat. no.: D3392-02);

[0140] 2XeasyTaq PCR SuperMix (Cat.no: AS111, Beijing TransGen Biotech Biotechnology Co., Ltd.);

[0141] PyroMark Gold Q24 Reagents (Ca...

Embodiment 3

[0148] A detection method for detecting CYP2C19*2 gene polymorphism using the kit provided in Example 1 of the present invention.

[0149] 1. Materials and Methods

[0150] 1.1 Research object

[0151] 96 unrelated EDTA anticoagulated blood samples from the inpatient department of Beijing Tiantan Hospital. Blood samples were stored at room temperature, and DNA extraction was completed within 4 hours after collection. All patients signed the informed consent. The 96 specimens were divided into 4 batches with 24 specimens in each batch. Each batch of 24 specimens is arranged in a 4×6 array, respectively labeled A1 to A6, B1 to B6, C1 to C6, and D1 to D6.

[0152] 1.2 Main instruments and equipment

[0153] High-speed centrifuge (Sigma, USA); polymerase chain reaction (PCR) instrument (thermal, USA); pyrosequencer (PyroMark Q24MDx, Qiagen, Germany); fully automatic hybridization instrument (Shanghai Bio Ao Technology Co., Ltd.).

[0154] 1.3 Main reagents

[0155] Reagents...

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Abstract

The invention relates to the technical field of gene polymorphism detection, and in particular relates to sequencing primers, 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 primers provided by the invention comprise a CYP2C19*2 sequencing primer of which the 3'-end base sequence comprises -ccactatcattgattatttc-3', and further comprises a CYP2C19*3 sequencing primer of which the 3'-end base sequence comprises -ttgtaagcaccccct-3'. The kit provided by the invention is short in detection time, low in detection cost and flexible in use, and the detection method 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 (CYP450) enzymes are important drug-metabolizing enzymes in the human body. Among them, CYP2C19 is an important isoenzyme of the CYP450 superfamily, and it...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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