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Method for detecting CYP2C9 gene exon 9 mononucleotide polymorphism

A technology of single nucleotide polymorphism and detection method, which is applied in the detection field of CYP2C9 gene exon 9 single nucleotide polymorphism, and can solve the problem of undiscovered exon single nucleotide polymorphism, etc.

Inactive Publication Date: 2009-05-20
SHANGHAI JIAO TONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] After searching the existing technical literature, it is found that there is no relevant report on the detection method of the single nucleotide polymorphism (SNP) of exon 9 of the CYP2C9 gene

Method used

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  • Method for detecting CYP2C9 gene exon 9 mononucleotide polymorphism
  • Method for detecting CYP2C9 gene exon 9 mononucleotide polymorphism
  • Method for detecting CYP2C9 gene exon 9 mononucleotide polymorphism

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Experimental program
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Embodiment

[0016] Step 1, nucleic acid isolation and sequencing

[0017] Primer design:

[0018] Using Primer 5.0 software, a pair of allele-specific nucleic acid primers were designed using the GenBank database CYP2C9 gene (NM_000771), the ninth exon and the exon-intron junction sequence as templates, and were synthesized by Invitrogen.

[0019] Primer information:

[0020] DNA sequence primer information for amplifying the SNP site of exon 9 of CYP2C9 gene:

[0021] Forward primer 5'-CTCTGTGCCGCCCTTCTAC-3';

[0022] Reverse primer 5'-TACCCTCTTCCTCTTTGTCC-3'.

[0023] PCR amplification conditions: all reagents in the system except DNA are provided by Bio-Asia,

[0024] The total volume of the reaction system is 15 μl, and the amount of each reaction reagent is expressed as follows:

[0025] wxya 2 O 10.0 μl

[0026] 10X Buffer 1.5μl, where the polymerization reaction buffer contains KCl Tris-HCl solution

[0027] DMSO 0.6μl

[0028] 2mM dNTP 1.5μl

[0029] 0.6 μl each of 10pM ...

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Abstract

The invention discloses a detection method for the CYP2C9 gene ninth exon single nucleotide polymorphism, belonging to the technical field of the genetic engineering. The method comprises the following steps: step one, by taking the CYP2C9 gene ninth exon in a GenBank database and the exon and intron joint part sequence as a template, a pair of allele-specific nucleic acid primers are designed; the primer pair is used for amplifying the DNA sequence of the CYP2C9 gene ninth exon; and then corresponding separated nucleic acids are obtained by conducting separation and purification on the amplified product; and step two, detection is carried out on the 1739-1740 nucleotides of the CYP2C9 gene ninth exon of the separated nucleic acids so as to fix whether single nucleotide polymorphism exists or not. The invention can be used for studying the relationship between the CYP2C9 gene polymorphism in the population of China and the clinical drug safety, and provides guiding basis for the research and development of new drugs.

Description

technical field [0001] The invention relates to a single nucleotide polymorphism detection method in the technical field of genetic engineering, in particular to a single nucleotide polymorphism detection method of the ninth exon of CYP2C9 gene. Background technique [0002] Cytochrome P450 2C9, or CYP2C9, is a member of cytochrome P450. CYP2C9 is abundant in human liver microsomes, accounting for about 20% of the total CYP450s, second only to CYP3A4. The human CYP2C gene is located on chromosome 10q24.2. So far, six CYP2C cDNAs have been isolated, namely CYP2C8, CYP2C9, CYP2C10, CYP2C17, CYP2C18 and CYP2C19. CYP2C9 and CYP2C10 are only different in 2 amino acids, and their catalytic activities are very similar, so CYP2C9 and CYP2C10 are often collectively referred to as CYP2C9 / 10 or CYP2C9. CYP2C9 contains 9 exons and 8 introns, with a total length of about 5.5kb. CYP2C9 5'-upstream 2.2kb contains several sequences consistent with glucocorticoid response elements, and t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 秦胜营贺林熊玉宇王鸣
Owner SHANGHAI JIAO TONG UNIV
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