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Kit for detecting SNP (Single Nucleotide Polymorphism) sites related to Warfarin individualized application, and multiplex PCR (Polymerase Chain Reaction) amplification method and detection method using same

A kit and site technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of decreased arachidonic acid metabolism, decreased CYP4F2 enzymatic activity, decreased arachidonic acid production, etc. , to achieve the effect of reducing detection cost and operation complexity, reducing operation steps and avoiding aerosol pollution

Inactive Publication Date: 2011-11-23
UNION STEMCELL & GENE ENG +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] CYP4F2 is the main metabolic enzyme that metabolizes arachidonic acid to produce tetraenoic acid. Recently, a V433M mutation in the CYP4F2 gene was found to reduce the metabolic capacity of arachidonic acid and reduce the production of tetraenoic acid. Enzymatic activity of CYP4F2
CYP4F2 is a monooxygenase of vitamin K, which can oxidize substrates to generate ω-hydroxyl derivatives, and different genotypes of human liver microparticles show different vitamin K metabolic activities, wild-type CC gene liver microparticles have the highest metabolic activity, while mutant pure The metabolic activity of hepatic microsomes with the TT gene is the lowest, which is 75% of the CC genotype, thereby reducing the metabolism of vitamin K and increasing the concentration of vitamin K, thereby affecting the effect of warfarin

Method used

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  • Kit for detecting SNP (Single Nucleotide Polymorphism) sites related to Warfarin individualized application, and multiplex PCR (Polymerase Chain Reaction) amplification method and detection method using same
  • Kit for detecting SNP (Single Nucleotide Polymorphism) sites related to Warfarin individualized application, and multiplex PCR (Polymerase Chain Reaction) amplification method and detection method using same
  • Kit for detecting SNP (Single Nucleotide Polymorphism) sites related to Warfarin individualized application, and multiplex PCR (Polymerase Chain Reaction) amplification method and detection method using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Step 1: Preparation of whole blood cell lysate

[0069] Take 300 μl of peripheral venous blood from the subject, add 700 μl of cell lysate, invert and mix 5 times, centrifuge at 12,000 rpm for 1 minute, discard the supernatant, and place the centrifuge tube upside down on clean absorbent paper for 2 minutes to ensure that the precipitate remains in the tube , add 300 μl double distilled water, vortex and shake to form a cell lysate suspension.

[0070] Step 2: PCR amplification reaction

[0071] 1. Configure the wild-type reaction system: add 4.8 μl of cell lysis suspension, 5 μl of 2× wild-type amplification buffer, and 0.2 μl of polymerase (2.5 U / μl) into the PCR tube to form an independent reaction system. Mix well after adding the sample. Mix well, centrifuge briefly, and finally add 20 μl liquid paraffin oil to seal the system gently along the tube wall, see the table below for details:

[0072] 2× wild-type amplification buffer solution

5μl

C...

Embodiment 2

[0088] Step 1: Extraction and dilution of whole blood genomic DNA

[0089] Take 300 μl of peripheral venous blood from the subject, and extract whole blood genomic DNA according to the instructions of the whole blood genomic DNA extraction kit. Measure the concentration of DNA with a spectrophotometer and dilute to 15-20 ng / μl.

[0090] Step 2: PCR amplification reaction

[0091] 1. Configure the wild-type reaction system: add 4.8 μl of cell lysis suspension, 5 μl of 2× wild-type amplification buffer, and 0.2 μl of polymerase (2.5 U / μl) into the PCR tube to form an independent reaction system. Mix well after adding the sample. Mix well, centrifuge briefly, and finally add 20 μl liquid paraffin oil to seal the system gently along the tube wall, see the table below for details:

[0092] 2× wild-type amplification buffer solution

5μl

whole blood DNA solution

2μl

double distilled water

2.8μl

Polymerase (2.5U / μl)

0.2μl

liquid par...

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Abstract

The invention discloses a kit for detecting SNP (Single Nucleotide Polymorphism) sites related to Warfarin individualized application by combining multiplex PCR (Polymerase Chain Reaction) technology and SNP sensitive molecule switch technology and a detection method using the same. The kit types four SNP sites related to Warfarin application, including rs9934438 SNP site on VKORC1 gene, rs1799853 and rs1057910 SNP sites on CYP2C9 gene, and rs2108622 SNP site on CYP4F2 gene. The kit comprises a wild type 2*amplification buffer, a mutant type 2*amplification buffer, polymerase, a cell lysis solution and glycerol, wherein the two buffers respectively contain corresponding SNP wild type and mutant phenotype sequence specific primers and beta-actin primers; and the four SNP sites can be typed in the two multiplex PCR reactions, thereby providing molecular biology references for reasonable application of Warfarin.

Description

technical field [0001] The invention relates to a kit for detecting SNP sites and a PCR amplification method thereof, in particular to a kit for detecting SNP sites related to warfarin individualized medication and a multiplex PCR amplification method thereof using multiplex PCR combined with molecular switch technology Detection method. Background technique [0002] Warfarin is a dicoumarin-based oral anticoagulant that inhibits the synthesis of coagulation factors II, VII, IX, and X by vitamin K in liver cells, thereby exerting an anticoagulant effect. It is mainly used clinically to prevent and treat thromboembolic diseases, such as treating thromboembolic phlebitis, reducing the morbidity and mortality of pulmonary embolism, and reducing venous thrombosis in major surgical operations, such as hip arthrodesis and artificial heart valve replacement. Incidence, and as an adjuvant drug for myocardial infarction. However, the effective therapeutic range of warfarin is narro...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 韩俊领杜宏伟周毓玲崔丽娟
Owner UNION STEMCELL & GENE ENG
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