CYP2C9*3 detection parting kit based on probe AllGlo and parting method of CYP2C9*3 detection parting kit

A kit and probe technology, applied in the field of single nucleotide polymorphism, can solve the problems of differences in effective treatment doses, reduce enzyme activity, etc., and achieve the effects of low detection price, improved Tm value, and good quenching effect

Inactive Publication Date: 2016-06-15
ZHONGSHAN HOSPITAL XIAMEN UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CYP2C9 is an enzyme that can inactivate warfarin. Studies have found that CYP2C9*3 can reduce the enzyme activity of CYP2C9 by 80%, which can lead to differences in the effective therapeutic dose of warfarin among different individuals (TakanashiK, TainakaH, KobayashiK, Yasumori T, Hosakawa M, Chiba K. CYP2C9Ile359 and Leu359 variants: enzyme kinetics study with seven substrates. Pharmacogenetics. 2000;10(2):95–104)

Method used

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  • CYP2C9*3 detection parting kit based on probe AllGlo and parting method of CYP2C9*3 detection parting kit
  • CYP2C9*3 detection parting kit based on probe AllGlo and parting method of CYP2C9*3 detection parting kit
  • CYP2C9*3 detection parting kit based on probe AllGlo and parting method of CYP2C9*3 detection parting kit

Examples

Experimental program
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Effect test

Embodiment 1

[0038] The CYP2C9*3 detection and typing kit based on the AllGlo probe of the present invention comprises the following components: real-time fluorescence quantitative PCR reagent, positive control and negative control.

[0039] ①Reagents for real-time fluorescent quantitative PCR include the following components: 10×Taq buffer (10×Taq buffer includes 100mmol Tris-HCl and 500mmol KCl), 10mmol MgCl 2 , 5u / μL TaqHotstart DNA polymerase, 10mmoldNTPs mixture, 50×LowROX, 100mL nuclease-free water, 10μmol / LCYP2C9*3 specific forward primer, 10μmol / LCYP2C9*3 specific reverse primer and AllGlo probe.

[0040] ② Positive controls include: positive homozygous control substance 1, positive homozygous control substance 2, and positive heterozygous control substance. The positive homozygous control substance 1 is a DNA sample of CYP2C9*3 type AA, the positive homozygous control substance 2 is a DNA sample of CYP2C9*3 type CC, and the positive heterozygous control substance is CYP2C9* 3 DNA...

Embodiment 2

[0043] The detection and typing of CYP2C9*3 in peripheral blood includes the following steps:

[0044] 1. Collect EDTA anticoagulated peripheral blood:

[0045] Take 2 mL of fresh blood samples and put them into EDTA anticoagulant tubes and divide them into multiple tubes. Each tube is divided into 400-500 μL, and 200 μL is used for DNA extraction. The rest of the whole blood samples are stored at -80°C.

[0046] 2. Extract DNA:

[0047] Use Genomic Blood DNA Extraction Kit (DP348) produced by Tiangen Biotechnology Co., Ltd. to extract DNA from the sample (whole blood) according to the operating instructions, 200μL of LEDTA anticoagulated whole blood according to the instructions, and finally use 50μL of elution buffer TB dissolves the DNA, and measures the concentration and purity on the nucleic acid spectrophotometer NanoDrop2000. The ratio of purity A260 / A280 is between 1.7 and 1.9, and the extracted DNA with a concentration of 10-50 ng / μL is used for SNP typing detection....

Embodiment 3

[0062] Example 3. SNP detection and typing of tissue samples

[0063] 1. Tissue sample processing:

[0064] Tissues (the amount of spleen tissue should be less than 10 mg) should be crushed and processed into a cell suspension, then centrifuged at 10,000 rpm (~11,200×g) for 1 min, and the supernatant was poured out, and 200 μL of buffer GA (Tiangen DNA Extraction Kit DP304) was added, and shaken until completely suspended;

[0065] 2. DNA extraction and enrichment of tissue samples:

[0066] Use the DNA extraction kit (DP304) produced by Tiangen Biotechnology Co., Ltd. to extract DNA from tissue samples according to the operating instructions, and finally dissolve the DNA with 50 μL of elution buffer TE, and measure the concentration on the nucleic acid spectrophotometer NanoDrop2000 and purity;

[0067] 3. SNP detection and typing of tissue samples.

[0068] For subsequent steps, refer to steps 3-4 of Example 2.

[0069] Applicable range:

[0070] The AllGlo probe-based...

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Abstract

The invention discloses a CYP2C9*3 detection parting kit based on a probe AllGlo and a parting method of the CYP2C9*3 detection parting kit and relates to single nucleotide polymorphism (SNP). The kit comprises a real-time fluorescence quantification PCR reagent, positive control and negative control. CYP2C9*3 is an SNP locus in a CYP2C9 gene of human chromosome 10 provided by NCBI. The parting method comprises the steps that firstly, DNA in an EDTA anti-freezing whole blood sample is extracted through a conventional method; secondly, the real-time fluorescence quantification PCR reagent provided by the CYP2C9*3 detection parting kit based on the probe AllGlo is used for conducting real-time fluorescence quantification PCR amplification on the DNA; thirdly, parting is conducted on the CYP2C9*3 locus of the human gene CYP2C9 according to detected fluorescence signals. On the premise of keeping high specificity and sensitivity of the probe AllGlo, the detecting price is lower than that of a direct sequencing method, the process is simpler, and consumed time is shorter.

Description

technical field [0001] The present invention relates to a single nucleotide polymorphism (SNP), in particular to an AllGlo probe-based CYP2C9*3 detection and typing kit and typing method thereof. Background technique [0002] Single nucleotide polymorphism (singlenucleotide polymorphism, SNP) mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. It is the most common type of heritable variation in humans. SNPs widely exist in the human genome, with an average of 1 in every 500-1000 base pairs, and it is estimated that the total number can reach 3 million or more. As a third-generation genetic marker, SNP is closely related to many phenotypic differences, drug susceptibility and disease susceptibility. The large number of SNPs in the genome makes it a powerful tool and plays a very important role in disease localization and cloning, drug design and testing, and basic biological research. [0003] Individualized d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q1/6851C12Q2531/113C12Q2563/107C12Q2545/113
Inventor 张忠英白永颖方宜臻
Owner ZHONGSHAN HOSPITAL XIAMEN UNIV
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