VKORC1 gene polymorphism detecting and genotyping kit based on AllGlo probe and genotyping method thereof

A technology of genetic polymorphism and kit, applied in the direction of microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of warfarin effective treatment dose difference, etc., and achieve low detection price, simple process, and time-consuming short effect

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

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Problems solved by technology

In the process of clinical medication, it was found that there are great differences in the effective therapeutic dose of warfarin among different individuals, especially the commonly used dose of Chinese people is significantly lower than that of Westerners.

Method used

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  • VKORC1 gene polymorphism detecting and genotyping kit based on AllGlo probe and genotyping method thereof
  • VKORC1 gene polymorphism detecting and genotyping kit based on AllGlo probe and genotyping method thereof
  • VKORC1 gene polymorphism detecting and genotyping kit based on AllGlo probe and genotyping method thereof

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Experimental program
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Effect test

Embodiment 1

[0038] The VKORC1 gene polymorphism 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 / LVKORC1(-1639A / G) specific forward primer, 10μmol / LVKORC1(-1639A / G) specific reverse primer Primers and AllGlo probes.

[0040] ② Positive controls include: positive homozygous control substance 1, positive homozygous control substance 2, and positive heterozygous control substance. The positive homozygous reference substance 1 is a DNA sample of VKORC1 (-1639A / G) typed as TT, and the positive homozygous control substance 2 is a DNA sample of VKORC1 (-1639A / G) type...

Embodiment 2

[0043] The detection and typing of VKORC1(-1639A / G) 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 de...

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, 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] A VKORC1 gene polymorphis...

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Abstract

The invention discloses a VKORC1 gene polymorphism detecting and genotyping kit based on an AllGlo probe and a genotyping method thereof and relates to single nucleotide polymorphisms. The kit comprises a real-time fluorescent quantitative PCR reagent, positive control and negative control. VKORC1(-1639A/G) provides an SNP locus in a VKORC1 gene of a chromosome 16 of a person for NCBI. The genotyping method includes the steps that firstly, DNA in an EDTA anticoagulant whole-blood sample is extracted through a conventional method; secondly, the real-time fluorescent quantitative PCR reagent provided by the detecting and genotyping kit performs real-time fluorescent quantitative PCR amplification on the DNA; thirdly, genotyping is performed on the single nucleotide polymorphisms locus VKORC1(-1639A/G) of the VKORC1 gene of a person according to a detected fluorescence signal. On the premise that high specificity and high sensitivity of the AllGlo probe are kept, the detection price is lower than that of a direction sequencing method, the process is simpler, and consumed time is shorter.

Description

technical field [0001] The invention relates to single nucleotide polymorphism (SNP), in particular to a VKORC1 gene polymorphism detection and typing kit and typing method based on AllGlo probe. 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
CPCC12Q1/6851C12Q1/6858C12Q2531/113C12Q2563/107C12Q2545/113
Inventor 张忠英方赞熙苏元晖方宜臻
Owner ZHONGSHAN HOSPITAL XIAMEN UNIV
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