Primer and method for detecting polymorphism of VKORC1 and CYP2C9 genes by adopting pyrosequencing method

A pyrosequencing method, CYP2C91075A technology, applied in the field of molecular biology testing, can solve the problems of difficult to achieve high-throughput detection, high equipment and technical requirements, slow detection speed, etc., to achieve short detection cycle and high specificity , the effect of simple operation

Pending Publication Date: 2017-09-26
GUIZHOU PROVINCIAL PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional method does not require high equipment and low cost, but the detection speed is slow, and it is difficult to achieve high-throughput detection; the new method can achieve high-throughput and automatic detection, but has high requirements for equipment and technology and high cost

Method used

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  • Primer and method for detecting polymorphism of VKORC1 and CYP2C9 genes by adopting pyrosequencing method
  • Primer and method for detecting polymorphism of VKORC1 and CYP2C9 genes by adopting pyrosequencing method
  • Primer and method for detecting polymorphism of VKORC1 and CYP2C9 genes by adopting pyrosequencing method

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Embodiment 1

[0057] A primer for detecting polymorphisms of VKORC1 and CYP2C9 genes by pyrosequencing, wherein the polymorphic site detected by VKORC1 is VKORC1-1639G>A, and the polymorphic site detected by CYP2C9 is CYP2C9430C>T or CYP2C9430C>T And / or CYP2C91075A>C, the primers include:

[0058] (1) The amplification primers for the VKORC1-1639G>A gene are:

[0059] Upstream primer: 5'-TGTTGGCCAGGCTTGTCTTAAACT-3'

[0060] Downstream primer: 5'-TGGGAAGTCAAGCAAGAGAAGACC-3'

[0061] The sequencing primer of VKORC1-1639G>A gene is: 5'-GCGTGAGCCACCGCA-3'

[0062] (2) The amplification primers for CYP2C9430C>T gene are:

[0063] Upstream primer: 5'-CTCATGACGCTGCGGAATTTT-3'

[0064] Downstream primer: 5'-CCACCCTTGGTTTTTCTCAACTC-3'

[0065] The sequencing primer of CYP2C9430C>T gene is: 5'-GGGCTTCCTCTTGAAC-3'

[0066] (3) The amplification primers for CYP2C91075A>C gene are:

[0067] Upstream primer: 5'-CCAGGAAGAGATTGAACGTGTGA-3'

[0068] Downstream primer: 5'-TTTGGGGACTTCGAAAACATG-3'

...

Embodiment 2

[0093] A primer for detecting polymorphisms of VKORC1 and CYP2C9 genes by pyrosequencing, wherein the polymorphic site detected by VKORC1 is VKORC1-1639G>A, and the polymorphic site detected by CYP2C9 is CYP2C9430C>T or CYP2C9430C>T And / or CYP2C91075A>C, the primers include:

[0094] (1) The amplification primers for the VKORC1-1639G>A gene are:

[0095] Upstream primer: 5'-TGTTGGCCAGGCTTGTCTTAAACT-3'

[0096] Downstream primer: 5'-TGGGAAGTCAAGCAAGAGAAGACC-3'

[0097] The sequencing primer of VKORC1-1639G>A gene is: 5'-GCGTGAGCCACCGCA-3'

[0098] (2) The amplification primers for CYP2C9430C>T gene are:

[0099] Upstream primer: 5'-CTCATGACGCTGCGGAATTTT-3'

[0100] Downstream primer: 5'-CCACCCTTGGTTTTTCTCAACTC-3'

[0101] The sequencing primer of CYP2C9430C>T gene is: 5'-GGGCTTCCTCTTGAAC-3'

[0102] (3) The amplification primers for CYP2C91075A>C gene are:

[0103] Upstream primer: 5'-CCAGGAAGAGATTGAACGTGTGA-3'

[0104] Downstream primer: 5'-TTTGGGGACTTCGAAAACATG-3'

...

Embodiment 3

[0131] A primer for detecting polymorphisms of VKORC1 and CYP2C9 genes by pyrosequencing, wherein the polymorphic site detected by VKORC1 is VKORC1-1639G>A, and the polymorphic site detected by CYP2C9 is CYP2C9430C>T or CYP2C9430C>T And / or CYP2C91075A>C, the primers include:

[0132] (1) The amplification primers for the VKORC1-1639G>A gene are:

[0133] Upstream primer: 5'-TGTTGGCCAGGCTTGTCTTAAACT-3'

[0134] Downstream primer: 5'-TGGGAAGTCAAGCAAGAGAAGACC-3'

[0135] The sequencing primer of VKORC1-1639G>A gene is: 5'-GCGTGAGCCACCGCA-3'

[0136] (2) The amplification primers for CYP2C9430C>T gene are:

[0137] Upstream primer: 5'-CTCATGACGCTGCGGAATTTT-3'

[0138] Downstream primer: 5'-CCACCCTTGGTTTTTCTCAACTC-3'

[0139] The sequencing primer of CYP2C9430C>T gene is: 5'-GGGCTTCCTCTTGAAC-3'

[0140] (3) The amplification primers for CYP2C91075A>C gene are:

[0141] Upstream primer: 5'-CCAGGAAGAGATTGAACGTGTGA-3'

[0142] Downstream primer: 5'-TTTGGGGACTTCGAAAACATG-3'

...

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Abstract

The invention discloses a primer and a method for detecting polymorphism of VKORC1 and CYP2C9 genes by adopting a pyrosequencing method, and belongs to the field of molecular biology examination. The primer comprises amplification upstream and downstream primers and sequencing primers of genes including VKORC1-1639G being more than A, CYP2C9430C being more than T, and CYP2C91075A being more than C. The method comprises specimen collection and DNA extraction, PCR (polymerase chain reaction) amplification reaction, gel electrophoresis on a PCR product and pyrosequencing, wherein a DNA extraction kit used in the specimen collection and DNA extraction step is prepared from lysis buffer liquid, adsorbent, rinsing buffer liquid and elution buffer liquid; the lysis buffer liquid is prepared from sodium chloride, sodium citrate, a denaturing agent, a reducing agent, ethylenediamine tetraacetic acid, ionic surfactant, absolute ethyl alcohol and ultra-pure water. In summary, the detection method provided by the invention has the advantages of an accurate detection result, high specificity, a short detection period, a simple operation, low cost and the like.

Description

technical field [0001] The invention belongs to the field of molecular biology testing, and in particular relates to a primer and a method for detecting VKORC1 and CYP2C9 gene polymorphisms by pyrosequencing. Background technique [0002] Pyrosequencing (Pyrosequencing) is a method of DNA sequencing (that is, to determine the sequence of nucleotides in DNA) based on the principle of aggregation. Quantity, high specificity, fast, intuitive and low cost advantages. The basic principle is that the enzyme cascade chemiluminescent reaction in the same reaction system catalyzed by four enzymes, in each round of sequencing reaction, only one dNTP is added, if the dNTP is paired with the template, the polymerase can convert it Incorporated into the primer chain and release an equimolar number of pyrophosphate groups (PPi), PPi can be finally converted into a visible light signal, and converted into a peak by PyrogramTM, the height of each peak is related to the nucleoside incorpora...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q1/6883C12Q2600/106C12Q2600/156C12Q2565/301
Inventor 黄盛文杨楠楠徐琴韩媛媛李頔
Owner GUIZHOU PROVINCIAL PEOPLES HOSPITAL
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