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Sequencing primer for qualitative detection of cytochrome oxidase CYP2C19 genetic typing and kit of sequencing primer

A technology of CYP2C19 and cytochrome, applied in the direction of DNA / RNA fragments, recombinant DNA technology, etc., to achieve good specificity, fast detection speed, and accurate qualitative results

Inactive Publication Date: 2013-02-06
CHANGSHA 3G BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] At present, there is no product on the market that uses pyrophosphate technology to detect CYP2C19 gene polymorphisms

Method used

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  • Sequencing primer for qualitative detection of cytochrome oxidase CYP2C19 genetic typing and kit of sequencing primer
  • Sequencing primer for qualitative detection of cytochrome oxidase CYP2C19 genetic typing and kit of sequencing primer
  • Sequencing primer for qualitative detection of cytochrome oxidase CYP2C19 genetic typing and kit of sequencing primer

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: the preparation of kit

[0051] 1. Design and synthesis of primers

[0052] Studies have shown that the current CYP2C19 gene polymorphisms are mainly CYP2C19*2 and CYP2C19*3 gene polymorphisms, and the CYP2C19*2 gene mutation mainly occurs at the 681st nucleotide of exon 5 where G is mutated to A. The CYP2C19*3 gene mutation mainly occurred in the 636th nucleotide of exon 4, where G was mutated into A. Using PyroMark Assay Design2.0 software, design and determine PCR amplification primers and pyrosequencing primers (Table 2) based on these two mutation sites (Table 1), the most important factor affecting the accuracy of the kit It is primers, including amplification primers and sequencing primers. In the early stage of design, we designed multiple sets of primers for comparison (see Figure 7 ); wherein the amplification primers and sequencing primers were first purified by PAGE, and then purified by HPLC, wherein CYP2C19*2 forward amplification primer (...

Embodiment 2

[0065] Embodiment 2: the use of kit

[0066] 1. sample amplification

[0067] Take the blank control, positive control and sample DNA respectively as PCR reaction templates, add UNG enzyme, Taq polymerase, specific PCR amplification primer 1 or 2, and PCR reaction solution 1 or 2 to form a PCR reaction system, and perform PCR Amplify.

[0068] The main components of the CYP2C19*2 system are as follows:

[0069]

[0070] The main components of the CYP2C19*3 system are as follows:

[0071]

[0072] 2. PCR reaction program

[0073] Set up the PCR reaction program, put the reaction tube into the fluorescent PCR instrument (ABI7500) to start amplification, the reaction program is as follows:

[0074] Table 5. PCR reaction program

[0075]

[0076] The annealing temperature of detection site 1 (CYP2C19*2) was set at 61°C, and the annealing temperature of detection site 2 (CYP2C19*3) was set at 59°C.

[0077] 1. Pyrosequencing

[0078] ①PCR amplification product pre...

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Abstract

The invention provides a sequencing primer for qualitative detection of cytochrome oxidase CYP2C19 genetic typing and a kit of the sequencing primer and belongs to the field of external nucleic acid detection. The kit comprises uracil DNA (Deoxyribonucleic Acid) glycosylase, Taq polymerase, PCR (Polymerase Chain Reaction) reaction liquor, PCR amplification primers, pyrophosphoric acid sequencing primers and positive control products. The kit is high in sensitivity and good in specificity, PCR products can be simply treated for a pyrophosphoric acid sequenator for sequencing, operation is simple, reaction time is short, the sensitivity of the kit is higher than that of a golden standard-capillary electrophoresis sequencing, and the kit is suitable for use for mutation analysis.

Description

technical field [0001] The present invention relates to the field of in vitro nucleic acid detection, in particular to a pair of sequencing primers for genotyping of two sites of the cytochrome oxidase CYP2C19 gene that is commonly used in drug metabolism in clinical samples and its kit, namely CYP2C19*2( 681G / A) and CYP2C19*3 (636G / A) genotyping. Background technique [0002] Cytochrome P450 (CytochromeP450, CYP450) is an isoenzyme encoded by a group of structurally and functionally related superfamily genes (Superfamily gene). In the human body, it mainly exists in liver cell particles. In the CYP450 superfamily, CYP2 is the largest family with 15 subfamilies, which metabolize about 20% of the drugs currently used clinically. Among them, CYP2C19 (S-Mephenytoin hydroxylase) is one of the main components of CYP450. The full-length cDNA of CYP2C19 gene is 1940, contains 9 exons, and the coding region is 1473bp. The encoded protein S-mephenytoin hydroxylase metabolizes a ser...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 周姗
Owner CHANGSHA 3G BIOTECH
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