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Primer composition for judging personalized medication type of captopril

A technique of primer composition and primer combination, which is applied in the field of molecular biology detection, and can solve problems such as difficulty in meeting fast and accurate requirements, cumbersome operations, and limitations of detection objects

Inactive Publication Date: 2020-05-01
BIOYONG TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection method and kit provided by this invention patent only target the polymorphic sites of key enzyme genes on the homocysteine ​​metabolic pathway, and the detection objects are limited, and the operation is cumbersome, and it is difficult to meet the requirements of clinical fast and accurate needs

Method used

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  • Primer composition for judging personalized medication type of captopril
  • Primer composition for judging personalized medication type of captopril
  • Primer composition for judging personalized medication type of captopril

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1: Primer Design and Synthesis

[0088] The surrounding sequences of the target SNP of this kit are queried in the db_SNP (build 132) and Hapmap (Rel 28, Phase II + III, Aug 10) databases, and these sequences are used to design multiple PCR primers and single base extension primers.

[0089] Corresponding specific PCR primer core sequences (SEQ1a to SEQ5a) and specific extension primer core sequences (SEQ1b to SEQ5b) were designed for five polymorphic sites related to the discrimination of drug types, including rs11209716, rs699, rs2016848, rs8012552, and rs2106809. 5 pairs of PCR primers and 5 extension primers form 3 independent reaction systems: SEQ1a / b to SEQ2a / b form the first reaction system, SEQ3a / b form the second reaction system, and SEQ4a / b to SEQ5a / b form the reaction system The third reaction system. In these 3 independent reaction systems, SEQ1a to SEQ2a, SEQ3a, SEQ4a to SEQ5a respectively participate in 3 independent multiplex PCR reactions, and S...

Embodiment 2

[0091] Embodiment 2: sample DNA extraction

[0092] A total of 10 DNA samples were collected from ordinary Chinese people, marked as A1-A10. Among them, sample collection, DNA extraction, etc. were collected in accordance with the requirements of the instructions, and human venous blood was collected with EDTA anticoagulant tubes. According to the instructions, the collected blood should not be stored at 2-8°C for more than one week, and at -20°C for no more than one month, and can be transported in a curling box with ice or a foam box with ice. It is recommended to use fresh blood as much as possible. Genomic DNA extraction. Since this kit does not provide human genomic DNA extraction reagents, a commercial nucleic acid extraction kit (such as QIAGEN’s DNeasy Blood and Tissuekit) was used to extract human genomic DNA from 200 μL of whole blood of each patient, and the DNA was extracted using a NanoDrop 2000 ( Thermo Company) quantified, and standardized to 30ng / μL (respecti...

Embodiment 3

[0093] Embodiment three: biological experiment

[0094] Using ABI 9700 type PCR instrument, according to the instructions, check the 5 polymorphic sites that are used to identify the drug type.

[0095] The components used in the kit for PCR, PCR product purification and single base extension are:

[0096] serial number component name main ingredient Specification 1 PCR Primer Mix PCR primers 24μl / tube x1 tube 2 PCR reaction solution Taq enzyme, dNTP 72μl / tube x1 tube 3 Enzyme digestion reaction solution SAP enzyme 48μl / tube x1 tube 4 Extension Primer Mix extension primer 24μl / tube x1 tube 5 Extension reaction solution Single base elongase, ddNTP 24μl / tube x1 tube 6 positive control Human Genomic DNA (30ng / μL) 10μL / tube x1 tube

[0097] The concentration of each primer pair is 500nmol / L.

[0098] According to the manual, the specific operation method is as follows:

[0099] 1. PCR amplification ...

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Abstract

The invention provides a primer composition for judging personalized medication types of an antihypertensive medicine, namely captopril. A preparation method comprises the following steps: respectively designing multiplex amplification primers and extension primers according to multiple target SNP (single nucleotide polymorphism) sites to be tested; preparing a multiplex amplification primer reaction system and an extension primer reaction system; in the reaction systems, simultaneously and respectively performing amplification and single base extension reaction on the multiple target SNP sites by using multiple sets of primers; and performing time-of-flight mass spectrometry analysis on products after the single base extension reaction, identifying genotypes of SNP related to different drug metabolism according to products (represented by mass spectrum peaks) of extension primers of different molecular weights, and instructing personalized medication of the antihypertensive medicine,namely captopril. The primer composition is capable of simultaneously detecting five SNP sites related to drug metabolism of the captopril, and in addition, and the advantages of being low in cost, free of probe synthesis, short in time, simple and convenient in result analysis and very wide in application field are achieved.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and relates to a method for detecting multiple PCR single-base extension products by using mass spectrometry characteristic peaks and its products. The method can simultaneously detect multiple PCR single-base extensions in two multiplex PCR reactions Amplified oligonucleotide products. More specifically, the method uses different time-of-flight mass spectrometry characteristic peaks generated by different purpose oligonucleotide fragments in the process of mass spectrometry typing, and simultaneously detects multiple target SNP sites to guide the antihypertensive drug Cato Puli's medication. Background technique [0002] Human genetic information is stored in the genome. In 2002, the Human Genome Project, an international cooperation project, was finally completed, drawing a fine map of the human genome structure and providing a reference sequence for related research. The human geno...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11C12Q1/6858
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2533/101C12Q2565/627
Inventor 马庆伟钟逾刘昕超
Owner BIOYONG TECH
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