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Primer system for detecting genetic polymorphic sites related to human cytochrome P450 and application of primer system

A cytochrome, polymorphic site technology, applied in DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of high cost of synthetic probes, complicated operation, complicated process, etc., to overcome the lack of SNP sites, detection High sensitivity and low cost effect

Inactive Publication Date: 2015-04-29
BIOYONG TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] Chinese patent application 200610072593.8, "An Oligonucleotide Probe and Gene Chip for Detecting Cytochrome P450 Enzyme Mutation Sites", involves a chip method. Since the oligonucleotide probe described in this method uses fluorescent labels or biological Because of the high cost and complicated process of synthetic probes, the above technical problems cannot be solved.
[0014] In summary, the current technical problems are: the lack of methods and products that can simultaneously detect multiple CYP450-related polymorphic sites at one time, and common detection technologies, such as sequencing, real-time fluorescent quantitative PCR, etc. Detecting one by one, when there are many sites, the operation is complicated and the cost is high. Therefore, a detection technology different from the past is needed at present, which can detect multiple SNPs of the same individual in the same system. Through the detection information of this technology, Provide reference for rational drug use

Method used

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  • Primer system for detecting genetic polymorphic sites related to human cytochrome P450 and application of primer system
  • Primer system for detecting genetic polymorphic sites related to human cytochrome P450 and application of primer system
  • Primer system for detecting genetic polymorphic sites related to human cytochrome P450 and application of primer system

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Example 1: Primer design and synthesis.

[0122] For CYP2C19 gene rs56337013 (CYP2C9*5), CYP2C19 gene rs4986893 (CYP2C19*3), CYP2C9 gene rs1799853 (CYP2C9*2), CYP2C19 gene rs12248560 (CYP2C19*17), 910rs105C79 (CYP2C9*3), CYP2C19 gene rs4244285 site (CYP2C19*2), CYP2C19 gene rs28399504 site (CYP2C19*4) and other 7 gene polymorphic sites related to human cytochrome P450, design corresponding specific PCR primer core sequences (SEQ ID No: 1 to SEQ ID No: 14) and specific extension primer core sequences (SEQ ID No: 15 to SEQ ID No: 21).

[0123] Among them, in order to prevent PCR primers from entering the detection window of the mass spectrometer and interfering with the detection effect, a certain number of bases can be added to the core sequence (SEQ ID No: 1 to SEQ ID No: 14) at the 5' end of each PCR primer , such as a 10bp tag (ACGTTGGATG), to increase the molecular weight of the PCR primers, thereby exceeding the detection window of the mass spectrometer.

[0124] ...

Embodiment 2

[0126] Embodiment 2: sample DNA extraction.

[0127] A total of 10 clinical patients were collected. 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 Tissue kit) was used to extract human genomic DNA from 200ul whole blood of each patient, and the DNA was extracted using NanoDrop2000 ( Thermo Company) quantified and standardized to 30ng / ul (C1-C10 respectively). Among them, the kit is recommend...

Embodiment 3

[0128] Embodiment three: Biological experiment.

[0129] Using ABI9700 PCR instrument, according to the instructions, the 7 gene polymorphic sites related to cytochrome P450 gene were tested.

[0130] The components used in the kit for PCR, PCR product purification and single base extension are:

[0131] No. Component Name main ingredient Specification 1 PCR mix dNTPs, MgCl2, PCR primers 360ul / tube x1 tube 2PCR enzyme Taq enzyme 24ul / tube x1 tube 3SAP Enzyme Mix SAP enzyme 24ul / tube x1 tube 4 extension primer mix extension primer 24ul / tube x1 tube 5 elongase mix iPLEX enzymes, ddNTPs 24ul / tube x1 tube 6 Positive controls Human Genomic DNA (30ng / ul) 40ul / tube x1 tube

[0132] According to the manual, the specific operation method is as follows:

[0133] 1. PCR amplification

[0134] 1.1 In the PCR preparation area, prepare 200ul PCR reaction tubes according to the number of samples to be tested (includin...

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Abstract

The invention discloses a primer system for detecting genetic polymorphic sites related to human cytochrome P450. Based on a product prepared by adopting the primer system, seven genetic polymorphic sites related to the human cytochrome P450 can be simultaneously detected. By using the product, through detecting the genetic polymorphic sites related to the human cytochrome P450, clinical medication scheme can be guided and regulated, the basis is provided for clinical personalized medicine, and adverse medicine effects are prevented. The primer system is capable of simultaneously detecting the seven genetic polymorphic sites on different genes in a reaction system, and has the advantages of being lower in cost and more convenient to operate, and increasing the accuracy and the sensitivity in comparison with technologies of sequencing and real-time fluorescence quantification PCR (Polymerase Chain Reaction).

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a detection method and product for determining polymorphic sites (SNPs) related to human cytochrome P450, specifically using multiplex PCR technology, single base extension technology and mass spectrometry Technology, a method for detecting 7 gene polymorphic sites related to human cytochrome P450 and corresponding kits. Background technique [0002] Cytochrome P450 enzyme (cytochrome P450, CYP), also known as mixed function oxidase (mixed function oxidase) and monooxygenase (monooxygenase), mainly exists in liver microsomes, other tissues in the human body such as: brain, lung, kidney , skin, placenta, gastrointestinal tract, mammary gland, etc. can also produce. Cytochrome P450 plays a very important role in the metabolism of exogenous substances (including drugs and poisons). Its activity determines the metabolic rate of drugs and is directly related to the clearance rate of drugs....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 马庆伟赵洪斌张海燕赵艳梅
Owner BIOYONG TECH