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Pair of specific primers and probe for detection of CYP2C19 gene chip

A CYP2C19, specific technology, applied in the field of molecular biology, can solve the problems of inaccurate detection results, long detection cycle, and difficulty in meeting clinical tests.

Inactive Publication Date: 2014-01-22
SHANGHAI BAIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current methods for determining CYP2C19 genotype mainly use methods such as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), manual or automatic sequencing, and PCR with sequence-specific primers. Difficult to be accurate and difficult to meet the requirements of clinical testing

Method used

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  • Pair of specific primers and probe for detection of CYP2C19 gene chip
  • Pair of specific primers and probe for detection of CYP2C19 gene chip
  • Pair of specific primers and probe for detection of CYP2C19 gene chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1 Preparation of gene chip

[0080] Aldehyde-modified glass slides (product number: BSM03011, Shanghai Bio Technology Co., Ltd.). The following probes were artificially synthesized (Shanghai Sangon Bioengineering Technology Service Co., Ltd.), dissolved in water to a concentration of 100pmol / ul, and then mixed in equal proportions with 2× spotting buffer (product number: BST02010, Shanghai Bio-Technology Co., Ltd.) . Then, use the GSM417 sample spotting instrument of Affymetrix Company to make points such as figure 1 array of . Leave overnight at room temperature.

[0081] The sequences of each probe are as follows (the SNP sites are underlined).

[0082] The specific oligonucleotide probe for detecting the 681A site is: NH2-TTTTTTTTTTTTTTTTATTATTTCCC A GGAACCCAT;

[0083] The specific oligonucleotide probe for detecting the 681G site is: NH2-TTTTTTTTTTTTTTTTATTATTTCCC G GGAACCC;

[0084] The specific oligonucleotide probe for detecting the 636G site is:...

Embodiment 2

[0088] Example 2 Preparation of chromosomal DNA

[0089] Use the blood DNA extraction kit from Shanghai Bio-Tech Co., Ltd. and follow the instructions as follows:

[0090] Adsorption column activation:

[0091] Put the adsorption column in the collection tube, add 500 μL buffer BH1, let it stand for 2-3 minutes, and centrifuge at 12,000 rpm (9,500×g) for 30 s; discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube. Add 500 μL of buffer BH2 ​​to the adsorption column, centrifuge at 12,000 rpm (9,500×g) for 30 s, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube for use.

[0092] Operating procedure:

[0093] (1) Add 20 μL of proteinase K to the bottom of a 1.5 mL centrifuge tube with a pipette.

[0094] (2) Add 200 μL of blood sample into the centrifuge tube.

[0095] (3) Add 200 μL buffer BL to the centrifuge tube, shake and mix for 15 seconds.

[0096] (4) Inc...

Embodiment 3

[0105] Example 3 Using the primers provided by the present invention to amplify the CYP2C19 gene fragment by PCR method

[0106] Entrust Shanghai Sangon Bioengineering Technology Service Co., Ltd. to synthesize primers, and the primer information is as follows.

[0107] When used to detect the 681G / A site, the sequence of the primer pair used for amplification is:

[0108] Upstream SEQ ID No.13: 5'-CTTGGCATATTGTATCTATACCTTT-3'

[0109] Downstream SEQ ID No.14: 5'-CAAAACACAAATGATGCCTACAAAT-3'

[0110] When used to detect the 636G / A site, the sequence of the primer pair used for amplification is:

[0111] Upstream SEQ ID No.19: 5'-CACCCTGTGATCCCACTTTC-3'

[0112] Downstream SEQ ID No.20: 5'-TCTGTCGGTACCCCACTTAT-3'

[0113] And the 5' end of the primer pair was modified with biotin.

[0114] Then dissolve and dilute to 10 pmol / μl with water. Prepare the PCR amplification system with the purchased Taq enzyme (TaKaRa), 10× buffer (TaKaRa), dNTP (Shanghai Sangon Bioengineering...

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Abstract

The invention relates to the molecular biology field, and discloses a specific oligonucleotide probe for detection of SNP loci of rs4244285681G / A and rs4986893636G / A of a CYP2C19 gene and a pair of specific primers for amplification of the CYP2C19 gene during detection of the above SNP loci. The probe can hybridize with different genotypes of the CYP2C19 gene specifically, and the genotypes comprise a CYP2C19*1 type, a CYP2C19*2 type and CYP2C19*3 type. The products can be used for detection of metabolic abilities for relevant medicines (such as plavix, omeprazole, voriconazole and the like) and of Chinese population, clinical medication schemes can be guided and adjusted, clinical personalized medication can be provided with a basis, the curative effects can be raised and the risks of toxic and side effects can be reduced.

Description

technical field [0001] The invention relates to the fields of molecular biology and nucleic acid detection, specifically designing specific primer pairs for amplifying CYP2C19 genes and specific probes for CYP2C19 gene chip detection. Background technique [0002] Cytochrome P450 isozymes, also known as drug enzymes, are a superfamily composed of a series of structurally and functionally related enzymes, and are the main enzyme system for drug metabolism in the human body. The composition of P450 enzyme system is complex and controlled by gene diversity. At least 12 subfamilies are known. Many P450 isoenzymes have genetic polymorphisms, which make the corresponding enzyme activities show differences, and the ability to metabolize drugs is also different. According to the differences in drug metabolism ability, the population is divided into four types: ultrafast metabolizers (UM), fast metabolizers (EM), intermediate metabolizers (IM), and slow metabolizers (PM). [0003]...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/106C12Q2600/156
Inventor 邢军芬朱滨孙悦张宇张莹
Owner SHANGHAI BAIO TECH
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