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Specific primer pair and specific probe for CYP2C9 (cytochrome P450 2C9) and (vitamin K epoxide reductase complex subunit 1) VKORC1 gene chip detection

A specific, primer pair technology, applied in the field of molecular biology, can solve the problems of cumbersome operation, difficult and accurate test results, and difficult to meet clinical tests.

Inactive Publication Date: 2014-04-09
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 CYP2C9 and VKORC1 genotypes mainly use methods such as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), manual or automatic sequencing, and PCR with sequence-specific primers. The test results are not easy to be accurate, and it is difficult to meet the requirements of clinical testing

Method used

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  • Specific primer pair and specific probe for CYP2C9 (cytochrome P450 2C9) and (vitamin K epoxide reductase complex subunit 1) VKORC1 gene chip detection
  • Specific primer pair and specific probe for CYP2C9 (cytochrome P450 2C9) and (vitamin K epoxide reductase complex subunit 1) VKORC1 gene chip detection
  • Specific primer pair and specific probe for CYP2C9 (cytochrome P450 2C9) and (vitamin K epoxide reductase complex subunit 1) VKORC1 gene chip detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081]Example 1 Preparation of gene chip

[0082] Artificially synthesized (Shanghai Sangon Bioengineering Technology Service Co., Ltd.) the following specific probes, dissolved in water to a concentration of 100pmol / ul solution, and then used 2× spotting buffer (product number: BST02010, Shanghai Bio-Tech Co., Ltd.), etc. Than mixed. Next, use the GSM417 sample spotting instrument of Affymetrix Company to spot on the aldehyde-modified glass slide (product number: BSM03011, Shanghai Bio Technology Co., Ltd.) according to the method described in the manual. figure 1 array of . Leave overnight at room temperature.

[0083] Each specific probe sequence is as follows:

[0084] The 430C site-specific oligonucleotide probe sequence for detecting CYP2C9 gene rs1799853 is:

[0085] NH 2 -TTTTTTTTTTTTTTTT-CATTGAGGACCGTGTTCAAG; The specific oligonucleotide probe sequence for detecting the 430T site of CYP2C9 gene rs1799853 is: NH 2 -TTTTTTTTTTTTTTTT-CATTGAGGACTGTGTTCAAGAG.

[008...

Embodiment 2

[0090] Example 2 Preparation of chromosomal DNA

[0091] Use the blood DNA extraction kit from Shanghai Bio-Tech Co., Ltd. and follow the instructions as follows:

[0092] Activation of the adsorption column: 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 seconds; discard the waste liquid in the collection tube, and put the adsorption column back In 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.

[0093] Operating procedures:

[0094] (1) Add 20 μL of proteinase K to the bottom of a 1.5 mL centrifuge tube with a pipette.

[0095] (2) Add 200 μL of blood sample into the centrifuge tube.

[0096] (3) Add 200 μL buffer BL to the centrifuge tube, shake and mix for 15 seconds.

[0097] (4) I...

Embodiment 3

[0106] Embodiment 3 uses primer provided by the present invention to amplify ALDH2 gene fragment by PCR method

[0107] Entrust Shanghai Sangon Bioengineering Technology Service Co., Ltd. to synthesize primers, and the primer information is as follows.

[0108] Primer pair for detection of CYP2C9 gene 430C / T:

[0109] SEQ ID No.19: Upstream 5'-CAGCAATGGAAAGAAATGGA-3',

[0110] SEQ ID No. 20: Downstream 5'-TGAAACGAAAAAACGAAAGT-3'.

[0111] Primer pair for detection of CYP2C9 gene 1075A / C:

[0112] SEQ ID No.25: Upstream 5'-TTGCTACAACAAATGTGCCA-3',

[0113] SEQ ID No. 26: Downstream 5'-ATTTGGAGATGGTAGTGGCC-3'.

[0114] Primer pair for detection of VKORC1 gene-1639G / A:

[0115] SEQ ID No.31: Upstream 5'-AGGGTTCAAGTGGTTCTCGT-3',

[0116] SEQ ID No. 32: Downstream 5'-TACTTAGGGACCTTGAACAC-3'.

[0117] Also, the 5' end of the above primer was modified with biotin.

[0118] The primer pairs were then dissolved in water and diluted to 10 pmol / μl. Prepare the PCR amplification ...

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Abstract

The invention relates to the field of molecular biology, and discloses a specific oligonucleotide probe for detection of SNP (single nucleotide polymorphism) sites of CYP2C9 (cytochrome P450 2C9) and (vitamin K epoxide reductase complex subunit 1) VKORC1, and the probe can hybridize with different genotypes of 430th and 1075th site of the CYP2C9 and -1639th site of the VKORC1. The present invention also discloses a specific primer pair for amplification of CYP2C9 and VKORC1 genes in the detection of the SNP (single nucleotide polymorphism) sites, and the primer pair can be used for specific amplification of target areas containing detection target sites in the CYP2C9 and VKORC1 genes. The specific primer pair and the specific probe can complete detection of three SNP site genotypes of the CYP2C9 and VKORC1 by one step, and provides information for people to right use warfarin.

Description

technical field [0001] The invention relates to the fields of molecular biology and nucleic acid detection, specifically designing specific primer pairs for amplifying CYP2C9 and VKORC1 genes and specific probes for detecting CYP2C9 and VKORC1 gene chips. Background technique [0002] Warfarin, a dicoumarin derivative, is a commonly used oral anticoagulant. There are large differences in its stable dose among different races and individuals, and the difference in stable dose among different individuals can reach more than 20 times. Studies have shown that the gene polymorphism of vitamin K epoxide reductase complex subunit 1 gene (VKORC1) and cytochrome P4502C9 gene (CYP2C9) are the two most important genetic factors affecting individual differences in warfarin dosage. Among them, the most common SNP site-1639G / A of VKORC1 was found to be significantly related to individual differences in warfarin dosage. CYP2C9 is the main metabolizing enzyme of warfarin. So far, 30 alle...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/106C12Q2600/156
Inventor 朱滨邢军芬孙悦张宇张佳琦张艳张莹
Owner SHANGHAI BAIO TECH
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