Specific primer, liquid-phase chip and method for SNP detection of CYP2C9 and VKORC1 genes

A technology of G-1639ASNP and detection solution, which is applied in the fields of medicine and biology, can solve the problems of poor repeatability of test results, low sensitivity, high false positive rate, etc., to avoid uncertain factors, avoid cross-reaction, and accurate test results Effect

Active Publication Date: 2010-09-08
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current products are mainly based on traditional solid-phase chips, which are expensive, and the sensitivity is not high, and the repeatability of the test results is poor.
However, other PCR-based SNPs detection technologies, such as direct sequencing, semi-quantitative PCR technology, PCR-single-strand conformation polymorphism (SSCP) detection, etc., have the disadvantages of low sensitivity, easy sample contamination, and high false positive rate.
Ordinary PCR methods and fluorescent quantitative PCR cannot meet clinical needs due to the limitation of detection throughput, while polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis technology and allelic difference analysis based on TaqMan technology The method can only detect one mutation at a time, which is time-consuming and laborious

Method used

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  • Specific primer, liquid-phase chip and method for SNP detection of CYP2C9 and VKORC1 genes
  • Specific primer, liquid-phase chip and method for SNP detection of CYP2C9 and VKORC1 genes
  • Specific primer, liquid-phase chip and method for SNP detection of CYP2C9 and VKORC1 genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 CYP2C9 and VKORC1 gene SNP detection liquid chip mainly includes:

[0032] 1. ASPE Primers

[0033] Specific primer sequences were designed for four common SNP sites of CYP2C9 and three common SNP sites of VKORC1 gene. ASPE primers consist of "Tag + specific primer sequence". ASPE primer sequences are shown in the table below:

[0034] Table 1 ASPE primer sequence (Tag+ specific primer sequence)

[0035]

[0036]

[0037] Each ASPE primer includes two parts, the 5' end is a specific tag sequence for the anti-tag sequence on the corresponding microsphere, and the 3' end is a mutant or wild-type specific primer fragment (as shown in the above table 1). All ASPE primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Each synthesized primer was prepared into a stock solution of 100 pmol / mL with 10 mmol / L Tris Buffer.

[0038] 2. Microspheres coated with anti-tag sequences

[0039] According to the designed ASPE-specific...

Embodiment 2

[0050] Example 2 Detection of clinical samples using CYP2C9 and VKORC1 gene SNP detection liquid chip The formulations of the various solutions are as follows:

[0051] 50mM MES buffer (pH5.0) formula (250ml):

[0052] Reagent

source

Final concentration

Dosage per 250ml

MES(2[N-Morpholino]

ethanesulfonic acid)

Sigma M-2933

0.05M

2.44g

5MNaOH

Fisher SS256-500

---

5 drops

[0053] 2×Tm hybridization buffer

[0054] Reagent

source

Final concentration

Dosage per 250ml

1M Tris-HCl, pH8.0

SigmaT3038

0.2M

50ml

5MNaCl

Sigma S5150

0.4M

20ml

Triton X-100

Sigma T8787

0.16％

0.4ml

[0055] Store at 4°C after filtration.

[0056] ExoSAP-IT kit was purchased from US USB Company.

[0057] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0058] 1. Sample DNA ...

Embodiment 3

[0119] Example 3 Detection of CYP2C9 and VKORC1 gene SNP sites by liquid chip with different ASPE primers

[0120] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0121] Taking the detection liquid chip of the CYP2C9*3 (A1075C) site mutation of CYP2C9 gene and the C1173T site mutation of VKORC1 gene as an example, the specific primers for the 3' end of ASPE primers were designed for the wild-type and mutant types of A1075C and C1173T, and ASPE The Tag sequence at the 5' end of the primer is selected from 12 of SEQ ID NO.1-SEQ ID NO.14. Correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO. 29 - SEQ ID NO. 42. The specific design is shown in the following table (Table 7). The synthesis of ASPE primers, Anti-tag sequence coated microspheres, amplification primers, detection methods, etc. are as described in Example 1 and Example 2.

[0122] ...

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Abstract

The invention discloses a specific primer, a liquid-phase chip and a method for SNP detection of CYP2C9 and VKORC1 genes. The liquid-phase chip comprises wild-type and mutable-type ASPE primer pairs and microspheres coated by a specific anti-tag sequence respectively, which are designed respectively aiming at each type of mutable loci, primers used for amplifying a CYP2C9 gene target sequence having CYP2C9*2, CYP2C9*3, CYP2C9*5 and CYP2C9*6SNP loci, and / or primers used for amplifying a VKORC1 gene target sequence having G1639A, G1173T and G3730A SNP loci. The liquid phase chip of the invention has a quite good signal-noise ratio, and the cross reaction does not happen between a designed probe and the anti-tag sequence basically; the ASPE primer designed by the invention has quite good specificity, and can accurately differentiate various types of mutable loci; and the detection method has the advantages that: a few steps are adopted, 7 types of SNP loci can be detected in one step, the operation is convenient, a lot of uncertain factors existing in a process of repeated operations can be avoided, and the detection accuracy is greatly improved.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a CYP2C9 and VKORC1 gene SNP detection specific primer, a liquid phase chip and a method. Background technique [0002] Warfarin (Warfarin) is a new generation of anticoagulant, which was officially approved by the US FDA in February 2000. Its mechanism of action is to competitively resist the effect of vitamin K, inhibit the synthesis of coagulation factors in liver cells, and reduce the platelet aggregation reaction induced by thrombin, thus having anticoagulant and antiplatelet aggregation functions. As the first oral anticoagulant, warfarin has dominated the postoperative antithrombotic, stroke and atrial fibrillation market for nearly a decade. In the U.S., more than 2 million prescriptions are filled annually for the drug. [0003] However, the pharmacological effects of warfarin are easily affected by many factors, with large indivi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森朱泽尧何嘉英曾涛
Owner SUREXAM BIO TECH
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