CYP2C19 and ABCB1 gene SNP detection liquid-phase chip and detection method thereof

A CYP2C19, detection liquid technology, applied in the field of molecular biology, can solve the problems of poor repeatability of detection results, can not meet clinical needs, easy to contaminate samples, etc., achieve accurate and reliable detection results, improve detection accuracy, and strong scalability. Effect

Active Publication Date: 2009-10-28
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, products capable of detecting CYP2C19 gene SNPs at home and abroad, such as CodeLink P450 of Amersham Bioscience (GE healthcare), AmpliChip P450 of Roche, etc., are mainly based on traditional solid-phase chips, which are expensive and have low sensitivity. poor repeatability
However, other PCR-based techniques for detecting SNPs, such as direct sequencing, semi-quantitative PCR, and PCR-single-strand conformational polymorphism (SSCP) detection, have l

Method used

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  • CYP2C19 and ABCB1 gene SNP detection liquid-phase chip and detection method thereof
  • CYP2C19 and ABCB1 gene SNP detection liquid-phase chip and detection method thereof
  • CYP2C19 and ABCB1 gene SNP detection liquid-phase chip and detection method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] CYP2C19, ABCB1 gene SNP detection liquid chip, mainly includes:

[0043] 1. ASPE Primers

[0044] Specific primer sequences were designed for two common SNP sites of CYP2C19 and C3435TSNP site of ABCB1 gene. ASPE primers consist of "Tag + specific primer sequence". ASPE primer sequences are shown in the table below:

[0045] Table 1 ASPE primer sequence

[0046]

[0047] Each ASPE primer includes two parts, the 5' end is a specific tag sequence for the anti-tag sequence on the corresponding microsphere (as shown in Table 2), and the 3' end is a mutant or wild-type specific primer fragment (such as shown in Table 1 above). 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.

[0048] Table 2 Tag sequence at the right end of the ASPE-specific primer

[0049]

[0050]

[0051] 2. Microspheres coated with anti-tag ...

Embodiment 2

[0063] Example 2 Detection of clinical samples using CYP2C19, ABCB1 gene SNP detection liquid chip

[0064] The formula of described various solutions is as follows:

[0065] 50mM MES buffer (pH5.0) formula (250ml):

[0066] Reagent

source

Final concentration

Dosage per 250ml

MES(2[N-Morpholino]

ethanesulfonic acid)

Sigma M-2933

0.05M

2.44g

5M NaOH

Fisher SS256-500

---

5 drops

[0067] 2×Tm hybridization buffer

[0068] Reagent

source

Final concentration

Dosage per 250ml

1M Tris-HCl, pH8.0

SigmaT3038

0.2M

50ml

5M NaCl

Sigma S5150

0.4M

20ml

Triton X-100

Sigma T8787

0.16％

0.4ml

[0069] Store at 4°C after filtration.

[0070] ExoSAP-IT kit was purchased from US USB Company.

[0071] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd. ...

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PUM

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Abstract

The invention discloses a CYP2C19 and ABCB1 gene SNP detection liquid-phase chip and a detection method thereof. The liquid-phase chip comprises microspheres and amplification primer, wherein the microspheres are coated with specific anti-tag sequences respectively; and the anti-tag sequences are selected from sequences from SEQ ID NO.7 to SEQ ID NO.12. As for three pairs of ASPE primers formed by specific sequences of SNP sites of a target gene and tag sequences of 5' ends, the specific sequences are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively. The fluorescent signal value detected by the liquid-phase chip is greatly improved, so that the detection sensitivity is further improved, the signal-to-noise ratio is strengthened, and the detection result is more accurate and reliable.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to medicine and biotechnology, in particular to a liquid phase chip for detecting CYP2C19 and ABCB1 gene SNP and a detection method thereof. Background technique [0002] Plavix (also known as Clopidogrel, Clopidogrel) is a new generation of platelet aggregation inhibitors, which was officially approved by the FDA in March 1998. Its combination therapy with aspirin is widely used in patients with acute coronary syndrome and percutaneous coronary intervention (PCI), and can effectively prevent myocardial infarction, stroke and vascular death. At present, tens of thousands of patients around the world are taking Plavix for a long time to prevent thrombosis after implanting coronary stents. [0003] Plavix is ​​a prodrug that needs to be metabolized by P450 enzymes to form active metabolites. Scientific research has proved that there are significant individual differences in the efficac...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 许嘉森何嘉英任立芬
Owner SUREXAM BIO TECH
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