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Specific primer, liquid phase chip and detection method for CYP3A5 gene SNP (Single Nucleotide Polymorphism) detection

A detection solution and specific technology, applied in the field of molecular biology, can solve the problems of poor repeatability of detection results, easy contamination of samples, and high price, and achieve the effects of accurate and reliable detection results, improved detection accuracy, and improved sensitivity.

Inactive Publication Date: 2012-08-22
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The methods reported to detect CYP3A5 polymorphism mainly include traditional solid-phase chip and real-time fluorescence quantitative PCR. Among them, the traditional solid-phase chip is expensive, the sensitivity is not high, and the reproducibility of the detection results is poor, while the real-time fluorescent quantitative PCR technology has sensitivity Low, easy sample contamination, high false positive rate, and due to limitations in detection throughput, it cannot meet the needs of detection applications

Method used

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  • Specific primer, liquid phase chip and detection method for CYP3A5 gene SNP (Single Nucleotide Polymorphism) detection
  • Specific primer, liquid phase chip and detection method for CYP3A5 gene SNP (Single Nucleotide Polymorphism) detection
  • Specific primer, liquid phase chip and detection method for CYP3A5 gene SNP (Single Nucleotide Polymorphism) detection

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Embodiment 1

[0030] Example 1 CYP3A5 gene SNP detection liquid chip mainly includes:

[0031] 1. ASPE Primers

[0032]Specific primer sequences were designed for five common SNPs of CYP3A5: A6986G, G14690A, 27131_27132insT, C3699T, and G19386A. ASPE primers consist of "Tag + specific primer sequence". ASPE primer sequences are shown in the table below:

[0033] Table 1 ASPE primer sequence (Tag+ specific primer sequence)

[0034]

[0035]

[0036] 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 sequence (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.

[0037] 2. Microspheres coated with anti-tag sequences

[0038] According to the designed ASPE-specific pri...

Embodiment 3

[0121] Example 3 Detection of CYP3A5 gene SNP detection gene mutation by liquid chip with different ASPE primers

[0122] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0123] Taking the detection liquid chip of the CYP3A5*6 (G14690A) site mutation of the CYP3A5 gene as an example, the specific primer sequence at the 3' end of the ASPE primer was designed for the wild type and mutant type of G14690A, and the Tag sequence at the 5' end of the ASPE primer was selected from For 6 of SEQ ID NO.1-SEQ ID NO.10, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected as SEQ ID NO.21-SEQ ID NO.30. The specific design is shown in the following table (Table 7). The synthesis of ASPE primers, microspheres coated with anti-tag sequences, amplification primers, detection methods, etc. are as described in Example 1 and Example 2.

[0124] Table 7 Design of liquid phase ...

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Abstract

The invention discloses a liquid phase chip for CYP3A5 gene SNP (Single Nucleotide Polymorphism) detection, which mainly comprises ASPE (Allele-Specific Primer Extension) primer pairs, microspheres and amplimers, wherein the ASPE primer pairs are respectively designed for the SNP loci of the CYP3A5 gene, and each ASPE primer comprises tag sequences on a 5' end and specific primer sequences on a 3' end to the target gene SNP loci; the microspheres are respectively coated by specific anti-tag sequences; and the amplimers are used for amplifying CYP3A5 gene target sequences with A6986G, G14690A,2713127132insT, C3699T and / or G19386A SNP loci. The invention also provides a detection method using the liquid phase chip and a specific primer for CYP3A5 gene SNP detection. The compatible rate of the detection method and a sequencing method is up to 100%. The prepared liquid phase chip for CYP3A5 gene SNP detection has favorable signal-to-noise ratio, no cross reaction exists between a designed probe and the anti-tag sequences, and parallel detection on multiple SNP loci can be realized.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a CYP3A5 gene SNP detection specific primer, a liquid phase chip and a detection method. technical background [0002] Cytochrome P450 enzyme system (CYPs) is an important enzyme system involved in the detoxification of exogenous compounds and the metabolism of endogenous compounds, mainly including three families of CYP1, CYP2 and CYP3. CYP3A5 is an important isozyme in the CYP3A subfamily. Its genetic polymorphism affects the oxidative metabolism of many drugs mediated by CYP3A, such as diltiazem and alfentanil, etc. in liver microsomes. There are obvious individual differences in the activity of this enzyme. Effects and adverse reactions and drug toxicity have an important impact, which is one of the reasons for the differences in the ability to metabolize the same substrate between individuals and races. It plays a crucial role in drug...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森朱泽尧
Owner SUREXAM BIO TECH
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