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CYP3A4 gene SNP detection specific primer, liquid-phase chip and method

A technology of CYP3A4 and detection solution, which is applied in the field of molecular biology, can solve the problems of poor repeatability of detection results, expensive solid-phase chips, and low sensitivity, so as to achieve accurate and reliable detection results, improve detection accuracy, and avoid cross-reactions Effect

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

[0006] At present, there are a few reports on methods for detecting CYP3A4 gene polymorphism at home and abroad, mainly based on traditional solid-phase chips and PCR. Solid-phase chips are expensive, and the sensitivity is not high, and the reproducibility of 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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  • CYP3A4 gene SNP detection specific primer, liquid-phase chip and method
  • CYP3A4 gene SNP detection specific primer, liquid-phase chip and method
  • CYP3A4 gene SNP detection specific primer, liquid-phase chip and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 CYP3A4 gene SNP detection liquid chip mainly includes:

[0032] 1. ASPE Primers

[0033] Specific primer sequences were designed for ten common SNP sites of CYP3A4. Among them, CYP3A4*1B is A392G mutation, which occurs in 5'UTR; CYP3A4*2 is T664C mutation, which occurs in exon 7; CYP3A4*3 is T1334C mutation, which occurs in exon 12; CYP3A4*4 is A352G mutation, which occurs in exon CYP3A4*5 is the C653G mutation, which occurs in exon 7; CYP3A4*6 is the 831insA mutation, which occurs in exon 9; CYP3A4*15 is the G485A mutation, which occurs in exon 6; CYP3A4*17 is the T566C mutation, which occurs in exon Exon 7; CYP3A4*18 is a T878C mutation, which occurs in exon 10; CYP3A4*19 is a C1399T mutation, which occurs in exon 12. 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]

[0038] Each ASPE prim...

Embodiment 2

[0052] Example 2: Detection of clinical samples using CYP3A4 gene SNP detection liquid chip

[0053] The formula of described various solutions is as follows:

[0054] 50mM MES buffer (pH5.0) formula (250ml):

[0055] 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

[0056] 2×Tm hybridization buffer

[0057] 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

[0058] Store at 4°C after filtration.

[0059]ExoSAP-IT kit was purchased from US USB Company.

[0060] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0061] 1. Sample DN...

Embodiment 3

[0122] Example 3 Detection of CYP3A4 gene SNP detection gene mutation by liquid chip with different ASPE primers

[0123] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0124] Taking the detection liquid chip of the CYP3A4*1B (A392G) site mutation of the CYP3A4 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 A392G, 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.20, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected as SEQ ID NO.41-SEQ ID NO.60. The specific design is shown in the following table (Table 8). 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.

[0125] Table 8 Design of liquid phase chi...

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Abstract

The invention discloses a CYP3A4 gene SNP detection specific primer, a liquid-phase chip and a method; and the liquid-phase chip comprises wild-type and mutant ASPE primers which are respectively designed for each type of mutation points, microspheres which are respectively coated with specific anti-tag sequences, and the primer which is used for amplifying a CYP3A4 gene target sequence with CYP3A4*1B, CYP3A4*2, CYP3A4*3, CYP3A4*4, CYP3A4*5, CYP3A4*6, CYP3A4*15, CYP3A4*17, CYP3A4*18 and / or CYP3A4*19SNP sites. The CYP3A4 gene SNP detection liquid-phase chip has very good signal-noise ratio, and a designed probe and the anti-tag sequences essentially have no cross reaction. The designed ASPE primers have very good specificity and can accurately distinguish all types of mutation points. The detection method has simple steps and ten types of SNP sites can be detected at one step, so that the operation is convenient, thereby preventing a plurality of uncertain factors in a plurality of operation processes, and greatly improving the detection accuracy rate.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a CYP3A4 gene SNP detection specific primer, a liquid phase chip and a method. Background technique [0002] Cytochrome P450 (cytochrome P450, cytochrome CYPs, P450 for short) is a class of proteases of the B family cytochrome superfamily with heme as the prosthetic group, and exists on the endoplasmic reticulum membrane of cells. P450 enzymes are the most important group of metabolic enzymes, involved in the metabolism of carcinogens and clinical therapeutic drugs. In the CYP450 superfamily, CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 are related to the metabolism and drug-drug interactions of more than 95% of the drugs currently on the market. [0003] CYP3A isoenzymes are involved in the metabolism of tobacco carcinogens and steroids, including CYP3A4, CYP3A5, CYP3A7 and CYP3A43, and there are significant inter-individual differen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森何嘉英曾涛
Owner SUREXAM BIO TECH
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