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Specific primmer, liquid phase chip and detection method for CYP2E1 (Cytochrome P450 2E1) gene SNP (Single Nucleotide Polymorphism) detection

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

Inactive Publication Date: 2012-10-24
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing detection technologies are mainly based on traditional solid-phase chips and PCR. Traditional solid-phase chips are expensive, and their sensitivity is not high, and the reproducibility of detection results is poor. Other detection technologies based on PCR mainly include fluorescence Quantitative PCR and direct sequencing methods, these technologies have the disadvantages of low sensitivity, easy sample contamination, and high false positive rate. At the same time, the detection throughput is limited and cannot meet the needs of detection applications.

Method used

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  • Specific primmer, liquid phase chip and detection method for CYP2E1 (Cytochrome P450 2E1) gene SNP (Single Nucleotide Polymorphism) detection
  • Specific primmer, liquid phase chip and detection method for CYP2E1 (Cytochrome P450 2E1) gene SNP (Single Nucleotide Polymorphism) detection
  • Specific primmer, liquid phase chip and detection method for CYP2E1 (Cytochrome P450 2E1) gene SNP (Single Nucleotide Polymorphism) detection

Examples

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Effect test

Embodiment 1

[0031] Example 1 CYP2E1 gene SNP detection liquid chip mainly includes:

[0032] 1. ASPE Primers

[0033] Specific primer sequences were designed for three common SNP sites of CYP2E1: G1168A, C1053T and G1293C. Among them, G1168A is a CYP2E1*2 mutation, located in exon 2; C1053T and G1293C are CYP2E1*5 mutations, located in the 5' non-coding region; the ASPE primer consists 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 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...

Embodiment 3

[0105] Example 3 Detection of CYP2E1 gene SNP detection gene mutation by liquid chip with different ASPE primers

[0106] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0107] Taking the detection liquid chip of the CYP2E1*2 (G1168A) site mutation of the CYP2E1 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 G1168A, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1-SEQ ID NO.6, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence selects SEQ ID NO.13-SEQ ID NO.18. The specific design is shown in the following table (Table 6). 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.

[0108] Table 6 Design of liquid phase chip preparation

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Abstract

The invention discloses a specific primmer, a liquid phase chip and a detection method for CYP2E1 (Cytochrome P450 2E1) gene SNP (Single Nucleotide Polymorphism) detection. The liquid phase chip comprises wild-type and mutant ASPE (Allele-Specific Primer Extension) primer pairs respectively designed to each type of mutant site, microspheres respectively encapsulated with specific anti-tag sequences, and primers for respectively amplifying a CYP2E1 gene target sequence with G1168A, C1053T and / or G1293C SNP site. The prepared liquid phase chip for CYP2E1 gene SNP detection has excellent signal-to-noise ratio, and the designed probe and the anti-tap sequence do not exit the cross reaction basically. The ASPE primers have excellent specificity and can exactly distinguish all kinds of mutant sites. The detection method has simple steps, the detection of three kinds of SNP bits can be finished once with simple operation, and a plurality of undetermined factors existing in multiple operation processes are avoided, thus the precision rate of the detection can be greatly improved.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a CYP2E1 gene SNP detection specific primer, a liquid phase chip and a detection method. technical background [0002] Dimethylnitrosamine D-demethylase (Cytochrome P450 2E1, CYP2E1) is a member of the cytochrome P450 superfamily that metabolizes endogenous and exogenous substances, and is mainly involved in nitrosamine and its The metabolism of the former carcinogens N-nitrosodimethylamine and N-nitroso tetrapyrrolidine is involved in the oxidative metabolism of alcohol and the generation of free radicals to initiate lipid peroxidation. CYP2E1 is more important than other CYP450 enzymes in detoxification and metabolism, and has specific metabolic effects on toxic substances such as ethanol, nitrosamines, benzene and carbon tetrachloride. At the same time, CYP2E1 gene polymorphism is closely related to the occurrence and development of vari...

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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