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EPHX1 (Microsomal epoxide hydrolase, mEH, EPHX1) gene detection specific primer and liquid phase chip

A detection solution and specificity technology, applied in the field of molecular biology, can solve the problems of easy contamination of samples, high false positive rate, low sensitivity, etc., and achieve the effect of avoiding uncertain factors, avoiding cross-reaction, and simple steps

Active Publication Date: 2011-04-13
成都益善医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, the detection products of EPHX1 polymorphism are generally based on PCR technology, such as fluorescent quantitative PCR method, RFLP method and DNA sequencing method, which have the disadvantages of low sensitivity, easy contamination of samples, and high false positive rate. At the same time, due to the limitation of detection throughput characteristics, can not meet the needs of practical applications

Method used

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  • EPHX1 (Microsomal epoxide hydrolase, mEH, EPHX1) gene detection specific primer and liquid phase chip
  • EPHX1 (Microsomal epoxide hydrolase, mEH, EPHX1) gene detection specific primer and liquid phase chip
  • EPHX1 (Microsomal epoxide hydrolase, mEH, EPHX1) gene detection specific primer and liquid phase chip

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Embodiment 1

[0022] Example 1 EPHX1 gene SNP detection liquid chip mainly includes:

[0023] 1. ASPE Primers

[0024] Specific primer sequences were designed for the SNP sites G357A (rs2292566), G 19512990A (rs4653436), T 337C (rs1051740) and A416G (rs2234922) of the EPHX1 gene. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0025] Table 1 ASPE primer sequence (Tag sequence + specific primer sequence)

[0026]

[0027]

[0028] 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.

[0029] 2. Microspheres coated with anti-tag sequences...

Embodiment 3

[0112] The liquid phase chip of embodiment 3 different ASPE primers is to the detection of EPHX1 gene SNP site

[0113] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0114] Taking the EPHX1 gene G357A site mutation detection liquid chip as an example, the specific primer sequence of the 3' end of the ASPE primer was designed for the wild type and mutant type of G357A, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1 - SEQ ID NO.8, correspondingly, the anti-tag sequence coated on the microspheres and complementary to the corresponding tag sequence is selected from SEQ ID NO.17-SEQ ID NO.24. 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.

[0115] Table 7 Design of liquid phase chip preparation

[0116]

[0117...

Embodiment 4

[0124] Example 4 Detection of EPHX1 Gene SNP by Different Spacer Arm Liquid Chips

[0125] 1. Design of liquid phase chip preparation (selection of spacer arm)

[0126] Taking the detection liquid chip of the T337C site mutation of the EPHX1 gene as an example, the influence of different spacer liquid chips on the detection of the SNP of the EPHX1 gene was explored. Design the specific primer sequence of the 3' end of the ASPE primer for the wild type and mutant type of T337C, and the Tag sequence at the 5' end of the ASPE primer is SEQ ID NO.5 and SEQ ID NO.6, correspondingly, coated on the microsphere The anti-tag sequences complementary to the corresponding tag sequences are SEQ ID NO.21 and SEQ ID NO.22 respectively. The influence of different spacer arm liquid phase chips on the detection of EPHX1 gene SNP was investigated, wherein the different spacer arms were (CH2)12 or 5-10 T, and the specific design was shown in the following table (Table 9). The synthesis of ASPE ...

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Abstract

The invention discloses an EPHX1 (Microsomal epoxide hydrolase, mEH, EPHX1) gene SNP (Single Nucleotide Polymorphism) detection liquid phase chip mainly comprising wild type and mutant type ASPE (Automated Solid Phase Extraction) primer pairs respectively designed aiming at the SNP sites of an EPHX1 gene, microspheres which are respectively coated with a specific anti-tag sequence and have different color coding and an amplification primer which is used for amplifying an EPHX1 gene target sequence with the SNP sites, wherein each ASPE primer comprises the tag sequence of a 5' end and the specific primer of a 3' end, which aims at a target gene mutation site; the specific primer is selected from SEQ ID NO.9-SEQ ID NO.16; the tag sequence is selected from SEQ ID NO.1-SEQ ID NO.8; and an anti-tag sequence can be correspondingly complemented and paired with the tag sequence. The detection liquid phase chip has very good signal-noise ratio, can avoid cross reaction and can realize the parallel detection of multiple SNP sites.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and specifically relates to EPHX1 gene detection specific primers and a liquid phase chip. Background technique [0002] Microsomal epoxide hydrolase (Microsomal epoxide hydrolase, mEH, EPHX1) is an important biotransformation phase II metabolic enzyme, which is encoded by the EPHX1 gene located on human chromosome 1q42.1 and is highly conserved. It catalyzes the hydrolysis of various epoxidation intermediates into more water-soluble trans-dihydrodiols and excreted from the body. Studies have shown that polymorphisms in the coding region of the EPHX1 gene alter the activity of the enzyme by affecting the stability of the protein. The T→C transition of exon 3 replaces Tyr113 with His (rs1051740, T337C), resulting in a 39% decrease in enzyme activity; the A→G transition of exon 4 replaces His139 with Arg (rs2234922, A416G), resulting in a decrease in enzyme a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森秦会娟余刚曾涛
Owner 成都益善医学检验所有限公司
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