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Liquid phase chip and specificity primer for SNP detection of CYP4F2 and EPHX1 genes

A CYP4F2, detection solution technology, applied in the field of molecular biology, can solve the problems of increased enzyme activity, high false positive rate, easy contamination of samples, etc., achieving the effect of simple steps, avoiding cross-reaction, and improving detection accuracy.

Active Publication Date: 2011-04-20
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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 activity Studies have shown that the decrease of EPHX1 enzyme activity is beneficial to the prevention of lung cancer, but this polymorphism is not associated with the risk of occasional colorectal cancer
[0004] At present, the detection products of CYP4F2 and EPHX1 polymorphisms 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. limitations and cannot meet the needs of practical applications

Method used

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  • Liquid phase chip and specificity primer for SNP detection of CYP4F2 and EPHX1 genes
  • Liquid phase chip and specificity primer for SNP detection of CYP4F2 and EPHX1 genes
  • Liquid phase chip and specificity primer for SNP detection of CYP4F2 and EPHX1 genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 CYP4F2 and EPHX1 gene SNP detection liquid chip mainly includes:

[0032] 1. ASPE Primers

[0033] Two common SNP sites G1297A (rs2108622) and T34G (rs3093105) of the CYP4F2 gene, and SNP sites G357A (rs2292566), G19512990A (rs4653436), T337C (rs1051740) and A416G (rs2234922) of the EPHX1 gene were specifically designed, respectively, primer sequence. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0034] Table 1 ASPE primer sequence (Tag sequence + 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 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 sol...

Embodiment 2

[0051] Example 2 Using the CYP4F2 and EPHX1 gene detection liquid chip described in Example 1 to detect samples The formulations of the various solutions are as follows:

[0052] 50mM MES buffer (pH5.0) formula (250ml):

[0053] Reagent

source

Final concentration

Dosage per 250ml

MES(2[N-Morpholino]

ethanesulfonic acid)

Sigma M-2933

0.05M

2.44g

5MNaOH

Fisher SS256-500

---

5 drops

[0054] 2×Tm hybridization buffer

[0055] 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

[0056] Store at 4°C after filtration.

[0057] ExoSAP-IT kit was purchased from US USB Company.

[0058] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0059] 1. ...

Embodiment 3

[0121] Example 3 Detection of CYP4F2 and EPHX1 gene SNP sites 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 CYP4F2 gene G1297A site and 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 the G1297A SNP site and the G357A SNP site, respectively, and the ASPE The Tag sequence at the 5' end of the primer is selected from SEQ ID NO.1-SEQ ID NO.12. Correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.25- SEQ ID NO. 36. 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 ...

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PUM

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Abstract

The invention discloses a specificity primer and a liquid phase chip for SNP detection of CYP4F2 gene. The liquid phase chip comprises an ASPE primer formed by the specificity primer aiming at a target gene SNP locus of the tag sequence on the 5' end and 3' end, wherein the specificity primer is SEQ ID NO.13 and SEQ ID NO.14 aiming at the G1297A SNP locus and / or SEQ ID NO.15 and SEQ ID NO.16 aiming at the T34G SNP locus, a microsphere and an amplification primer. The invention also provides a liquid phase chip for SNP detection of CYP4F2 and EPHX1 genes, which comprises the corresponding compositions of the liquid phase chip for the SNP detection of the CYP4F2 and EPHX1 genes, an ASPE primer pair aiming at the G357A, G19512990A, T337C and / or A416G SNP locus of the EPHX1 gene, microsphere and amplification primer. The detection liquid phase chip has an excellent signal-noise ratio, can avoid cross reaction and can realize the parallel detection of a plurality of SNP loca.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a CYP4F2 and EPHX1 gene detection liquid chip and specific primers. Background technique [0002] Cytochrome P450 4F2 (Cytochrome P450, CYP4F2) is a member of the cytochrome P450 superfamily of environmental metabolic enzymes. acid, 20-HETE) is the most important enzyme, 20-HETE has an important role in constricting blood vessels and regulating ion homeostasis. Insufficient synthesis of 20-HETE in the kidney is closely related to the occurrence of hypertension, so CYP4F2 has become an important influence on the occurrence of hypertension factor. Studies have shown that the W12G and V433M mutations in CYP4F2 will cause 20-HETE to decrease to 56%-66% compared to the control. Among them, W12G (rs3093105, T34G) of CYP4F2 is CYP4F2*2, and V433M (rs2108622, G1297A) occurs in exon 2. [0003] Microsomal epoxide hydrolase (mEH, EPHX1) is an impor...

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 SUREXAM BIO TECH
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