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Specific primers and liquid-phase chip for SNP (Single Nucleotide Polymorphism) detection of MTHFR and FGF5 genes

A technology of G121ASNP and detection solution, applied in the field of molecular biology, can solve the problem that gene mutation detection cannot be used, and achieve the effect of avoiding uncertain factors, consistent detection effect and simple steps.

Active Publication Date: 2014-01-01
广州益善医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, the methods for detecting and analyzing the polymorphisms of MTHFR and FGF5 genes mainly include: PCR-RFLP analysis method and traditional solid-phase chip and other methods, among which the most commonly used method is PCR-RFLP analysis method, PCR-RFLP method is based on Changes in the recognition sites of restriction endonucleases caused by gene mutations, such as loss of sites or creation of new sites, amplify a specific fragment by PCR, then digest the amplified product with restriction endonuclease, and observe the fragment by electrophoresis The size of this method is used to detect gene mutations with changes in restriction sites, and can directly determine the genotype, but this method cannot be used for the detection of gene mutations without new restriction sites. Again, the detection method based on PCR technology (PCR-RFLP) has limitations in the detection throughput, and only one mutation can be detected at a time, which cannot meet the needs of practical applications

Method used

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  • Specific primers and liquid-phase chip for SNP (Single Nucleotide Polymorphism) detection of MTHFR and FGF5 genes
  • Specific primers and liquid-phase chip for SNP (Single Nucleotide Polymorphism) detection of MTHFR and FGF5 genes
  • Specific primers and liquid-phase chip for SNP (Single Nucleotide Polymorphism) detection of MTHFR and FGF5 genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 MTHFR and FGF5 gene SNP detection liquid chip mainly includes:

[0025] 1. ASPE Primers

[0026] Specific primer sequences were designed for the wild type and mutant type of the three common genotypes G121A, A105G and G111A of the MTHFR gene and the wild type and mutant type of the common genotype T196A of the FGF5 gene. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0027] Table 1 ASPE primer sequences of MTHFR and FGF5 genes (Tag sequence + specific primer sequence)

[0028]

[0029] 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 100 pmol / mL stock solution w...

Embodiment 2

[0041] Example 2 Detection of samples using MTHFR and FGF5 gene detection liquid chip

[0042] The formula of described various solutions is as follows:

[0043] 50mM MES buffer (pH5.0) formula (250ml):

[0044]

[0045] 2×Tm hybridization buffer:

[0046] Reagent

[0047] Store at 4°C after filtration.

[0048] ExoSAP-IT kit was purchased from US USB Company.

[0049] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0050] 1. Sample DNA extraction

[0051] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0052] 2. PCR amplification of samples to be tested

[0053] Two pairs of primers were designed, and multiplex PCR was used to amplify three common genotypes G121A, A105G and G111A containing the MTHFR gene, and four target sequences of the common genotype T196A containing the FGF5 gene in one step. The sequence sizes were 326bp, 352bp, 249bp and 3...

Embodiment 3

[0110] Example 3 Detection of MTHFR and FGF5 gene SNP sites by liquid chip with different ASPE primers

[0111] 1. Design of liquid phase chip preparation (selection of tag sequence and anti-tag sequence)

[0112] Taking the G121A site of the MTHFR gene and the T196A mutation detection liquid chip of FGF5 as examples, the specific primer sequences at the 3' end of the ASPE primers were designed for the wild type and mutant types of G121A and T196A, respectively, and the tag sequences at the 5' end of the ASPE primers were selected. From SEQ ID NO.1-SEQ ID NO.8, correspondingly, the anti-tag sequence coated on the microsphere 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.

[0113] Table 7 Design of li...

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Abstract

The invention discloses specific primers and a liquid-phase chip for SNP (Single Nucleotide Polymorphism) detection of MTHFR and FGF5 genes. The liquid-phase chip comprises an ASPE primer, anti-tag sequence coated microspheres, and an amplification primer, wherein the ASPF primer is composed of a 5'-terminal tag sequence and 3'-terminal specific primers for target gene mutation, and the specific primers are respectively SEQ ID NO. 9 and SEQ ID NO. 10 specific for a G121A SNP site, SEQ ID NO. 11 and SEQ ID NO. 12 specific for an A105G SNP site, SEQ ID NO. 13 and SEQ ID NO. 14 specific for a G111A SNP site, and / or SEQ ID NO. 15 and SEQ ID NO. 16 specific for a T196A SNP site of the FGF5 gene. According to the invention, the coincidence rate between the detection result of the liquid-phase chip for SNP detection of the MTHFR and FGF5 gene and the detection result of a sequencing method is up to 100%.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a specific primer for detecting MTHFR and FGF5 gene SNP and a liquid phase chip. Background technique [0002] N 5，10 -Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folic acid metabolism and is also a key enzyme required for the remethylation of homocysteine ​​(Hcy) to methionine. The main biochemical function is to catalyze the reduction reaction from 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate mediated by reduced nicotinamide adenine dinucleotide phosphate (NADPH) to generate the most important The methyl donor, 5-methyltetrahydrofolate, is the main form of folic acid in tissues and serum, and participates in many important biochemical reactions in the body (such as the biosynthesis of purine and thymine, etc.). MTHFR is located on chromosome lp36.3. The gene includes 11 exons and 10 introns. The lengths o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 许嘉森秦会娟郭婧刘志明
Owner 广州益善医学检验所有限公司
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