Method for detecting FOXH1 gene SNP locus rs750472 genotype

A genotype and locus technology, applied in the field of detection of the FOXH1 gene SNP locus rs750472 genotype, can solve the problems of heavy economic burden on children's families, increase in abnormal cardiac function, etc., and achieve rapid detection, high sensitivity, and good specificity Effect

Inactive Publication Date: 2016-03-30
SUZHOU BAIYUAN GENT CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although most VSDs can be corrected by surgery, surgical treatment will bring a heavy economic burden to the children's families and society, and the probability of abnormal cardiac function in VSD patients is much higher than that of normal people

Method used

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  • Method for detecting FOXH1 gene SNP locus rs750472 genotype
  • Method for detecting FOXH1 gene SNP locus rs750472 genotype
  • Method for detecting FOXH1 gene SNP locus rs750472 genotype

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Preparation of Example 1FOXH1rs750472 Standard

[0066] To establish an HRM analysis method, the external standard required by the method must first be prepared. The standard should contain highly conserved and specific sequences, and high specificity of the reaction must be ensured. In this study, the wild-type and homozygous mutant DNA sequences containing the FOXH1rs750472 (c.-314T>G) site were synthesized and connected to the plasmid vector pUC57 by gene recombination technology to construct the wild-type and pure mutants of the recombinant plasmid pUC57-rs750472. The mutants were identified by PCR and sequencing, and finally quantified as the standard for the method to be established, laying the foundation for the next method and evaluation.

[0067] 1. Construction and transformation of recombinant negative and positive plasmid pUC57-rs750472

[0068] 1. Synthesize FOXH1rs750472 (c.-314T>G) wild-type and homozygous mutant DNA sequences. The sequence synthesized i...

Embodiment 2

[0106] Example 2 Establishment of HRM-PCR detection method for FOXH1rs750472

[0107] 1. Preparation of samples to be tested

[0108] Genomic DNA from peripheral blood was extracted from 30 samples and used as a template for PCR amplification of FOXH1 gene.

[0109] (1) Take 1ml whole blood, add 3ml TE, invert and mix well, let stand for 10min, centrifuge at 8000rpm for 5min, discard the supernatant;

[0110] (2) Repeat the above steps 2-3 times until the precipitate is white;

[0111] (3) Add 900 μl of 10% SDS and 10 μl of 10 mg / ml proteinase K, and bathe in water at 55°C for 1 hour;

[0112] (4) Cool the centrifuge tube to room temperature, add an equal volume of phenol / chloroform / isoamyl alcohol mixture (the volume ratio of phenol, chloroform and isoamyl alcohol is 25:24:1), mix well, and centrifuge at 12000rpm for 10min;

[0113] (5) After carefully aspirating the supernatant, add an equal volume of phenol / chloroform / isoamyl alcohol mixture (the volume ratio of phenol, ...

Embodiment 3

[0170] Example 3 Application of detection method of the present invention to detect sample gene mutation

[0171] Using the optimized detection conditions in Step 4 of Example 2, HRM analysis was performed on 30 samples, compared with the Tm value of the positive plasmid, and the mutation type in the samples was determined accordingly. After the HRM analysis, agarose gel electrophoresis was performed, and one sample was randomly selected for each genotype according to the HRM analysis results for Sanger sequencing verification at Sangon Biotech (Shanghai).

[0172] The results of HRM analysis are shown in Table 3 and image 3 , the data showed that among the 30 samples, 5 cases of FOXH1 gene rs750472 were T>C heterozygous mutant, 3 cases were T>C homozygous mutant, and the remaining 22 cases were wild type.

[0173] For Sanger sequencing results, see Figure 4 ; Wherein, a is the sequencing result of sample number 4, b is the sequencing result of sample number 16, and c is the...

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Abstract

The invention provides a method for detecting an FOXH1 gene SNP locus rs750472 genotype. The method comprises the following steps: (1) constructing a recombinant negative plasmid and a recombinant positive plasmid; (2) extracting a peripheral blood genomic DNA of a to-be-detected sample, carrying out PCR amplification and HRM analysis of the recombinant negative plasmid, the recombinant positive plasmid and the peripheral blood genomic DNA of the to-be-detected sample, and collecting data; and (3) drawing high resolution melting curves according to the collected data, and judging the FOXH1 gene SNP locus rs750472 genotype of the to-be-detected sample according to the melting curves of the recombinant negative plasmid and the recombinant positive plasmid. The method can effectively detect the FOXH1 gene SNP locus rs750472 genotype, and has the advantages of high sensitivity, good specificity, and rapid detection; and an analysis result with application of the method is entirely consistent to a sequencing result, and the method can be used for assistant judgment of ventricular septal defect heart disease.

Description

technical field [0001] The invention relates to a method for detecting the genotype of the FOXH1 gene SNP site rs750472. Background technique [0002] The forkhead box (FOX) gene family, as a transcription factor, widely exists in organisms. Members of this gene family have different functions, but they all have a highly conserved forkhead DNA domain, which includes a helix-turn-helix motif composed of more than 100 amino acids, a core region composed of three α-helices and A wing-like structure composed of 2 loops that interact with target DNA molecules. This family of genes plays a key regulatory role in the development, differentiation, and metabolism of the organism by activating or inhibiting the transcription and expression of target genes. All FOX gene family members can bind DNA through the DNA domain, and its functions involve the maintenance of cell differentiation state, tissue-specific gene expression, embryonic development, cell cycle regulation, carbohydrate ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q1/6883C12Q2600/156C12Q2527/107C12Q2563/107
Inventor 谢小冬车团结尤崇革沈颂东李琳李亚鹏
Owner SUZHOU BAIYUAN GENT CO LTD
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