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Kit for detecting genotyping of folic acid metabolizing genes

A folic acid metabolism gene and genotyping technology, applied in the field of molecular biology, to reduce the possibility of late contamination and improve detection efficiency

Inactive Publication Date: 2019-01-18
WUHAN CMLABS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Patent No. 201410330456.4 developed a PCR-reverse dot hybridization method to detect folic acid metabolism-related genes. This patent uses RNase H2 and PCR technology to develop a new type of detection of folic acid metabolism-related genes. It was found that this method was applied to the detection of genes related to folic acid metabolism

Method used

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  • Kit for detecting genotyping of folic acid metabolizing genes
  • Kit for detecting genotyping of folic acid metabolizing genes
  • Kit for detecting genotyping of folic acid metabolizing genes

Examples

Experimental program
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Embodiment 1

[0045] This embodiment designs folic acid metabolism genotyping primer probes, using the sequence shown in SEQ ID NO. The sequence shown in SEQ ID NO.2 at the 1298 site of the gene is used as a template to design specific primers for polymorphism detection at the 1298 site A / C, and the sequence shown in SEQ ID NO.3 at the 66 site of the MTRR gene is used as a template Design specific primers for polymorphism detection of 677 site A / G. The specific sequence is as follows:

[0046] 5'-CTCCTGACTGTCATCCCTATTG-3', the sequence is shown in SEQ ID NO.4;

[0047] 5'-AAGCTGCGTGATGATGAAATCGgCTCC-x, the sequence is shown in SEQ ID NO.5;

[0048] 5'-AAGCTGCGTGATGATGAAATCGaCTCC-x, the sequence is shown in SEQ ID NO.6;

[0049] 5'-ATGTCCACAGCATGGAGG-3', the sequence is shown in SEQ ID NO.7;

[0050] 5'-GAGGAGCTGACCAGTGAAGaAAGT-x, the sequence is shown in SEQ ID NO.8;

[0051] 5'-GAGGAGCTGACCAGTGAAGcAAGT-x, the sequence is shown in SEQ ID NO.9;

[0052] 5'-GCCTTGAAGTGATGAGGAG-3', the s...

Embodiment 2

[0061] In this example, a folic acid metabolism genotyping kit was prepared.

[0062] Since the same probe and different specific primers are used for the SNP site, the same SNP site cannot be detected in the same reaction well, otherwise it cannot be distinguished. Therefore, the present invention divides the detection of these three SNP sites into two groups, each group only detects one polymorphism of each SNP site, and the specific design of the kit is as follows:

[0063] PCR reaction premix 1: combine the primers shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.10 and SEQ ID NO.11, wherein , the concentration of primers for SEQ ID NO.4, SEQ ID NO.7, and SEQ ID NO.10 is 0.25 μM; the concentration of primers for SEQ ID NO.5, SEQ ID NO.8, and SEQ ID NO.11 is 0.1 μM. The sequences shown in the probes SEQ ID NO.13-SEQ ID NO.15 are combined together, and the concentration of the probes is 0.1 μM. It is prepared by adding universal human-derived internal...

Embodiment 3

[0073] In this example, the folic acid metabolism genotyping kit prepared in Example 2 is used to detect samples, and the specific detection steps are as follows.

[0074] (1) Genomic DNA template extraction: Genomic DNA in blood or saliva was extracted using a universal kit.

[0075] (2) PCR amplification detection: use the genomic DNA extracted in step (1) as a template, and use the kit provided by the present invention to perform corresponding amplification detection.

[0076] Make two reaction wells for each sample, take PCR reaction master mix 1 and 21 μl of PCR reaction master mix respectively and add them to different reaction wells, then add 2 μl of the same template and 2 μl enzyme mixture, and make a negative control well and a well at the same time For positive control wells, add positive quality control product 1 to PCR reaction master mix 1 as the positive control of the reaction system, add positive quality control product 2 to PCR reaction master mix 2 as the po...

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Abstract

The invention provides a kit for detecting the genotyping of folic acid metabolizing genes, and belongs to the technical field of molecular biology. Specifically, RNase H2 and Taq DNA polymerase biphasic enzyme systems were used to detect the folate metabolism-related gene loci. The kit provided by the invention has the advantages of high sensitivity, fast detection, good stability and closed detection, and reduces the possibility of later pollution.

Description

technical field [0001] The invention relates to the technical field of molecular biology, belongs to the detection method of SNP polymorphism in the field of in vitro diagnostic reagents, in particular to a kit for genotyping folic acid metabolism genes. Background technique [0002] China is one of the countries with a high incidence of birth defects in the world, and the number of children with birth defects every year accounts for about 20% of the world. Common birth defects in my country include neural tube defects, congenital heart disease, cleft lip and palate, and hypospadias. According to statistics from the Chinese Center for Disease Control and Prevention, there are 800,000 to 1.2 million birth defects every year, and an average of one defect is born every 30 seconds. Among them, in addition to 20%-30% of children who can obtain a better quality of life through early diagnosis and treatment, 30%-40% of children will die after birth, and about 40% will become perma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6876C12N15/11
CPCC12Q1/6876C12Q2600/156
Inventor 徐志勇秦伟
Owner WUHAN CMLABS CO LTD
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