Method for detecting folic acid metabolism related gene mutation through combination of overlap extension PCR and Sanger sequencing

A folic acid metabolism and sequencing technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of limited read length of Sanger sequencing, reduce the number of PCR reactions and sequencing The effect of detecting cost and simplifying operation steps

Active Publication Date: 2019-05-28
GUANGZHOU HYBRIBIO MEDICINE TECH LTD +3
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Sanger sequencing is a classic method for detecting single nucleotide polymorphism sites, but due to the limited read length of Sanger sequencing, a sequencing reaction can only detect about 800bp, usually only one site can be detected, and each site needs to be detected Perform a PCR reaction

Method used

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  • Method for detecting folic acid metabolism related gene mutation through combination of overlap extension PCR and Sanger sequencing
  • Method for detecting folic acid metabolism related gene mutation through combination of overlap extension PCR and Sanger sequencing
  • Method for detecting folic acid metabolism related gene mutation through combination of overlap extension PCR and Sanger sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 A kit for detecting single nucleotide polymorphisms in folic acid metabolism-related gene loci based on Sanger sequencing method

[0056] 1. Composition

[0057] The nucleotide sequence of the primer combination shown in SEQ ID No. 1 to 8, the nucleotide sequence of the sequencing primer shown in SEQ ID No. 7 to 8, the reagents for PCR reactions and the reagents for Sanger sequencing. Reagents include: GeneAmp TM 10×PCR Buffer I(ABI TM 4379876), 25mM Mg 2+ , 25mM dNTPs, Roche Taq enzyme (Cat. No.: 03501221190), H 2 O; The reagent for Sanger sequencing is BigDye Terminator v3.1 Cycle Sequencing Kit (including: BigDye, 5×seq Buffer).

[0058] 2. How to use

[0059] S1. Collect samples with EDTA anticoagulant blood;

[0060] S2. DNA extraction by column method;

[0061] S3. Use the primer combination with nucleotide sequence as shown in SEQ ID No. 1-8 for PCR reaction,

[0062] The PCR reaction is a 50μl system, as follows:

[0063]

[0064] The PCR reaction procedure is as ...

Embodiment 2

[0075] Example 2 Detection of mutation sites of folate metabolism-related genes

[0076] 1. Experimental method

[0077] A kit for detecting single nucleotide polymorphisms in folic acid metabolism-related gene loci in Example 1 based on the Sanger sequencing method was used to detect C677T, A1298C of the MTHFR gene and A66G of the MTRR gene.

[0078] 2. Experimental results

[0079] Such as Figure 1 to Figure 4 As shown, C677T has no mutations normally, A1298C has no mutations normally, and A66G has heterozygous mutations.

Embodiment 3

[0081] 1. Experimental method

[0082] Using a Sanger sequencing-based detection kit for detecting single nucleotide polymorphisms in folate metabolism-related gene sites in Example 1, 20 samples and fluorescent quantitative PCR were used to analyze the C677T, A1298C and MTRR genes of the MTHFR gene. A66G is tested to determine consistency.

[0083] 2. Experimental results

[0084]

[0085]

[0086]

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Abstract

The invention discloses a method for detecting folic acid metabolism related gene mutation through combination of overlap extension PCR and Sanger sequencing and primer combination for detecting folicacid metabolism related gene mutation. A nucleotide sequence of the primer combination is shown as SEQ ID NO.1 to 8, and the primer combination is utilized to establish the detection method disclosedby the invention; according to the method disclosed by the invention, the overlap extension PCR and the Sanger sequencing are combined to detect folic acid metabolism related gene locus single nucleotide polymorphism, only once PCR reaction and once sequencing reaction are needed in the whole process, PCR reaction times and sequencing reaction times are remarkably reduced, and 6 mutation sites related to folic acid metabolism of MTHFR and MTRR genes can be detected; the method can be further utilized to find new variation points in amplified fragments. As the detection technology is applied to detecting folic acid metabolism related gene locus single nucleotide polymorphism, important clinical significance is achieved; thus, not only are operation steps simplified and is detection cost reduced, but also the detection efficiency is improved; the method is worth of popularizing.

Description

Technical field [0001] The present invention relates to the technical field of gene detection, and more specifically, to a method for detecting mutations of folate metabolism-related genes by overlapping extension PCR combined with Sanger sequencing. Background technique [0002] Folic acid is a water-soluble B vitamin. As the most important carrier of one carbon unit in the body, its main physiological function is to participate in many cell metabolism processes in the body. Folic acid is essential in the metabolism of Met. It works in the form of 5-MTHF. The lack of folic acid will cause great harm to the human body, especially the development of pregnant women and fetuses. [0003] Early pregnancy is a critical period for the differentiation of the fetal organ system and the formation of the placenta. Cell growth and division are very vigorous. At this time, folic acid deficiency can cause fetal malformations, and it may also cause early spontaneous abortion. In the second and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12N15/11
Inventor 郑焱陈佩璇谢龙旭翁丹容邱美兰徐爱娟
Owner GUANGZHOU HYBRIBIO MEDICINE TECH LTD
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