Method and kit for detecting genetic information of folate metabolism related genes

Abstract

The invention discloses a method and a kit for detecting genetic information of folate metabolism related genes, and relates to three sites, namely an MTHFR gene rs1801133 site, an rs1801131 site andan MTRR gene rs1801394 site. The method and kit provided by the invention, on the basis of the comprehensive use of such technologies as direct PCR, nested PCR, multiple PCR, created restriction sitemethod PCR, PCR-RFLP and the like, can implement PCR amplifications by two turns in a mode of directly using cells without extracting DNA, and the second turn of PCR product of each site can use cheaprestriction endonuclease to conduct enzyme digestion typing; in accordance with actual demands, the three sites can be simultaneously detected within a same tube, or two sites can be simultaneously detected within a same tube, or any one of the sites can be independently detected. All PCR products take enzyme digestion internal control sequences as quality control of enzyme digestion, so that theaccuracy of result judging. The method is simple and convenient in operation of a whole process and is low cost; and accuracy is quite guaranteed.

Description

technical field [0001] The invention belongs to the field of molecular detection, and in particular relates to a method and a kit for detecting the genotypes of the rs1801133 site [C677T], rs1801131 site [A1298C] and MTRR gene rs1801394 site [A66G] of the folic acid metabolic pathway MTHFR gene. Background technique [0002] Folic acid is an essential element for the synthesis of nucleic acid, an essential substance for cell growth and tissue repair, and an indispensable nutrient for embryonic development. 5,10-Methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) are the two most important enzymes involved in folic acid metabolism, [0003] A large number of studies in recent years have confirmed that folic acid deficiency is the main cause of birth defects such as Down syndrome, cleft lip and palate, and neural tube defects. In addition to preventing fetal neural tube defects, the clinical function of folic acid can also reduce the incidence...

AI Technical Summary

Problems solved by technology

In addition, the primer design of the above literature has certain disadvantages. Among them, the PCR products amplified by the primers at the MTHFR C677T and MTRR A66G sites will be digested by enzymes, which may lead to genotype misjudgment. See the analysis in the above table

However, the MTHFR A1298C site selection restriction endonuclease MboII cannot be typed correctly at all, because when the allele is A, GAAGA【 figure 2 , 16681-16685 bases] is the recognition site of MboII, TCTTC not far downstream of the polymorphic site [ figure 2 , 16700-16704 bases] is also the recognition site of MboII, the two cleavage sites are only separated by one base, it is impossible to judge whether the cleavage is caused by GAAGA or TCTTC, that is, the TCTTC sequence will seriously interfere with the judgment of the result

Nevertheless, many researchers choose MboII to type the MTHFR A1298C site. Although the size of the PCR product is somewhat different, the interference of the TCTTC sequence near the polymorphic site cannot be avoided.

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