Abstract
Background5,10‐Methylentetrahydrofolate reductase (MTHFR) C677T polymorphism is one of the most studied genetic variations in the human genome. Polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) is one of the most used techniques to characterize the point mutations in genomic sequences because of its suitability and low cost. The most widely used method for the MTHFR C677T polymorphism characterization was developed by Frosst et al. (1995) but appears to have some technical limitations. The aim of this study was to propose a novel PCR‐RFLP method for the detection of this polymorphism.MethodsIn order to retrieve all published articles possibly describing any PCR‐RFLP methods useful to analyze MTHFR C677T polymorphism, we performed systematic queries on PubMed, using a combination of Boolean operators (AND/OR) and MeSH terms. Amplify software was used in order to design a new primer pair following the optimal standard criteria. Primer‐BLAST software was used to check primer pair's biological specificity.ResultsThe analysis of previous literature showed that PCR‐RFLP method remains the most used technique. None of the 108 primer pairs described was ideal with regard to main accepted primer pair biochemical technical parameters. The new primer pair amplifies a DNA‐fragment of 513 base pair (bp) that, in the presence of the polymorphism, is cut by Hinf I enzyme in two pieces of 146 bp and 367 bp and clearly visible on 2% agarose gel. The level of expertise and the materials required are minimal and the protocol takes one day to carry out.ConclusionOur original PCR‐RFLP strategy, specifically designed to make the analysis optimal with respect to PCR primers and gel analysis, fits the ideal criteria compared to the widely used strategy by Frosst et al (1995) as well as any other PCR‐RFLP strategies proposed for MTHFR C677T polymorphism genotyping to date.
Highlights
The C677T numbering is based on the single nucleotide polymorphism (SNP) location attributed by Goyette and coll. (Goyette et al, 1994) even though the substitution is on exon 5 in position 894 of the current messenger RNA reference sequence (NM_005957.4)
Many kinds of approaches were used to study the MTHFR C677T polymorphism (Table S1), but the systematic bibliographic search performed in the present work has showed that RFLP method is still widely used throughout the world
2,257 out of 4,913 articles published from 1995 to 2018 used PCR‐RFLP method (45.9%) to study the MTHFR mutation and 79 articles were published in the last 2‐year period (2017–2018)
Summary
5,10‐Methylentetrahydrofolate reductase (MTHFR) gene (On‐line Mendelian Inheritance in Man ‐ OMIM accession number: 607093) is located on the short arm of chromosome 1 (1p36.22); it contains 13 exons and is 20,373 base pair (bp) long. All the other methods were described in a smaller number of articles, for example some authors used DNA sequencing with different approaches or SNaPshot for genotyping, which is the best solution for multiple‐SNPs association studies, or the DNA strip technology, but all of them are very expensive and require test‐specific technical expertise All these methods and their references are available in the Table S1. The forward primer overlaps with the recognition site for Hinf I and could hide potential mutations in the restriction site, leading to false results This is needed for the screening of methionine synthase reductase (MTRR) polymorphism A66G (I22M). This work Forward primer TGTGGTCTCTTC ATCCCTCGC 66°C 0°C 21 Yes 57% No No Yes No Yes
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
