title: Triplex tetra-primer ARMS-PCR method for the simultaneous detection of MTHFR c.677C>T and c.1298A>C, and MTRR c.66A>G polymorphisms of the folate-homocysteine metabolic pathway.
abstract: The folate-homocysteine metabolic pathway was shown to play an important role in several diseases such as cancers, cardiovascular diseases, and neurodegenerative diseases. The c.677C>T and c.1298A>C polymorphisms of the Methylenetetrahydrofolate reductase (MTHFR) gene, and c.66A>G of the Methionine synthase reductase (MTRR) gene are the most commonly investigated polymorphisms in the folate-homocysteine metabolic pathway. The currently used methods for the detection of the three polymorphisms are either slow and laborious or extremely expensive. In this paper, a new highly optimized method for the simultaneous detection of the three single nucleotide polymorphisms is described. The proposed method utilizes 12 primers in a single PCR reaction to detect the three polymorphisms simultaneously based on the principle of tetra-primer ARMS-PCR (also known as PCR-CTPP). The proposed method offers extremely fast, economical, and simple detection. Validation by PCR-RFLP showed 100% concordance in genotype assignment. The proposed method was successfully applied to a sample of the Syrian population (n=126), which was not previously genotyped for any of the three SNPs. The variant allele frequencies were found to be 31, 29, and 43% for the c.677C>T, c.1298A>C, and c.66A>G polymorphisms, respectively. The proposed method is the first to detect three SNPs in a single PCR reaction based on tetra-primer ARMS-PCR or PCR-CTPP. We suggest that the use of Betaine may play an important role in multiplex tetra-primer ARMS-PCR or PCR-CTPP based on its potential capacity to close the gap in melting temperature between different primers.
authors.affiliation: Department of Analytical Chemistry, Faculty of Pharmacy, University of Aleppo, Aleppo, Syria. BassamL7@yahoo.co.uk
authors.forename: Bassam
authors.initials: B
authors.lastname: Lajin
authors.forename: Amal
authors.initials: A
authors.lastname: Alachkar
authors.forename: Amir Alhaj
authors.initials: AA
authors.lastname: Sakur
mesh_terms: D001622:Betaine / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D005287:Ferredoxin-NADP Reductase / Q000097:blood / Q000235:genetics*; D005492:Folic Acid / Q000378:metabolism; D005787:Gene Frequency / Q000235:genetics; D006710:Homocysteine / Q000378:metabolism; D006801:Humans; D053858:Metabolic Networks and Pathways; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000097:blood / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*
chemical_list: D017931:DNA Primers; D006710:Homocysteine; D001622:Betaine; D005492:Folic Acid; C111241:methionine synthase reductase; D005287:Ferredoxin-NADP Reductase; D042965:Methylenetetrahydrofolate Reductase (NADPH2)