Multiplex PCR for simultaneous detection of 677 C-->T and 1298 A-->C polymorphisms in methylenetetrahydrofolate reductase gene for population studies of cancer risk.
Methylenetetrahydrofolate reductase (MTHFR) plays a pivotal role in folate metabolism by regulating the diversion of folate metabolites toward DNA methylation or toward DNA synthesis. Because aberrations in both of these pathways can be tumor promoting, the two common polymorphisms in the MTHFR gene, 677 C-->T and 1298 A-->C, have been implicated as risk factors for several cancers. Homozygosity for the 677 C-->T polymorphism and compound heterozygosity for 677 C-->T and 1298 A-->C polymorphisms both reduce enzyme activity by more than 50% and can promote oncogenic alterations in DNA methylation especially when folate status is low. Thus, rapid identification of both polymorphisms in MTHFR gene would be of importance in understanding the genetics of abnormal folate metabolism as related to human cancer risk. Here we describe a multiplex polymerse chain reaction/restriction fragment length polymorphism procedure in which two sets of primers are used to amplify simultaneously the DNA regions spanning 677 and 1298 loci in one PCR reaction. The amplified products are digested by HinfI or MboII followed by agarose gel electrophoresis for simultaneous detection of the 677 C-->T and 1298 A-->C polymorphisms in the same gel.
Yi
Ping
P
Division of Biochemical Toxicology, Food and Drug Administration - National Center for Toxicological Research, Jefferson, AR, USA.
Pogribny
Igor
I
Jill James
S
S
D001483:Base Sequence; D006801:Humans; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D008969:Molecular Sequence Data; D009369:Neoplasms / Q000209:etiology* / Q000235:genetics; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D012306:Risk
D000587:Oxidoreductases Acting on CH-NH Group Donors; D042965:Methylenetetrahydrofolate Reductase (NADPH2)