Development of a highly accurate, rapid PCR-RFLP genotyping assay for the methylenetetrahydrofolate reductase gene.
The C677T mutation of the methylenetetrahydrofolate (MTHFR) gene is a nutrient-oriented, "eco" genetic mutation that is associated with elevated levels of homocysteine and an increased risk for coronary heart disease. The purpose of this study was to optimize and automate an assay for PCR-restriction fragment length polymorphism (RFLP) genotyping of the MTHFR gene. We developed an automated assay for the PCR-RFLP genotyping of the MTHFR gene. The DNA was amplified with MTHFR-specific PCR primers, and the resulting PCR product was then digested using the restriction enzyme Hinf I. We then analyzed the restriction fragments on the ABIPrism310 Genetic Analyzer, an automated capillary electrophoresis instrument. Reaction conditions were optimized to achieve an approximately equal ratio of the expected 198-bp and 175-bp Hinf I restriction fragments obtained from a DNA sample that was heterozygous for the C677T mutation. We have developed a high-throughput, accurate method to genotype DNA obtained from blood samples or other sources in less than 1 day. Automation of the PCR-RFLP genotyping of the MTHFR gene can be achieved successfully using the capillary electrophoresis-based ABIPrism310 Genetic Analyzer. In conclusion, automation of the PCR-RFLP genotyping of the MTHFR gene will support the screening of large sample sizes, either for routine clinical diagnostic testing or for large population studies.
Sell
S M
SM
University of Alabama at Birmingham, Department of Nutrition Sciences 35294, USA. sells@admin.shrp.uab.edu
Lugemwa
P R
PR
D004252:DNA Mutational Analysis; D005838:Genotype; D006801:Humans; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length
D000587:Oxidoreductases Acting on CH-NH Group Donors; D042965:Methylenetetrahydrofolate Reductase (NADPH2)