ABSTRACT
An improved assay for genotyping the common Alu insertion in the tissue-type plasminogen activator (PLAT) locus is described in this report. The assay is a valuable asset to clinical researchers interested in exploring disease associations with this allele. The automation and improved accuracy will facilitate future population-based studies, as well as clinical screening.
Subject(s)
Alu Elements/genetics , Polymerase Chain Reaction/methods , Tissue Plasminogen Activator/genetics , Automation , GenotypeABSTRACT
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.
