Summary

Thrombophilia is a multigenic disease in which the combination ofgenetic polymorphisms increases the risk of deep vein thrombosis(DVT). The rapid identification of these genetic combinations requireshigh-throughput analysis of single nucleotide polymorphisms (SNPs).The TaqMan® fluorogenic 5’→ 3’ nuclease assay (PE/Applied Biosystems,Foster City, CA) with custom-designed primers, probes andcontrols has provided a highly efficient platform for high throughput.This assay was used to rapidly detect two SNPs, FV Leiden (G1691A)and FV A4070G (R2 allele), in a study of 6295 subjects. With onethermal cycler, we completed sample set-up, PCR and analysis on84 samples in 3 h with an additional 12 wells containing 4 "no templatecontrols" (NTC), 4 "allele-1 controls", and 4 "allele-2 controls" in a96-well plate. When additional thermal cyclers were used and moreassays were set up while the initial sets of reactions were in the PCRmachines, the output could correspondingly be increased. The Taq-Man® assay was extremely accurate, avoided contamination by usinguracil-N-glycolase (UNG) in a single, closed tube, and offered thepossibility for additional automation with robotic equipment to implementthe PCR. This TaqMan® assay facilitates high throughput toscreen large populations quickly and economically while utilizing asimple protocol that requires minimal expenditure of personnel time.Our results demonstrated a prevalence of the R2 allele of 11.9% in U.S.Caucasians, 5.6% in African-Americans, 13.4% in Asian or PacificIslanders and 11.3% in Hispanics. No association between venousthromboembolism and the R2 allele was noted, and furthermore nointeraction with FV Leiden was observed.