PCR-RFLP Genotyping Assay for the Bcl I Polymorphism of the β-Fibrinogen Gene

Abstract

Polymorphisms of the β-fibrinogen gene have been shown to affect plasma fibrinogen levels and risk of coronary artery disease (CAD). We were interested in developing an automated, PCR-based genotyping assay for the purpose of exploring relationships between CAD and CAD-associated aortic stiffness and the Bcl I allele of the β-fibrinogen gene. We have developed a rapid PCR-RFLP assay for the Bcl I polymorphism of the β-fibrinogen gene. We carried out direct PCR of genomic DNA to facilitate sequencing of the flanking region of the β-fibrinogen gene. Using this new sequence information, primers were designed which border the site of the Bcl I polymorphism. One of the primers was labeled with a fluorophore to facilitate detection of the fragments. DNA fragment analysis was carried out using an automated capillary electrophoresis instrument (ABI310). We have developed an improved PCR-RFLP high-sample-throughput assay for the semiautomated detection of the Bcl I polymorphism of the β-fibrinogen gene. This assay will support screening of large sample sizes required for population studies.