Rapid, Homogeneous Genotyping of the 4G/5G Polymorphism in the Promoter Region of the PAI1 Gene by Fluorescence Resonance Energy Transfer and Probe Melting Curves

Abstract

AbstractBackground: Many studies have convincingly shown that survivors of myocardial infarction have impaired fibrinolytic activity because of increased concentrations of plasma plasminogen activator inhibitor-1 (PAI-1). A single guanosine insertion/deletion polymorphism in the promoter region of the PAI1 gene, commonly called 4G/5G, has been shown to be associated with plasma PAI-1 activity. Our aim was to develop and validate a homogeneous assay for rapid genotyping of the 4G/5G polymorphism.Methods: In this report we present a single-tube method for genotyping of the 4G/5G polymorphism that combines both rapid-cycle PCR with real-time monitoring of the amplification process and generation of allele-specific fluorescent probe melting profiles on the LightCyclerTM. Two fluorescently labeled hybridization probes recognizing adjacent sequences in the amplicon were present in the reaction mixture. The shorter detection probe spanned the polymorphic site, perfectly matching the 5G allele. After annealing, the fluorophores were in resonance energy transfer, providing real-time monitoring of the amplification process. At the completion of the PCR, fluorescence was monitored as the temperature increased through the Tm of the probe/product duplex, and a characteristic melting profile for each genotype was obtained.Results: With this method, 32 samples were genotyped within 30 min without the need of any post-PCR sample manipulation. The genotypes of 100 DNA samples determined with the LightCycler were identical to those obtained with conventional PCR and restriction fragment length analysis.Conclusion: The genotyping of the 4G/5G polymorphism with the LightCycler is a rapid, reliable method that is suitable for typing both small and large numbers of samples.