Rapid and reliable genotyping for the Toll-like receptor 4 A896G polymorphism using fluorescence-labeled hybridization probes in a real-time polymerase chain reaction assay

Abstract Background : The Toll-like receptor 4 (TLR4) is involved in immune response to endotoxin as well as the pathogenesis of atherosclerosis. The TLR4 gene was shown to carry a single-nucleotide polymorphism (A896G). We developed a rapid-cycle polymerase chain reaction (PCR) which allows for rapid genotyping and, therefore, may be useful in clinical risk assessment. Methods : Fluorescently labeled oligonucleotide hybridization probes were designed for the LightCycler™ instrument. A PCR product was generated in a single reaction followed by melting point analysis. Ninety-three German Caucasians were genotyped. The interleukin-1β (IL-1β) response to endotoxin was assessed after whole blood stimulation with endotoxin according to TLR4 genotypes. Results : The method suggested by us is a time-saving technique requiring no additional manual steps. Frequencies of the A and G allele were 0.95 and 0.05, respectively. The study population was in Hardy–Weinberg equilibrium. The specificity of the method was confirmed by sequencing. The IL-1β levels were lower in carriers of the G allele. Conclusions : This PCR assay is a rapid and reliable technique for TLR4 genotyping.