Rapid genotyping of tumor necrosis factor alpha with fluorogenic hybridization probes on the LightCycler

Genotyping of tumor necrosis factor alpha (TNF-alpha) has become an important procedure in the selection of high-risk population of septic shock and prevention from death due to septic shock. We present a single-tube method for TNF-alpha genotyping that performed on the LightCycler by melting curve analysis with allele-specific fluorescent probe. A fragment covering the polymorphic site is amplified in the presence of two fluorescently labeled hybridization probes. During amplification, probe hybridization is observed as fluorescence increases every cycle as the product accumulates during amplification. A single base mismatch resulted in a melting temperature (Tm) shift of 7-8 degrees C, allowing for the easy distinction of a common type allele from the polymorphic allele. Using this method, genotyping of 104 samples was completed within 1 h without the need for any post-PCR sample manipulation, thereby eliminating the risks of end-product contamination and sample tracking errors. The genotypes determined with the LightCycler were identical when compared with a conventional sequencing. The simplicity, speed, and accuracy of real-time PCR analysis using FRET probes make it the method of choice in the clinical laboratory for genotyping of a variety of human DNA polymorphisms and mutations.