The use of real-time PCR and fluorogenic probes for rapid and accurate genotyping of newborn mice

Real-time PCR and fluorogenic probes were combined in a simple, rapid and s ensitive method to genotype murine breeding stocks and their progeny for a point mutation. DNA from tail biopsies of newborn mice was mixed with ampli fication primers and fluorogenic hybridization probes in a PCR mixture. The primers were designed to amplify a region of the Fas-Ligand gene including the site for the gld natural point mutation. The fluorogenic hybridization probes overlaid this target sequence and were used to detect amplification of the PCR fragment as well as determine the presence of the point mutatio n using fluorescence resonance energy transfer (FRET). Both mutated and wil d-type forms of the gene fragment were amplified as detected with real-time PCR. Melting curve profiles completed on each amplified sample revealed th e genotype for each mouse. These genotypes were confirmed by sequencing tl- le amplified fragments. These results suggest real-time spectrofluorometric PCR techniques incorporating FRET-based hybridization probes may be used f or rapid, sensitive, inexpensive and reliable genotyping. (C) 2001 Academic Press.