Real-time PCR integrates microvolume rapid-cycle PCR with fluorometry, allowing real time fluorescent monitoring of the amplification reaction for quantitative PCR and/or characterization of PCR products for rapid genotyping, precluding any post-PCR sample manipulation. The LightCycler® (Roche Molecular Biochemicals) is one such system. The detec tion of potential sequence differences for genotyping applications (usually Single Nucleotide Polymorphisms, SNPs) employs the use of two fluorescent probes which hybridize to adjacent internal sequences within target amplified DNA, one of which covers the region expected to contain the mutation(s). Close proximity of annealed probes facilitates fluorescence resonance energy transfer (FRET) between them. The probes are designed to have different melting temperatures (Tm) whereby one with the lower Tm spans the mutation site(s). Monitoring of the emitted fluorescent signals as the temperature increases will detect loss of fluorescence (F) as the lower Tm probe melts off the template. A single base mismatch under this probe results in a Tm shift of 5-10°C, allowing easy distinction between wild-type and mutant alleles. The ability to detect base mismatches under the low Tm probe (mutation detection probe) and the use of two different colored probes (LightCycler® system 1.0 or 1.5) allows more than one mutation to be screened in a single PCR reaction.
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