Instrumentation for unlabeled probe genotyping

In contrast to mutation scanning and amplicon genotyping, high-resolution melting analysis is not required with unlabeled probes. Results are best on the HR-1™ and LightScanner™, but the LightTyper™ and the LightCycler® produce adequate genotyping results (Zhou et al., 2004a). Although other real-time PCR instruments have not been tested, unlabeled probe geno-typing should be possible with LCGreen™ PLUS. Recommended melting rates for unlabeled probe genotyping are 0.1°C for the LightScanner™ and 0.3°C/sec for the HR-1™. Background fluorescence from single strands (primers and single stranded product) is prominent at low temperatures. However, conversion of the original data directly to derivative curves reveals the melting transitions as peaks on a downward slope (Figure 9.4).

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