Unlabeled probe genotyping can be combined with amplicon scanning in the same reaction (Zhou et al., 2005). Both probe melting (for genotyping) and the amplicon melting (for scanning) are analyzed from the same melting curve. Reaction conditions and design are the same as with unlabeled probes with the exception that denaturation and cooling after PCR is required for heteroduplex formation. High-resolution instrumentation is required. Multiple unlabeled probes can be used in the same reaction, as long as the Tm values of all alleles are unique. The melting temperature of different probe/allele duplexes can be adjusted by probe length, mismatch position, and probe dU vs. dT content. Combined scanning and genotyping with LCGreen™ dyes presents a powerful closed-tube genetic analysis system. The method is only as complex as PCR, yet promises to vastly reduce the need for re-sequencing.
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