Ever since the introduction of the LightCycler® in 1996 (Wittwer et al., 1997b), melting analysis has been an integral part of real-time techniques. SYBR® Green I allows both quantification without probes (Wittwer et al., 1997a) and verification of product identity by melting analysis (Ririe et al., 1997). However, fine sequence discrimination (e.g. SNP genotyping) has usually required labeled probes. Melting analysis for SNP typing was first achieved with one labeled primer and one labeled probe (Lay and Wittwer, 1997) and later with two labeled probes, each with a single label (Bernard et al., 1998). These techniques are widely used today with conventional realtime instrumentation (Wittwer and Kusukawa, 2004, Gingeras et al., 2005).
New instruments and saturating double-stranded DNA binding dyes for high-resolution melting enable mutation scanning and genotyping without probes. High-resolution melting was first reported with labeled primers (Gundry et al., 2003) for distinguishing different heterozygotes and homozygotes. This was rapidly followed by the introduction of saturating dyes, eliminating any need for fluorescently labeled oligonucleotides (Wittwer et al., 2003). Because no separation steps are required, mutation scanning by melting is inherently simple and inexpensive. The sensitivity and specificity for detecting heterozygous SNPs is at least as good as alternative, more complicated techniques (Reed and Wittwer, 2004, Chou et al., 2005). High-resolution melting for mutation scanning has been reported for c-kit (Willmore et al., 2004), medium chain acyl-CoA dehydrogenase (McKinney et al., 2004), SLC22A5 (Dobrowolski et al, 2005), and BRAF (Willmore-Payne et al., 2005) genes. The method can be used for transplantation matching by establishing sequence identity among related donors within HLA genes (Zhou et al., 2004b). Complete genotyping is possible for most SNPs (Liew et al., 2004, Graham et al., 2005) and bacterial speciation has been reported (Odell et al., 2005). Unlabeled probes can be added when regions of high sequence complexity are analyzed (Zhou et al., 2004a).
Because of its simplicity and power, we suspect high-resolution melting techniques will become integrated into real-time platforms, perhaps even by the publication of this book. In what follows, we briefly describe the instrumentation and saturation dyes that make high-resolution melting analysis possible. Next we present experimental details for mutation scanning, followed by amplicon genotyping and unlabeled probe genotyp-ing. Finally, we compare high-resolution melting analysis to other options for scanning and homogeneous genotyping.
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