Melting curve analysis of SNPs (McSNP (R)): A gel-free and inexpensive approach for SNP genotyping

High-throughput methods for assaying DNA variation require two important st eps: (i) discriminating the variation and (ii) detecting the signal. In thi s report, we describe a novel SNP genotyping method that we refer to as mel ting curve analysis of SNPs (McSNP(R)). McSNP combines a classic approach f or discriminating allees, restriction enzyme digestion, with a more recent method for detecting DNA fragments, melting curve analysis. Melting curve a nalysis is performed by slowly hearing DNA fragments in the presence of the dsDNA-specific fluorescent dye SYBR(R) Green I. As the sample is heated, f luorescence rapidly decreases when the melting temperature of a particular fragment is reached. We show that it is possible to determine the compositi on of simple mixtures of DNA fragments, such as those that result from rest riction enzyme digestions of short PCR products. McSNP is well suited for h igh-throughput genotyping because 96 samples can be analyzed and automatica lly scored in 20 min Our results clearly demonstrate that McSNP is a simple , inexpensive, and accurate means of genotyping SNP variation.