The recent development of the SYBR® Green melting curve analysis assay has allowed allelic discrimination assays to be performed much more easily and less expensively than other methods. Both alleles of the gene of interest can be co-amplified in a single PCR reaction based on one primer discriminating the polymorphic site (allele specific PCR). The discrimination between the two alleles is based on one of these primers also being much longer (additional 10 bp at least) resulting in a size difference for the amplicons. The introduction of a SYBR® Green melting curve analysis assay has helped considerably in resolving the difference in size between the allele specific amplicons by providing a rapid mean of analysis length using Tm. There are numerous examples in the literature (Donohoe et al., 2000, Beuret, 2004) and a theoretical illustration of a basic allelic discrimination assay using SYBR® Green detection method is given in Figures 8.7A and 8.7B.
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