Genotyping using DNA melting profiles

Genotyping by direct analysis of DNA has become increasing important in the breeding of plants and animals, for the diagnosis and study of genetic disorders in humans as well as in forensic science. One limitation of the current methodology is the lack of a very rapid test that can be performed in the order of minutes. DNA detection methods which are dependant upon separation or hybridisation such as gel electrophoresis or ELISA are usually lengthy, complex, multi-step processes and are not suited to very rapid assays. The melting profiles of dsDNA have been investigated as an alternative rapid detection method for use in genotyping. Subjecting PCR products to a temperature ramp in the presence of a dsDNA binding dye may generate DNA melting profiles which is characteristic of a particular genotype. A rapid decrease in fluorescence accompanies the conformation change from double to single strands. The plot of fluorescence versus temperature is dependent upon the fragment length; sequence and GC content and may distinguish it from other fragments differing in these properties. Detection is simple and can be performed in around 5 minutes in some cases. Both amplification and detection is possible in a single reaction vessel by the addition of a dye to the PCR reaction mix. We have used this approach to discriminate between admixtures of cereal crops and between cereal varieties.