High-resolution melting has proven to be a highly sensitive method for mutation discovery. PCR is performed in the presence of the double-strand DNA binding dye LCGreen® Plus. Samples are then denatured on the Idaho Technology (www.idahotech.com) LightScanner® instrument. Nucleic acid melting is tracked by monitoring the fluorescence of the samples across a defined temperature range, generating melting profiles that can be used to identify the presence of sequence variation within the amplicon. Single point mutations, small insertions, and deletions can all be identified using high-resolution melting. The technique has been shown to have near 100% sensitivity and specificity when used on products up to 400 base pairs in length.
LunaProbes™, an extension of the high-resolution DNA melting technique, provides a simple and inexpensive alternative for genotyping. An unlabeled oligonucleotide probe, blocked at the 3´ end to prevent extension, is designed to sit over a SNP of interest and is included in the PCR reaction prior to amplification.
Genotyping is accomplished by monitoring the melting of the probe-target duplex post-PCR. Key to the success of this method is the use of asymmetric PCR, where one primer is used in excess, resulting in the overproduction of the target strand recognized by the probe and the use of LCGreen Plus dye, which is capable of producing a strong fluorescent signal from the probe-target interaction.
Genotyping by melting analysis is beneficial to investigators primarily due to the cost-effectiveness of the technique. In the past, using PCR methods for genotyping has required fluorescently labeled oligonucleotides. These methods can be costly and time consuming. With LunaProbes, all three possible genotypes that result from a given polymorphism can be detected with a single unlabeled probe.
Described is the assessment of a common polymorphism (c1155C>G) in the phenylalanine hydroxylase (PAH) gene using high-resolution melting, LCGreen Plus dye, and a LunaProbe.