The LightCycler® 480 High Resolution Melting Master for Gene Scanning

Roche has announced that its High Resolution Melting (HRM) is a close-tube, post-PCR method enabling genomic researchers to analyze genetic variations (SNPs, mutations, methylations) in PCR amplicons prior to or as an alternative to sequencing. It can provide high specificity, sensitivity and convenience at higher speed and lower cost much than other established.

The LightCycler® 480 High Resolution Melting Master contains a special generic DNA dye which binds to DNA in a saturating fashion and without preference for certain regions or conformations. It gives rise to very sharp and distinct melting signals which, when acquired at high resolution, can reveal very subtle differences between.

The dye contained in the LightCycler® 480 High Resolution Melting Master is chemically different from other HRM dyes like LC Green Plus (Idaho Tech) or SYTO9 (Invitrogen). It works at much lower concentrations (~ten fold) than other dyes and can therefore be used in a PCR master mix without interfering with PCR performance. It also shows improved stability and signal dynamics and gives rise to melting curves whose shape can be easily analyzed and interpreted.

The main application for the HRM method is gene scanning – the search for unknown genetic variations (SNPs, mutations, deletions) in PCR amplicons (the detection and calling of already known mutations is referred to as genotypin). Gene scanning on the LightCycler® 480 System is based on the analysis of melting curve data obtained at high resolution (HRM).

With HRM, unknown sequence variations in diploids become apparent in heterozygous samples because they have differently shaped melting curves compared to those derived from homozygous (wildtype or mutant) samples. The LightCycler® 480 Instrument is the first plate-based real-time PCR instrument facilitating such analysis due to its high-performance thermal control, and optical components.