LightCycler® Real-time PCR Systems from Roche (www.roche.com) are efficient platforms for genetic variation research. They feature the temperature homogeneity and the optical characteristics required for high-performance melting curve analysis. In addition, the plate-based LightCycler 480 System facilitates advanced applications at high resolution in the emerging field of amplicon scanning for new mutations.
Melting curve analysis is a well-established method for characterizing amplicons (e.g., for microbiological identification or for detecting mutations and SNPs). The goal of SNP studies is either the detection of different allelic variants of a given SNP or the identification of polymorphic positions at which previously unknown SNPs occur without determining the allele. Due to the modular design of the LightCycler 480 System, both approaches can be pursued. A broad range of detection chemistries and data analysis algorithms adds flexibility.
Homozygous and heterozygous samples can be differentiated easily using certain saturating DNA-binding dyes that do not interfere with amplification even at high concentrations and give sharp melting profiles (Figure 1). Once SNPs are known, their alleles are best analyzed by melting in the presence of sequence-specific labeled probes that bind with different affinities to different alleles, or allele combinations, in the SNP-containing region (Figure 2).
Melting curve raw data is generally represented by plotting fluorescence over temperature, with the data resolution and temperature range being higher with high-resolution dyes than with labeled probes (Figures 1a, 2a). To make analysis more convenient, the negative first derivatives
(-dF/dT) are often used, revealing melting temperatures at peaks (Figure 1b, 2b).
Data derived from homozygous wild types, homozygous mutants, and heterozygous samples can differ regarding peak number, peak position, or a combination of both. With high-resolution dyes, differences between melting peaks are often detectable but not always large enough to enable clear differentiation of homozygotes (Figure 1b). Using sequence-specific probes allows for a more reliable separation (Figure 2b). The LightCycler 480 Genotyping Software uses the latter approach for identifying SNP alleles.