The LightCycler® 480 real-time PCR system: a versatile platform for genetic variation research

Abstract

Real-time PCR is a well established technique for studying genetic variation using various probe-based methods for genotyping as well as high-resolution analysis of whole amplicons melted in the presence of saturating DNA dyes. The latter, relatively new, method allows screening for unknown mutations or DNA modifications. The LightCycler ® 480 real-time PCR system is a multiwell plate-based instrument that provides integrated applications for detecting and characterizing genetic variation using all these methodological approaches. From endpoint fluorescence to melting curve analysis Today's research on somatic, genetic and epigenetic variation in eukary-otic cells requires fast, accurate and cost-effective methods for screening large numbers of samples or loci in parallel. Variations identified by genomic sequencing or array studies need to be subsequently confirmed and validated. Real-time PCR has become a well established technology for this purpose. The plate-based LightCycler 480 system offers a broad selection of methods and applications (Fig. 1). The basic method (endpoint genotyping) is highly suitable for simple experimental setups when well characterized single-nucleotide poly-morphisms (SNPs) and regions need to be analyzed, especially when commercial primers and probes are available. Allele-specific signals can be generated and acquired upon the completion of PCR, using two differently labeled hydrolysis probesone for each allelein the same reaction. Primers and probes can be readily combined with LightCycler 480 reagents, and the results can be analyzed with the LightCycler 480 Endpoint Genotyping Software module. For more advanced setups, melting curves obtained with fluorescent hybridization probes provide additional information about the sequence under study. Known SNPs present in PCR amplicons may be investigated with sequence-specific, labeled probes (known as HybProbe probes) that bind with different strengths to different alleles or allele combinations in the SNP-containing region, depending on whether there are mismatches that keep the probes from fully matching the target sequence. Because signal generation and analysis are done after amplification, there is no influence on PCR efficiency (for example, when DNA amounts or purity are an issue), making the method more robust than endpoint analysis. Only one fluorescence channel is acquired per locus, allowing investigation of several SNPs in parallel in multiplex assays if a differe…