High-Resolution Assessment of Copy Number Variation

PCR revolutionized genetic analysis by enabling selective amplification of targeted sequences that, as a consequence of massive enrichment, could undergo genetic analysis by a variety of methodologies. The subsequent addition of a double-stranded DNA intercalating dye to the master mix was a key adaptation to PCR that allowed monitoring of amplification in real time, thus enabling quantification of the template [quantitative PCR (qPCR)]4 (1). Importantly, qPCR usually eliminated the necessity for further downstream analysis, since the determination of a quantitation cycle (Cq) value was an end in itself. The use of double-stranded DNA intercalating dyes also enabled post-PCR melting analysis.Melting analysis was originally introduced to determine the specificity of the PCR (2). However, the subsequent development of high-resolution melting (HRM) brought melting analysis to the fore as a technique in its own right. HRM relied on the availability of intercalating dyes such as LC green that bind double-stranded DNA to saturation, as well as more sophisticated fluorescence monitoring instrumentation [reviewed in (3)]. As with qPCR, analysis could be done in the same tube in which PCR amplification was performed, in this case by programming a melting analysis to follow the PCR. PCR could thus be monitored in real time and then undergo HRM without any operator intervention. In many cases, this was sufficient for a full analysis, such as in single nucleotide polymorphism genotyping or methylation detection (4, 5). The PCR products could be discarded without the tubes ever having been opened (thus reducing PCR contamination issues) or selectively undergo further analysis such as DNA sequencing.The analysis of copy number variation has emerged …