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)] (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 bound 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. The PCR reaction 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 as a major need in both human genetic variation and disease, as well as in the analysis of cancer (reviewed in (6 )). Genomic approaches to identify copy number alterations include array comparative genomic hybridization, single nucleotide polymorphism arrays, molecular inversion probe arrays, and massively parallel sequencing. However, particularly as the genomic assays are costly and time consuming and often require a considerable amount of input DNA, there is a need for reliable, lowcost, locus-specific assays to detect specific copy number alterations or validate genome-wide copy number analyses. When single regions are being analyzed for deviation from diploidy, it becomes necessary to compare the target to reference sequences that are not expected to show variation. PCR-based locus-specific methods in current use include qPCR, which quantifies copy number based on real-time amplification. However, qPCR has many parameters that may affect readout and thus must be performed according to stringent guidelines (7 ). In particular, it is necessary to assess amplification in the exponential phase. Droplet digital PCR directly counts copy number using Poisson statistics based on partitioning of PCRs, enabling limiting dilution of templates, and is less influenced by technical issues such as PCR inhibition and amplification efficiency (8 ). However, protocols for copy number variation determination by HRM have remained elusive. Zhou et al. now address these protocols in this issue of Clinical Chemistry (9 ). The authors chose multiplex PCR for relative quantification. This has the advantage that both the regions of interest (target) and the reference region are amplified from the same DNA sample, and thus intertube variation is eliminated. In this case, HRM was chosen to differentiate the amplicons. Accordingly, amplicons were selected with melting temperatures that were readily distinguishable by melting analysis. In addition, amplicons were designed with small sizes, single melting domains, no internal sequence variation, and no sequence homologs. Importantly, the intensity and position of the reference peaks on the melting curves were normalized, allowing direct visual estimation of the target copy number. The authors explored several options for restricting PCR so that the primer did not become limiting. After varying cycle numbers, the amount of polymerase, and the amount of deoxyribonucleotide triphosphates (dNTPs), it was found that restricting PCR amplification was best done by using a dNTP concentration of 3–12 mol/L. Although amplification was markedly inhibited 1 Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, Australia; School of Cancer Medicine, La Trobe University, Bundoora, Victoria, Australia; Department of Pathology, University of Melbourne, Parkville, Victoria, Australia. * Address correspondence to this author at: OliviaNewton-JohnCancer Research Institute, Studley Road, Heidelberg, 3084, Australia. E-mail [email protected]. Received March 3, 2015; accepted March 6, 2015. Previously published online at DOI: 10.1373/clinchem.2015.239368 2 Nonstandard abbreviations: qPCR, quantitative PCR; Cq, quantitation cycle; HRM, highresolution melting; dNTP, deoxyribonucleotide triphosphate. Clinical Chemistry 61:5 000–000 (2015) Editorials