Improved Signal-to-Noise Ratios in qPCR Assays for Genomic Copy Number Variation (CNV) Determination Using Dual-Quenched BHQ® Probes

Copy Number Variation (CNV) is an important form of genetic diversity with medical and agricultural significance. CNV determination with quantitative PCR requires a sensitive and specific detection chemistry that is quantitative across a broad dynamic range.  In this study, we introduce a dual-quenched probe format with a unique configuration of the Black Hole Quencher® (BHQ) dye to improve quenching efficiency and enhance signal-to noise values. Probe-based qPCR assays were designed using LGC Biosearch Technologies RealTimeDesign™ software to target specific regions in human, plant, and animal genomes that are known to harbor sequence rearrangements, producing variation in copy number.  Each CNV assay was paired into a duplexed reaction with a reference assay to a genomic region that remains a constant copy number, to act as a normalizer for the variable target assay. The differential of cycle threshold values provides an accurate determination of copy number per genome. Quantification is initially demonstrated by supplying template at fixed ratios, and further verified in the context of wheat and bovine samples. These results reveal lower background fluorescence accompanied by enhanced signal release compared to standard probes containing only a single quencher.  Dual-quenched BHQ probes are a robust tool for all manner of qPCR applications such as copy number variation.