FRET-based quantitative discrimination of bisulfite-untreated DNA from bisulfite-treated DNA

We report a sensitive and reliable FRET-based nanotechnology assay for efficient detection and quantification of bisulfite-unmodified or modified DNA. Bisulfite-untreated DNA or bisulfite-treated DNA is subjected to PCR amplification with biotin-conjugated primers so that the amounts of bisulfite-untreated and treated DNA can be differentiated. Streptavidin-coated quantum dots (QDs) are used to capture biotinylated PCR products intercalated with SYBR Green, enabling FRET measurement. Key features of our method include its low intrinsic background noise, high resolution, and high sensitivity, enabling detection of as little as 1.75 ng of bisulfite-untreated DNA in the presence of an approximately 1,000-fold excess of bisulfite-untreated DNA compared to bisulfate-treated DNA, with the use of PCR reduced (as low as 15 cycles). SYBR Green as an intercalating dye as well as a FRET acceptor allows for a single-step preparation without the need for primers or probes to be chemically conjugated to an organic fluorophore. Multiple acceptors per FRET donor significantly enhance the signal-to-noise ratio as well. In consideration of the high relevance of bisulfite treatment to DNA methylation quantitation, our system for FRET measurement between QDs and intercalating dyes can be generally utilized to analyze DNA methylation and to potentially benefit the scientific and clinical community.