Bisulfite-free and quantitative detection of 5-formylcytosine in DNA through qPCR

An easily operated bisulfite-free method was presented to detect and quantify 5fC through quantitative real-time PCR. Malononitrile can selectively label 5fC under mild reaction conditions causing a C-to-T conversion that affects the nick ligation of the complementary pairing oligos, and then the ligation product is amplified and visualized by qPCR.