A Facile Clamp-Assisted Ligation Strategy for Direct Discrimination and Background-Free Quantification of Site-Specific 5-Formylcytosine

Quantification of site-specific 5-formylcytosine (5fC) in DNA is highly significant to better understand its biological functions. However, it is still a big challenge to precisely discriminate 5fC from cytosine (C), 5-hydroxymethylcytosine (5hmC), 5-methylcytosine (5mC) and 5-carboxycytosine (5caC) owing to their similar structures that will interfere the quantification of 5fC. To solve this issue, a novel peptide nucleic acid (PNA) clamp-assisted ligation amplification strategy coupled with a 5fC-selective chemical conversion route is employed, through which 5fC can be precisely quantified with other interfering signals completely suppressed. As a result, as low as 200 aM of site-specific 5fC-containing DNA target can be accurately determined at single-base resolution in a background-free manner.