Detection of PNA/DNA hybrid molecules by antibody Fab fragments isolated from a phage display library

We have isolated Fab fragments that specifically recognize duplexes formed between DNA and PNA (peptide nucleic acid) from an immunized murine phage display library. Rearranged murine Fd- and Kappa chains were assembled by PCR and cloned into a phagemid expression vector. Subsequently, affinity selection on immobilized PNA/DNA duplexes of the Fab-displaying phages resulted in the isolation of clones that uniquely recognized PNA/DNA duplexes. One of these clones was characterized in detail, and its recognition of PNA/DNA duplexes was relatively sequence independent, taking place equally well with sticky-end and blunt end PNA/DNA duplexes. Duplexes smaller than 15-mers could not be detected. The selected clone recognized neither single-stranded DNA and PNA, nor double-stranded DNA and PNA. Binding of the Fab fragments to immobilized PNA/DNA duplexes could be inhibited by PNA/DNA duplex molecules in solution, with an apparent affinity in the nanomolar range. The use of this anti PNA/DNA Fab-phage as an immunochemical reagent was demonstrated in dot blot assays.