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 fo rmed between DNA and PNA (peptide nucleic acid) from an immunized muri ne phage display library. Rearranged murine Fd- and Kappa chains were assembled by PCR and cloned into a phagemid expression vector. Subsequ ently, affinity selection on immobilized PNA/DNA duplexes of the Fab-d isplaying phages resulted in the isolation of clones that uniquely rec ognized PNA/DNA duplexes. One of these clones was characterized in det ail, and its recognition of PNA/DNA duplexes was relatively sequence i ndependent, taking place equally well with sticky-end and blunt end PN A/DNA duplexes. Duplexes smaller than 15-mers could not be detected. T he selected clone recognized neither single-stranded DNA and PNA, nor double-stranded DNA and PNA. Binding of the Fab fragments to immobiliz ed 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 demonstr ated in dot blot assays.