Automated detection of point mutations by electrophoresis in peptide-nucleic acid-containing gels

Polymerization of electronically, essentially neutral peptide nucleic acids (PNA) into polyacrylamide gels creates a medium in which the salt-independent properties of PNA/DNA interactions are used to achieve hybridization with target DNA during affinity electrophoresis. Such physical entrapment of PNA has been used to differentiate between a retarded, complementary DNA strand and a non-retarded sequence differing by a single point mutation. Analysis of fluorescent PCR products--from both model mismatches and clinically relevant point mutations using a conventional automated DNA sequencer--allows one to follow this hybridization in real time and to distinguish homo- and heterozygous mutants visually. It has been shown that parameters affecting the resolution of these species include not only temperature and concentration (as a function of the [GC] content of the PNA), but also the position of the PNA binding sequence within the interacting DNA segment. Under conditions optimized in terms of temperature and PNA concentration, the maximum separation of retarded from non-retarded DNA single strands is obtained when the PNA binding sequence is close to either DNA terminus. Strategies of PNA and PCR primer design that permit a diagnostic application ranging over 85-451 DNA base pairs are proposed.