Characterization of imidazole as a DNA denaturant by using TGGE of PCR products from a random pool of DNA

Perpendicular temperature gradient gel electrophoresis (TGGE) profiles were analyzed for PCR products from a random pool of DNA [60 nts random region flanked by two primer (20 nts) sites], Besides a normal transition profile of a homoduplex, unique mobility transition profiles of two kinds of hetero duplex with a big internal loop were observed, representing the successive helix-coil transitions of the DNAs. As the appearance of the heteroduplex b and is an estimator of the complexity of a random pool, it will be applicab le to monitor the extent of the selection process in the in vitro selection method. When imidazole was added to the electrophoretic buffer, the transi tion pattern shifted to the low temperature side. At a concentration of 1 M , imidazole lowered the melting temperature (T-m) of DNA by 13+/-2 degrees C for all the three chain separation transitions observed, Thus imidazole i s a stronger denaturant than urea, at least at dilute concentration. Depend ence of T-m on concentration of imidazole and the mobility change suggested that imidazole binds to nucleotide in the single-stranded state.