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 heteroduplex with a big internal loop were observed, representing the successive helix-coil transitions of the DNAs. As the appearance of the heteroduplex band is an estimator of the complexity of a random pool, it will be applicable to monitor the extent of the selection process in the in vitro selection method. When imidazole was added to the electrophoretic buffer, the transition pattern shifted to the low temperature side. At a concentration of 1 M, imidazole lowered the melting temperature (Tm) of DNA by 13+/-2 degrees C for all the three chain separation transitions observed. Thus imidazole is a stronger denaturant than urea, at least at dilute concentration. Dependence of Tm on concentration of imidazole and the mobility change suggested that imidazole binds to nucleotide in the single-stranded state.
Eli
P
P
Department of Functional Materials Science, Saitama University, Urawa, 338-8570, Japan.
Ariyama
T
T
Nishigaki
K
K
Husimi
Y
Y
D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000187:drug effects* / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D019020:Directed Molecular Evolution; D004591:Electrophoresis, Polyacrylamide Gel; D007093:Imidazoles / Q000494:pharmacology*; D009691:Nucleic Acid Denaturation / Q000187:drug effects*; D016133:Polymerase Chain Reaction; D013696:Temperature
D017931:DNA Primers; D007093:Imidazoles; C029899:imidazole; D004247:DNA