DNA studies: a novel structural transition, relaxation of secondary structure by TOPO I, and resolution of a PCR problem

Abstract

Both theory and experiments were employed previously to investigate the effects of small neutral osmolytes on the twist energy parameter ( ET) and average intrinsic twist (l 0) of p30delta DNA (4932 bp). The results for the supercoiling free energy and intrinsic twist at 37°C, in ethylene glycol (EG) and acetamide (Ac) suggested, and were interpreted in terms of a two-state model. The DNA was assumed to undergo a conformational transition between two states that had different numbers of waters 'associated' with the duplex. Those previous studies, however, only extended to 20 (w/v)% ethylene glycol where the transition is only about half-completed. Hence, quantitative assessments of the twist energy parameter, which governs the supercoiling free energy, and of the intrinsic twist of the more dehydrated state were lacking. In the present experiments, the effects of EG over the range from 0 to 40 (w/v) % are examined and certain properties of the more dehydrated state ascertained for the first time.The torsion elastic constants of certain DNAs have exhibited a temporal evolution, on a time scale of two weeks or more, subsequent to a change in deformational strain, which implies that an intensive variable, namely the superhelix density, has traversed one or more structural transition boundaries, leaving the DNA in a metastable state. The observed time-scale for these structural transitions is consistent with a model, wherein, any given short sequence of DNA embedded in a longer DNA transiently fluctuates among two (or more) distinct secondary structures that extend over much larger domains of variable position and size, and whose relative stabilities depend on distant as well as close-lying base pairs. In general, one expects to observe very slow kinetics and metastable states for highly cooperative structural transitions, due to the difficulty of nucleating a small island of one structure in the midst of a large domain of the other.While attempting to amplify the sequence of DNA from base pairs 233-432 of the plasmid pBR322, several problems and puzzles intrinsic to the Polymerase Chain Reaction (PCR) technique were encountered. Considerable evidence was collected which suggests that the mechanism of amplification for some sequences, including this one, may differ from that of the standard model and indicates that the process may be more complex than previously thought for sequences with non-randomly distributed and high percentages of GC's.