Supercoiled plasmid sequencing one buffer for template denaturation, primer annealing, and synthesis of labeled strands by reverse transcriptase

We describe a simple modification of the direct plasmid sequencing procedure. In this procedure, denaturation of plasmid DNA, primer annealing and synthesis of labeled strands are carried out in a one-buffer system. The simple heat-denaturation step allows rapid preparation of supercoiled DNA for primer annealing. The use of reverse transcriptase minimizes artifacts encountered when using the Klenow polymerase to synthesize the labeled strands.