Direct Dideoxy DNA Sequencing

Advances in DNA sequencing techniques have made it possible to routinely sequence long DNA molecules (1 ,2 ). Recently double-stranded DNA has been directly sequenced with chain terminators (3 ), and this eliminates the tedious task of subcloning the fragments into single-stranded M13 vectors. The procedure requires a plasmid DNA template that is denatured to separate the DNA strands and a primer complementary to one of the strands of the plasmid vector adjacent to the cloning site that has been used. Thus by using primers complementary to each strand, the sequence of the insert can be read from both ends. In the presence of the four deoxynucleoside triphosphates, one of which is radioactively labeled, and the Klenow fragment of Escherichia coli DNA polymerase I (or reverse transcriptase, as described in this chapter), primed synthesis occurs across the region of interest. In the presence of competing dideoxynucleoside triphosphates (ddNTPs), specific termination occurs at each of the four different nucleotides, and if this is carried out separately with each of the ddNTPs, then the four sets of reaction products can be analyzed on DNA sequencing gels.