Rapid plasmid DNA sequencing with multiple octamer primers.

Oligonucleotide walking is an important technique for generating new DNA sequence information. We have developed an efficient sequencing method based on the use of octamer primers, which can potentially simplify and reduce the cost of sequencing. In this procedure the plasmid DNA is thoroughly denatured in the presence of heat and NaOH. Following cooling, the mixture is aliquoted into tubes containing the different octamer primers and neutralized by the addition of HCl. The mixture is then sequenced by a modified Sequenase protocol that involves performing the initial labeling reactions at low temperature (on ice) and the termination reactions at high temperatures (43 degrees C). Analysis of sequencing gels revealed octamer sequencing to be as effective as sequencing with 17-mer primers.