RAPID PLASMID DNA-SEQUENCING WITH MULTIPLE OCTAMER PRIMERS

Oligonucleotide walking is an important technique for generating new D NA sequence information. We have developed an efficient sequencing met hod based on the use of octamer primers, which can potentially simplif y and reduce the cost of sequencing. In this procedure the plasmid DNA is thoroughly denatured in the presence of heat and NaOH. Following c ooling, the mixture is aliquoted into tubes containing the different o ctamer primers and neutralized by the addition of HCI. The mixture is then sequenced by a modified Sequenase (R) protocol that involves perf orming the initial labeling reactions at low temperature (on ice) and the termination reactions at high temperatures (43 degrees C). Analysi s of sequencing gels revealed octamer sequencing to be as effective as sequencing with 17-mer primers.