Abstract: A novel method of primer walking using octamer oligonucleotides to prime DNA sequencing reactions is described. Octamer sequencing is compatible with isotopic and fluorescent sequencing chemistry, reaction conditions are optimized such that the samples can be processed in parallel and the procedures are automated. This strategy is faster than the traditional primer walking sequencing strategy as the existence of a primer library allows immediate access to a primer for the next sequencing reaction, eliminating delays associated with designing and synthesizing gene specific primers. The octamer library is comprised of optimized sequencing primers, such that octamer sequencing yields results equivalent to or better than traditional primer walking.