A Dye Terminator Method for Automated DNA Sequencing Using Four Fluorescent Dideoxynucleosides and Thermal Cycling

Abstract

Automated DNA sequencers have been developed rapidly, but dye terminator sequencing using the single-dye method remains inefficient. We have successfully established a new sequencing system using a thermostable DNA polymerase and a thermal cycling approach, combined with the use of four fluorescent dideoxynucleosides on an automated DNA sequencer. Using double-strand DNAs as templates and synthesized oligonucleotides as primers, this method gave 550-900bp read lengths (with a mean read length of 755bp). This dye terminator method is both reliable and economical and should become a general technique for walking sequencing using automated DNA sequencers.