A DYE TERMINATOR METHOD FOR AUTOMATED DNA-SEQUENCING USING 4 FLUORESCENT DIDEOXYNUCLEOSIDES AND THERMAL CYCLING

Automated DNA sequencers have been developed rapidly, but dye terminat or 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 550-900 bp read lengths (with a mean rea d length of 755 bp). This dye terminator method is both reliable and e conomical and should become a general technique for walking sequencing using automated DNA sequencers.