Sequencing multimegabase-template DNA with BigDye terminator chemistry

Abstract

Using the recently introduced BigDye® terminators, large-template DNA can be directly sequenced with custom primers on automated instruments. Cycle sequencing conditions are presented to sequence DNA samples isolated from a number of microbial genomes including 750-kb Ureaplasma urealyticum, 1.2-Mb Mycoplasma fermentans, 2.3-Mb Streptococcus pneumoniae, and 4.6-Mb Escherichia coli. Average read lengths of > 700 bp from unique primer annealing sites are often sufficient to fill final gaps in microbial without additional manipulations of template DNA. The technique can also be applied to sequence-targeted regions, thereby bypassing tedious subcloning steps.