Sequencing Multimegabase-Template DNA with BigDye Terminator Chemistry

摘要: In microbial genome or large-insert clone sequencing projects that use the predominant random subclone strategy, progress tends to decrease dramatically at late stages as one confronts gaps. At these points, DNA is under-represented unstable in subclones (E.Y. Chen et al. 1996; Chissoe 1997). Further with additional then inefficient best, and must frequently employ alternative cloning systems methods like long-range PCR recover missing (C.N. 1996). The variability of performance necessity for custom-tailored work tend hamper efforts. contrast, if can sequence directly from genomic (or clones such BACs PACs) walking primers, cumbersome fill gaps could be completed a much shorter time. As an example, recent project 750-kb Ureaplasma urealyticum (J. Glass, prep.) assemblage ∼13,000 reads combinatorial reactions join contigs left two No λ pUC, M13 were recovered spanned gaps, nor products derived any several sets flanking primers. difficulty segments probably attributable repeated sequences near but high sensitivity recently introduced BigDye terminator (Rosenblum 1997) permitted direct gap regions on U. templates. Using conditions described this report, 259 121 bp sequenced both strands primers complete 751,723 bp. Direct was further tested larger templates, good results reproducibly obtained 1.2-Mb Mycoplasma fermentans, 2.3-Mb Streptococcus pneumoniae, 4.6-Mb Escherichia coli (see example Fig. ​Fig.1).1). addition, difficult BAC clones, ranging size 140 250 kb, have also been filled manner. Essentially method applicable whenever 2–3 μg high-quality large-template available. Figure 1 Sequencing E. K12 strain terminators. Approximately 3 apaG gene primer (5′-GTTCCCACACTCATTCATTA) using text.