An improved method for purifying bacterial genomic DNAs for direct sequencing by capillary automated sequencer

Abstract

Since the first publication of the dideoxy sequencing method (Ref. [1] ), technologies have developed for both labeling and separating molecules. Fluorescent dye labels, among others, have enabled automated sequencing with increased capacity and throughput. In addition, the enhanced sensitivity conferred by energy-transfer dyes like BigDye (PE Biosystems, Foster City, CA, USA) and DYEnamic ET (Amersham Pharmacia Biotech, Buckinghamshire, UK) raises the possibility of sequencing directly from very large plasmids such as BACs, PACs and P1s, and even from bacterial genomes (Ref. [2] ). This would provide a means of closing gaps between contigs generated by a whole genome shotgun approach, and of identifying transposon insertion sites without any intermediate PCR step. For successful direct sequencing, however, high-quality genomic DNA is necessary, requiring time-consuming proteinase K digestion and subsequent phenol–chloroform extraction. Here, I present a simple and reliable method for preparing bacterial genomic templates for direct sequencing. This method uses a chaotropic reagent and silica matrix for purifying needle-sheared DNA. It can be used for both Gram-positive and Gram-negative bacteria, and DNA sequences can be determined using a capillary sequencing machine [e.g. 310 Genetic Analyzer (PE Biosystems)].