EFFICIENT LARGE-SCALE SEQUENCING OF THE ESCHERICHIA-COLI GENOME - IMPLEMENTATION OF A TRANSPOSON-BASED AND PCR-BASED STRATEGY FOR THE ANALYSIS OF ORDERED LAMBDA PHAGE CLONES

We have developed a strategy for efficient sequence analysis of the genome of E.coli K-1 2 using insertions of a Tn5-derived mini-transposon into overlapping ordered lambda phage clones to provide universal primer-binding sites, and PCR amplification of DNA segments adjacent to the insertions. Transposon-containing clones were selected by blue plaque formation on a dnaB(amber) lacZ(amber) E.coli strain. Insertion points every 0.5-1 kb were identified by 'analytical PCR' and segments between the transposon inserts and phage arms were amplified by 'preparative PCR' using one biotinylated and one non-biotinylated primer. Single strands of amplified DNA fragments were coupled to Streptoavidin-coated paramagnetic beads (Dynabeads M280) through their biotin tails, purified magnetically, and used as templates for fluorescence-based automatic nucleotide sequencing.