title: Efficient large-scale sequencing of the Escherichia coli genome: implementation of a transposon- and PCR-based strategy for the analysis of ordered lambda phage clones.
abstract: We have developed a strategy for efficient sequence analysis of the genome of E. coli K-12 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 dnaBamber lacZamber 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.
authors.affiliation: Postgraduate School, Faculty of Science, Kobe University, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Kasai
authors.forename: S
authors.initials: S
authors.lastname: Isono
authors.forename: M
authors.initials: M
authors.lastname: Kitakawa
authors.forename: J
authors.initials: J
authors.lastname: Mineno
authors.forename: H
authors.initials: H
authors.lastname: Akiyama
authors.forename: D M
authors.initials: DM
authors.lastname: Kurnit
authors.forename: D E
authors.initials: DE
authors.lastname: Berg
authors.forename: K
authors.initials: K
authors.lastname: Isono
mesh_terms: D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics*; D016680:Genome, Bacterial; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004251:DNA Transposable Elements; D009838:Oligodeoxyribonucleotides