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.
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.
Kasai
H
H
Postgraduate School, Faculty of Science, Kobe University, Japan.
Isono
S
S
Kitakawa
M
M
Mineno
J
J
Akiyama
H
H
Kurnit
D M
DM
Berg
D E
DE
Isono
K
K
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*
D004251:DNA Transposable Elements; D009838:Oligodeoxyribonucleotides