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Nucleic Acids Research, 1992, Vol. 20, No. 24 6509-6515
© 1992

MOLECULAR BIOLOGY

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

H. Kasai1, S. Isono2, M. Kitakawa2, J. Mineno3, H. Akiyama4, D.M. Kurnit5, D.E. Berg6 and K. Isono1,2,*

1Postgraduate School, Kobe University Rokkodai, Kobe 657, Japan 2Department of Biology, Faculty of Science, Kobe University Rokkodai, Kobe 657, Japan 3 Takara Shuzo, Ohtsu, Japan 4 Toray, Kamakura, Japan 5Howard Hughes Medical Institute, University of Michigan Medical Center Ann Arbor, 48109-0650, USA 6Department of Molecular Microbiology and Genetics, Washington University Medical School St Louis, MO 63130, USA

*To whom correspondence should be addressed at: Department of Biology, Faculty of Science, Kobe University, Rokkodai, Kobe 657, Japan

Received September 29, 1992. Accepted November 10, 1992.

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 dnaBamber IacZamber 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.


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