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. 1992 Dec 25;20(24):6509–6515. doi: 10.1093/nar/20.24.6509

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 Kasai 1, S Isono 1, M Kitakawa 1, J Mineno 1, H Akiyama 1, D M Kurnit 1, D E Berg 1, K Isono 1
PMCID: PMC334565  PMID: 1336178

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.

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Selected References

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  1. Bachmann B. J. Linkage map of Escherichia coli K-12, edition 8. Microbiol Rev. 1990 Jun;54(2):130–197. doi: 10.1128/mr.54.2.130-197.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Daniels D. L., Plunkett G., 3rd, Burland V., Blattner F. R. Analysis of the Escherichia coli genome: DNA sequence of the region from 84.5 to 86.5 minutes. Science. 1992 Aug 7;257(5071):771–778. doi: 10.1126/science.1379743. [DOI] [PubMed] [Google Scholar]
  3. Hultman T., Bergh S., Moks T., Uhlén M. Bidirectional solid-phase sequencing of in vitro-amplified plasmid DNA. Biotechniques. 1991 Jan;10(1):84–93. [PubMed] [Google Scholar]
  4. Hultman T., Ståhl S., Hornes E., Uhlén M. Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support. Nucleic Acids Res. 1989 Jul 11;17(13):4937–4946. doi: 10.1093/nar/17.13.4937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kersulyte D., Krishnan B. R., Berg D. E. Nonrandom orientation of transposon Tn5supF insertions in phage lambda. Gene. 1992 May 1;114(1):91–96. doi: 10.1016/0378-1119(92)90712-x. [DOI] [PubMed] [Google Scholar]
  6. Kohara Y., Akiyama K., Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. doi: 10.1016/0092-8674(87)90503-4. [DOI] [PubMed] [Google Scholar]
  7. Komine Y., Adachi T., Inokuchi H., Ozeki H. Genomic organization and physical mapping of the transfer RNA genes in Escherichia coli K12. J Mol Biol. 1990 Apr 20;212(4):579–598. doi: 10.1016/0022-2836(90)90224-A. [DOI] [PubMed] [Google Scholar]
  8. Krishnan B. R., Kersulyte D., Brikun I., Berg C. M., Berg D. E. Direct and crossover PCR amplification to facilitate Tn5supF-based sequencing of lambda phage clones. Nucleic Acids Res. 1991 Nov 25;19(22):6177–6182. doi: 10.1093/nar/19.22.6177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kröger M., Wahl R., Schachtel G., Rice P. Compilation of DNA sequences of Escherichia coli (update 1992). Nucleic Acids Res. 1992 May 11;20 (Suppl):2119–2144. doi: 10.1093/nar/20.suppl.2119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kulakauskas S., Wikström P. M., Berg D. E. Efficient introduction of cloned mutant alleles into the Escherichia coli chromosome. J Bacteriol. 1991 Apr;173(8):2633–2638. doi: 10.1128/jb.173.8.2633-2638.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kurnit D. M., Seed B. Improved genetic selection for screening bacteriophage libraries by homologous recombination in vivo. Proc Natl Acad Sci U S A. 1990 Apr;87(8):3166–3169. doi: 10.1073/pnas.87.8.3166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Phadnis S. H., Huang H. V., Berg D. E. Tn5supF, a 264-base-pair transposon derived from Tn5 for insertion mutagenesis and sequencing DNAs cloned in phage lambda. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5908–5912. doi: 10.1073/pnas.86.15.5908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Phadnis S. H., Kulakauskas S., Krishnan B. R., Hiemstra J., Berg D. E. Transposon Tn5supF-based reverse genetic method for mutational analysis of Escherichia coli with DNAs cloned in lambda phage. J Bacteriol. 1991 Jan;173(2):896–899. doi: 10.1128/jb.173.2.896-899.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ponce M. R., Micol J. L. PCR amplification of long DNA fragments. Nucleic Acids Res. 1992 Feb 11;20(3):623–623. doi: 10.1093/nar/20.3.623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Singer M., Baker T. A., Schnitzler G., Deischel S. M., Goel M., Dove W., Jaacks K. J., Grossman A. D., Erickson J. W., Gross C. A. A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli. Microbiol Rev. 1989 Mar;53(1):1–24. doi: 10.1128/mr.53.1.1-24.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Smith C. L., Econome J. G., Schutt A., Klco S., Cantor C. R. A physical map of the Escherichia coli K12 genome. Science. 1987 Jun 12;236(4807):1448–1453. doi: 10.1126/science.3296194. [DOI] [PubMed] [Google Scholar]
  17. Umeda M., Ohtsubo E. Mapping of insertion element IS5 in the Escherichia coli K-12 chromosome. Chromosomal rearrangements mediated by IS5. J Mol Biol. 1990 May 20;213(2):229–237. doi: 10.1016/S0022-2836(05)80186-X. [DOI] [PubMed] [Google Scholar]
  18. Umeda M., Ohtsubo E. Mapping of insertion elements IS1, IS2 and IS3 on the Escherichia coli K-12 chromosome. Role of the insertion elements in formation of Hfrs and F' factors and in rearrangement of bacterial chromosomes. J Mol Biol. 1989 Aug 20;208(4):601–614. doi: 10.1016/0022-2836(89)90151-4. [DOI] [PubMed] [Google Scholar]
  19. VanBogelen R. A., Neidhardt F. C. The gene-protein database of Escherichia coli: edition 4. Electrophoresis. 1991 Nov;12(11):955–994. doi: 10.1002/elps.1150121114. [DOI] [PubMed] [Google Scholar]
  20. Wiegand T. W., Reznikoff W. S. Characterization of two hypertransposing Tn5 mutants. J Bacteriol. 1992 Feb;174(4):1229–1239. doi: 10.1128/jb.174.4.1229-1239.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ye S. Y., Hong G. F. Heat-stable DNA polymerase I large fragment resolves hairpin structure in DNA sequencing. Sci Sin B. 1987 May;30(5):503–506. [PubMed] [Google Scholar]
  22. Yura T., Mori H., Nagai H., Nagata T., Ishihama A., Fujita N., Isono K., Mizobuchi K., Nakata A. Systematic sequencing of the Escherichia coli genome: analysis of the 0-2.4 min region. Nucleic Acids Res. 1992 Jul 11;20(13):3305–3308. doi: 10.1093/nar/20.13.3305. [DOI] [PMC free article] [PubMed] [Google Scholar]

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