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The Isolation of DNA Sequences Flanking Tn5 Transposon Insertions by Inverse PCR

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PCR Cloning Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 192))

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Abstract

Because of its versatility, the Tn5 transposon has become a powerful tool in the classical genetic studies of Gram-negative bacteria. The Tn5 transposon is functional in a broad range of Gram-negative bacteria and transposes at a high frequency with low specificity of insertion (1,2), allowing it to insert in a large number of locations in bacterial genomes. The initial use of the Tn5 transposon was in the identification of disrupted genes by insertion muatgenesis and phenotypic screening, however, derivatives of this mobile element were later developed for use in reporter gene fusion (3) and promoter probing experiments (4).

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References

  1. Goryshin, I. Y., Miller, J. A., Kil, Y. V., Lanzov, V. A., and Reznikoff, W. S. (1998) Tn5/ IS50 target recognition. Proc. Natl. Acad. Sci. USA 95, 10,716–10,721.

    Article  CAS  PubMed  Google Scholar 

  2. Berg, D. E. (1989) Transposon Tn5 in Mobile DNA, (Berg, D. E. and Howe, M. M., eds.), American Society for Microbiology, WA, pp. 163–184.

    Google Scholar 

  3. De Lorenzo, V., Herrero, M., Jakubzik, U., and Timmis, K. N. (1990) Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J. Bacteriol. 172, 6568–6572.

    PubMed  Google Scholar 

  4. Simon, R., Ouandt, J., and Klipp, W. (1989) New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in Gram-negative bacteria. Gene 80, 161–170.

    Article  CAS  PubMed  Google Scholar 

  5. DeBruijn, F., J. and Rossbach, S. (1993) Transposon mutagenesis in Methods for General and Molecular Bacteriology, (Gerhardt, P., ed.), American Society for Microbiology, WA, pp. 387–405.

    Google Scholar 

  6. Dennis, J. J. and Zylstra, G. J. (1998) Plasposons: Modular self-cloning minitransposon derivatives for rapid genetic analysis of gram-negative bacterial genomes. Appl. Environ. Microbiol. 64, 2710–2715.

    CAS  PubMed  Google Scholar 

  7. Hozbor, D. F., Pich Otero, A. J. L., Wynne, M. E., Petruccelli, S., and Lagares, A. (1998) Recovery of Tn5-flanking bacterial DNA by vector-mediated walking from the transposon to the host genome. Analyt. Biochem. 259, 286–288.

    Article  CAS  PubMed  Google Scholar 

  8. Ribot, E. M., Quinn, F. D., Bai, X., and Murtagh, J. J. (1998) Rapid amplification of transposon ends for the isolation, cloning and sequencing of transposon-disrupted chromosomal genes. Biotechniques 24, 16–22.

    CAS  PubMed  Google Scholar 

  9. Prod’hom, G., Lagier, B., Pelicic, V., Hance, A. J., Gicquel, B., and Guilhot, C. (1998) A reliable amplification technique for the characterization of genomic DNA sequences flanking insertion sequences. FEMS Microbiol. Lett. 158, 75–81.

    Article  CAS  Google Scholar 

  10. Karlyshev, A. V., Pallen, M. J., and Wren, B. W. (2000) Single-primer PCR procedure for rapid identification of transposon insertion sites. Biotechniques 28, 1078–1081.

    CAS  PubMed  Google Scholar 

  11. Subramanian, P. S., Versalovic, J., McCabe, E. R., and Lupski, J. R. (1992) Rapid mapping of Escherichia coli:Tn5 insertion mutations by REP-Tn5 PCR. PCR Meth. Appl. 1, 187–192.

    CAS  Google Scholar 

  12. Rich, J. J. and Willis, D. K. (1990) A single oligonucleotide can be used to rapidly isolate DNA sequences flanking a transposon Tn5 insertion by the polymerase chain reaction. Nucl. Acid Res. 18, 6673–6676.

    Article  CAS  Google Scholar 

  13. Martin, V. J. J. and Mohn, W. W. (1999) An alternative inverse PCR (IPCR) method to amplify DNA sequences flanking Tn5 transposon insertions. J. Microbiol. Meth. 35, 163–166.

    Article  CAS  Google Scholar 

  14. Huang, G., Zhang, L., and Birch, R. G. (2000) Rapid amplification and cloning of Tn5 flanking fragments by inverse PCR. Lett. Appl. Microbiol. 31, 149–153.

    Article  CAS  PubMed  Google Scholar 

  15. Sambrook, J. and Russell, D. W. (2001) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press.

    Google Scholar 

  16. Upcroft, P. and Healey, A. (1987) Rapid and efficient method for cloning of blunt-ended DNA fragments. Gene 51, 69–76.

    Article  CAS  PubMed  Google Scholar 

  17. Mitchell, D. and Smit, J. (1990) Identification of genes affecting production of the adhesion organelle of Caulobacter crescentus CB2. J. Bacteriol. 172, 5425–5431.

    CAS  PubMed  Google Scholar 

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Martin, V.J.J., Mohn, W.W. (2002). The Isolation of DNA Sequences Flanking Tn5 Transposon Insertions by Inverse PCR. In: Chen, BY., Janes, H.W. (eds) PCR Cloning Protocols. Methods in Molecular Biology™, vol 192. Humana Press. https://doi.org/10.1385/1-59259-177-9:315

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  • DOI: https://doi.org/10.1385/1-59259-177-9:315

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-969-8

  • Online ISBN: 978-1-59259-177-0

  • eBook Packages: Springer Protocols

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