Skip to main content

PCR Screening in Signature-Tagged Mutagenesis of Essential Genes

  • Protocol
Book cover PCR Cloning Protocols

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

  • 3670 Accesses

Abstract

Signature tagged-mutagenesis (STM) is a functional genomics technique that identifies microbial genes required for infection within an animal host, or within host cell (1,2). As first described by Hensel et al., 1995 (3), transposon mutants are generated and each one tagged with a unique DNA sequence. Originally, STM used comparative hybridization to isolate mutants unable to survive in specified environmental conditions and to identify genes critical for survival in the host (3). The original STM has been modified to use defined oligonucleotides for tag construction into mini-Tn5 and to use polymerase chain reaction (PCR) instead of hybridization for rapid screening of bacterial mutants in vivo (4). The modified STM technique has been called PCR-based signature-tagged mutagenesis (PBSTM).

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Shea, J. E., Santangelo, J. D., and Feldman, R. G. (2000) Signature-tagged mutagenesis in the identification of virulence genes in pathogens. Curr. Opin. Microbiol. 3, 451–458.

    Article  CAS  PubMed  Google Scholar 

  2. Lehoux, D. E. and Levesque, R. C. (2000) Detection of genes essential in specific niches by signature-tagged mutagenesis. Curr. Opin. Biotechnol. 11, 434–439.

    Article  CAS  PubMed  Google Scholar 

  3. Hensel, M., Shea, J. E., Gleeson, C., Jones, M. D., Dalton, E., and Holden, D. W. (1995) Simultaneous identification of bacterial virulence genes by negative selection. Science 269, 400–403.

    Article  CAS  PubMed  Google Scholar 

  4. Lehoux, D. E., Sanschagrin, F., and Levesque, R. C. (1999) Defined oligonucleotide tag pools and PCR screening in signature-tagged mutagenesis of essential genes from bacteria. Biotechniques 26, 473–478, 480.

    CAS  PubMed  Google Scholar 

  5. Chiang, S. L., Mekalanos, J. J., and Holden, D. W. (1999) In vivo genetic analysis of bacterial virulence. Annu. Rev. Microbiol. 53, 129–154.

    Article  CAS  PubMed  Google Scholar 

  6. Dieffenbach, W. C. and Dveksler, G. S., eds. (1995) PCR Primer: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p. 714.

    Google Scholar 

  7. Breslauer, K. J., Frank, R., Blocker, H., and Marky, L. A. (1986) Predicting DNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. USA 83, 3746–3750.

    Article  CAS  PubMed  Google Scholar 

  8. Rychlik, W. (1993) Selection of primer chain reaction, in PCR protocols: current application, W.H. Press, Editor.: Totowa NJ.

    Google Scholar 

  9. Kwok, S., Kellogg, D. E., McKinney, N., Spasic, D., Goda, L., Levenson, C., and Sninsky, J. J. (1990) Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucl. Acids Res. 18, 999–1005.

    Article  CAS  PubMed  Google Scholar 

  10. 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 

  11. Simon, R., Priefer, U., and Pühler, A. (1983) A broad range mobilization system for in vitro genetic engineering: transposon mutagenesis in gram negative bacteria. Bio/Technology 1, 784–791.

    Article  CAS  Google Scholar 

  12. Sambrook, J., Fritsch, E. F., and Maniatis, T., (eds) (1989) Molecular Cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

  13. Dewar, K., Sabbagh, L., Cardinal, G., Veilleux, F., Sanschagrin, F., Birren, B., and Levesque, R. C. (1998) Pseudomonas aeruginosa PAO1 bacterial artificial chromosomes: strategies for mapping, screening, and sequencing 100 kb loci of the 5.9 Mb genome. Microb. Comp. Genom. 3, 105–117.

    CAS  Google Scholar 

  14. Darwin, A. J. and Miller, V. L. (1999) Identification of Yersinia enterocolitica genes affecting survival in an animal host using signature-tagged transposon mutagenesis. Mol. Microbiol. 32, 51–62.

    Article  CAS  PubMed  Google Scholar 

  15. Camacho, L. R., Ensergueix, D., Perez, E., Gicquel, B., and Guilhot, C. (1999) Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon mutagenesis. Mol. Microbiol. 34, 257–267.

    Article  CAS  PubMed  Google Scholar 

  16. Chiang, S. L. and Mekalanos, J. J. (1998) Use of signature-tagged transposon mutagenesis to identify Vibrio cholerae genes critical for colonization. Mol. Microbiol. 27, 797–805.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2002 Humana Press Inc.

About this protocol

Cite this protocol

Lehoux, D.E., Levesque, R.C. (2002). PCR Screening in Signature-Tagged Mutagenesis of Essential Genes. In: Chen, BY., Janes, H.W. (eds) PCR Cloning Protocols. Methods in Molecular Biology™, vol 192. Humana Press. https://doi.org/10.1385/1-59259-177-9:225

Download citation

  • DOI: https://doi.org/10.1385/1-59259-177-9:225

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-969-8

  • Online ISBN: 978-1-59259-177-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics