Skip to main content

Flexible Genetic Engineering Using RecA Protein

  • Protocol
DNA Repair Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 152))

  • 495 Accesses

Abstract

The analysis or manipulation of small segments of DNA in plasmids has long been routine, but standard methods fail or are very time consuming when applied to long segments or complex mixtures of DNA. At present, RecA protein-based techniques usually have been used for making defined changes in large DNA constructs or for precise physical mapping of large regions of genomic DNA from several organisms. The techniques offer a rapid and versatile “cut and paste” approach to several other problems in molecular biology, and their use will probably increase as the goals of experiments with DNA and chromosomes evolve in sophistication. For example, they can now be used to analyze rearranged DNA in individual tumors or genetic deletions in patients with inherited or acquired syndromes. A new variation involving sequencespecific ligation may also eventually be used to rapidly screen libraries, label DNA fragments, or amplify large segments of DNA (1).

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 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
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. Ferrin, L. J. and Camerini-Otero, R. D. (1998) Sequence-specific ligation of DNA using RecA protein. Proc. Natl. Acad. Sci. USA 95, 2152–2157.

    Article  PubMed  CAS  Google Scholar 

  2. Ferrin, L. J. and Camerini-Otero, R. D. (1994) Long-range mapping of gaps and telomeres with RecA-assisted restriction endonuclease (RARE) cleavage. Nat. Genet. 6, 379–383.

    Article  PubMed  CAS  Google Scholar 

  3. National Institutes of Health and Institute of Molecular Medicine Collaboration. (1996) A complete set of human telomeric probes and their clinical application. Nat. Genet. 14, 86–89.

    Article  Google Scholar 

  4. Reston, J. T., Hu, X., Macina, R. A., et al. (1995) Structure of the terminal 300 kb of DNA from human chromosome 21q. Genomics 26, 31–38.

    Article  PubMed  CAS  Google Scholar 

  5. Feder, J. N., et al. (1996) A novel MHC class 1-like gene is mutated in patients with hereditary haemochromatosis. Nat. Genet. 13, 399–408.

    Article  PubMed  CAS  Google Scholar 

  6. Lauer, P., Schneider, S. S., and Gnirke, A. (1998) Construction and validation of yeast artificial chromosome contig maps by RecA-assisted restriction endonuclease cleavage. Proc. Natl. Acad. Sci. USA 95, 11,318–11,323.

    Article  PubMed  CAS  Google Scholar 

  7. Boren, J., Lee, I., Callow, M. J., et al. (1996) A simple and efficient method for making site-directed mutants, deletions, and fusions of large DNA such as P1 and BAC clones. Genome Res. 6, 1123–1130.

    Article  PubMed  CAS  Google Scholar 

  8. Nielsen, L. B., Kahn, D., Duell, T., et al. (1998) Apolipoprotein B gene expression in a series of human apolipoprotein B transgenic mice generated with recAassisted restriction endonuclease cleavage-modified bacterial artificial chromosomes. J. Biol. Chem. 273, 21,800–21,807.

    Article  PubMed  CAS  Google Scholar 

  9. McClelland, M. and Nelson, M. (1992) Effect of site-specific methylation on DNA modification methyltransferases and restriction endonucleases. Nucleic Acids Res. 20S, 2145–2157.

    Google Scholar 

  10. Smith, C. L., Klco, S., Zhang, T., et al. (1993) Analysis of megabase DNA using pulsed-field gel electrophoresis techniques. Methods in Molecular Genetics, Volume 2B (Adolph, K. W., ed.), Academic, San Diego, CA, pp. 155–194.

    Google Scholar 

  11. McClelland, M. (1987) Site-specific cleavage of DNA at 8-,9-, and 10-bp sequences. Methods in Enzymology, Volume 155 (Wu, R., ed.), Academic, San Diego, CA, pp. 22–32.

    Google Scholar 

  12. Carle, G. F. and Olson, M. V. (1987) Orthogonal-field-alternation gel electrophoresis, in Methods in Enzymology, Volume 155 (Wu, R., ed.), Academic, San Diego, CA, pp. 468–482.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2000 Humana Press Inc.

About this protocol

Cite this protocol

Ferrin, L.J. (2000). Flexible Genetic Engineering Using RecA Protein. In: Vaughan, P. (eds) DNA Repair Protocols. Methods in Molecular Biology™, vol 152. Humana Press. https://doi.org/10.1385/1-59259-068-3:135

Download citation

  • DOI: https://doi.org/10.1385/1-59259-068-3:135

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-643-7

  • Online ISBN: 978-1-59259-068-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics