Construction of MMR Plasmid Substrates and Analysis of MMR Error Correction and Excision

  • Huixian Wang
  • John B. Hays
Part of the Methods in Molecular Biology™ book series (MIMB, volume 314)


We describe simple and efficient construction of mismatch repair (MMR) substrates, by generation of gapped plasmids using one sequence-specific nicking endonuclease (N.BstNBI), ligation of synthetic oligomers into the gaps, and introduction of defined single nicks for initiation of MMR excision using a second such endonuclease (N.AlwI). We further describe measurement of completed mismatch correction and a sensitive quantitative assay for MMR excision intermediates. These methods can be easily adapted for construction of substrates containing defined DNA lesions, for analysis of MMR responses to DNA damage and for studies of other DNA repair pathways.

Key Words

DNA lesion error correction excision mismatch repair N.AlwN.BstNBI 


  1. 1.
    Wang, H. and Hays, J. B. (2002) Mismatch repair in human nuclear extracts: Quantitative analysis of excision of nicked mismatched DNA substrates, constructed by a new technique employing synthetic oligonucleotides. J. Biol. Chem. 277, 26,136–26,142.PubMedCrossRefGoogle Scholar
  2. 2.
    Seidman, C. E., Sheen, J., and Jessen, T. (1998) Introduction of plasmid DNA into cells, in: Current Protocols in Molecular Biology. Vol. 1, pp. 1.8.1–1.8.10.Google Scholar
  3. 3.
    Heilig, J. S., Elbing, K. L., and Brent, R. (1998) Large-scale preparation of plasmid DNA, in: Current Protocols in Molecular Biology. Vol. 1, pp. 1.7.1–1.7.16.Google Scholar
  4. 4.
    Gillam, I., Millward, S., Blew, D., von Tigerstrom, M., Wimmer, E., and Tenner, G. M. (1967) The separation of soluble ribonucleic acids on benzoylated diethylaminoethyl cellulose. Biochemistry 6, 3043–3056.PubMedCrossRefGoogle Scholar
  5. 5.
    Challberg, M. D. and Kelly, T. J. (1979) Adenovirus DNA replication in vitro: origin and direction of Daughter strand synthesis. J Mol Biol. 135, 999–1012.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Huixian Wang
    • 1
  • John B. Hays
    • 1
  1. 1.Department of Environmental and Molecular ToxicologyOregon State UniversityCorvallisOR

Personalised recommendations