Molecular Biotechnology

, Volume 15, Issue 2, pp 97–104 | Cite as

Preparation of DNA substrates for in vitro mismatch repair



Analyses in vitro of correction of DNA mismatches have been pivotal in biochemical dissection of mismatch repair pathways. However, the complex procedures needed to prepare DNA substrates for mismatch repair have posed substantial barriers to investigators who wish to pursue such analyses. Here we describe a simple, efficient way to prepare a variety of mismatched DNA substrates. We use in our procedure high-copy-number pUC19-derived plasmids, and a newly commercially available endonuclease N.BstNBI that makes site-specific single-strand nicks. The ability to prepare large substrate quantities in a relatively short time and to construct wider ranges of different mismatches in various sequence contexts will facilitate future research.

Index Entries

Mismatch repair substrate N.BstNBI 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Potter, D. (1999) Colorectal cancer: molecules and populations. J. Natl. Cancer Inst. 91, 916–932.PubMedCrossRefGoogle Scholar
  2. 2.
    Branch, P., Aquilina, G., Bignami, M., and Karran, P. (1993) Defective mismatch binding and a mutator phenotype in cells tolerant to DNA damage. Nature 362, 652–654.PubMedCrossRefGoogle Scholar
  3. 3.
    Kat, A., Thilly, W. G., Fang, W.-H., Longley, M.J., Li, G.-M., and Modrich, P. (1993) An alkylation-tolerant, mutator human cell line is deficient in strand-specific mismatch repair. Proc. Natl. Acad. Sci. USA 90, 6424–6428.PubMedCrossRefGoogle Scholar
  4. 4.
    Koi, M., Umar, A., Chauhan, D., Cherian, S. P., Carethers, J. M., Kunkel, T. A. and Boland, C. R. (1994) Human chromosome 3 corrects mismatch repair deficiency and microsatellite instability and reduces N-methyl-N′-nitro-N-nitrososguanidine tolerance in colon tumor cells with homozygous hMLH1 mutation. Cancer Res. 54, 4308–4312.PubMedGoogle Scholar
  5. 5.
    Risinger, J. I., Umar, A., Barrett, J. C., and Kunkel, T. A. (1995) A hPMS2 mutant cell line is defective in strand-specific mismatch repair. J. Biol. Chem. 270, 18183–18186.PubMedCrossRefGoogle Scholar
  6. 6.
    Aquilina, G., Hess, P., Fiumicino, S., Ceccotti, S. and Bignami, M. (1993) A mutator phenotype characterizes one of two complementation groups in human cells tolerant to methylation damage. Cancer Res. 55, 2569–2575.Google Scholar
  7. 7.
    Hampson, R., Humbert, O., MacPherson, P., Aquilina, G., and Karran, P. (1997) Mismatch repair defects and 0 6-methylguanine-DNA methyltransferase expression in acquired resistance to methylating agents in human cells. J. Biol. Chem. 272, 28596–28606.PubMedCrossRefGoogle Scholar
  8. 8.
    Umar, A., Koi, M., Risinger, J. I., Glaab, W. E., Tindall, K. R., Kolodner, R. D., et al. (1997) Correction of hypermutability, MNNG resistance and defective DNA mismatch repair by introducing chromosome 2 into tumor cells with mutations in MSH2 and MSH6. Cancer Res. 57, 3949–3955.PubMedGoogle Scholar
  9. 9.
    Corrette-Bennett, S. E. and Lahue, R. S. (1999) Mismatch repair assay. In DNA Repair Protocols: Eukaryotic Systems (Henderson, D.S., ed), Totowa, NJ; Humana Press, pp 121–132.CrossRefGoogle Scholar
  10. 10.
    Holmes, J., Clark, S., and Modrich, P. (1990) Strand-specific mismatch correction in nuclear extracts of human and Drosophila melanogaster cell lines. Proc. Natl. Acad. Sci. USA 87, 5837–5841.PubMedCrossRefGoogle Scholar
  11. 11.
    Modrich, P. (1997) Strand-specific mismatch repair in mammalian cells. J. Biol. Chem. 272, 24727–24730.PubMedCrossRefGoogle Scholar
  12. 12.
    Lanue, R. S. and Modrich, F. (1989) DNA mismatch correction in a defined system. Science 245, 160–164CrossRefGoogle Scholar
  13. 13.
    Parsons, R., Li, G-M., Longley, M. J., Fang, W-H., Rapadopoulos, N., Jen, J., et al. (1993) Hypermutability and mismatch repair deficiency in RER+ tumor cells. Cell 75, 1227–1236PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 2000

Authors and Affiliations

  1. 1.Department of Environmental and Molecular ToxicologyOregon State UniversityCorvallis

Personalised recommendations