Preparation of DNA substrates for in vitro mismatch repair
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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 EntriesMismatch repair substrate N.BstNBI
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- 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
- 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
- 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