Molecular Biotechnology

, Volume 15, Issue 2, pp 97–104

Preparation of DNA substrates for in vitro mismatch repair

Research

DOI: 10.1385/MB:15:2:97

Cite this article as:
Wang, H. & Hays, J.B. Mol Biotechnol (2000) 15: 97. doi:10.1385/MB:15:2:97

Abstract

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 repairsubstrateN.BstNBI

Copyright information

© Humana Press Inc 2000

Authors and Affiliations

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