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.
Wang
H
H
Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis 97331, USA.
Hays
J B
JB
D001482:Base Composition; D020137:Base Pair Mismatch; D001483:Base Sequence; D004247:DNA / Q000032:analysis / Q000235:genetics*; D004260:DNA Repair; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D017353:Gene Deletion; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics*; D010957:Plasmids
D009692:Nucleic Acid Heteroduplexes; D004247:DNA; D004262:DNA Restriction Enzymes