Analysis of Mismatch Repair in Human Nuclear Extracts

This unit describes an assay for completed mismatch correction along with a precise and sensitive assay for mismatch-repair excision intermediates and a simple and efficient procedure for construction of mismatch repair substrates. These substrates are derived by ligating synthetic oligomers into a gapped plasmid generated using the sequence-specific N.BstNBI nicking endonuclease, then the sequence-specific nicking endonuclease N.AlwI, to introduce single nicks for initiation of excision. These methods can be easily adapted for construction and analysis of DNA-lesion-containing substrates to study DNA-repair pathways other than mismatch repair. Keywords: mismatch repair; error correction; excision; N.AlwI; N.BstNBI