An oligo-swapping method: preparation of mismatch repair-monitoring substrate using a nicking endonuclease

Mismatch repair (MMR) contributes to accurate DNA replication by eliminating mismatched bases during DNA synthesis. Its importance is underscored by Lynch syndrome, a common hereditary colorectal cancer syndrome caused by MMR gene mutations. Therefore, quantification of MMR activity in human cells is important for diagnostic and therapeutic purposes. To monitor MMR, we established a novel DNA plasmid, pBluescript II NLS-MC-EGFP-tdTomato (pBET2). After processing using our protocol called the oligo swapping method, the mismatch included in the pBET2 enables us to evaluate the MMR proficiency of living cells by detecting specific fluorescent markers. Since the method simply "swaps" short single-stranded DNA to create a single mismatch on a specific site of the plasmid, it is significantly easier and more user-friendly. Briefly, the nicking endonuclease Nt.<i>Bbv</i>CI produces a short single-stranded DNA region in the pBET2. Swapping is achieved by filling the region with a mismatched oligonucleotide, and T4 DNA ligase seals the gap. To isolate the mismatch substrate, the restriction enzyme <i>Spe</i>I-HF and T5 exonuclease clean up all contaminants, such as non-mismatch and/or non-covalently closed circular DNA substrates. Finally, the nicking endonuclease Nt.<i>Bsp</i>QI induces a nick on the final mismatch substrates that enhances the MMR reaction specifically on the target strand. ● This experimental method allows for the easy preparation of mismatched DNA substrates, in contrast to conventionally complex methods. ● This method enables the specific and efficient evaluation of MMR activity in living cells by using the mismatch substrate, a fluorescent marker. ● This method is applicable not only to MMR but also to the preparation of substrates for other DNA repair pathways, making it a valuable resource for both <i>in vitro</i> and <i>in vivo</i> experiments.