Journal: MethodsX

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

doi: 10.1016/j.mex.2025.103715

Figure Lengend Snippet: Schematic diagram of the mismatch substrate pBET2 C/A synthesized from the reporter plasmid pBET2 by the oligo-swapping method. The plasmid DNA, pBET2 (for sequence, see Addgene ID 240,411), was designed with two nicking sites for the endonuclease Nt. Bbv CI to enable the oligo-swapping method. Using this method, the mismatched substrate pBET2 C/A is synthesized from pBET2. Successful repair of the C/A mismatch via MMR in living cells results in the detection of GFP protein within the nucleus. The co-expressed dtTOMATO protein is used as a control to confirm successful transfection.

Article Snippet: The plasmid DNA, pBET2 (for sequence, see Addgene ID 240,411), was designed with two nicking sites for the endonuclease Nt.

Techniques: Synthesized, Plasmid Preparation, Sequencing, Control, Transfection

Journal: MethodsX

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

doi: 10.1016/j.mex.2025.103715

Figure Lengend Snippet: Aliquots from various steps of the purification were analyzed on 0.8 % agarose gel, and the DNA substrates were visualized via EtBr staining. A) Lane 1, pBET2 (Method details, step 1); lane 2, Nt. Bbv CI-treatment (Method details, step 2); lane 3, T4 DNA ligase-treatment (Method details, step 4); lane 4, Spe I-HF- treatment (Method details, step 5); and lane 5, T5 exonuclease-treatment (Method details, step 6). Open circular DNA (OC), linear DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrows. B) DNA conformation in each lane. In lane 1, the purified pBET2 plasmid is a closed circular DNA. In lane 2, gapped pBET2 is an open circular DNA. In lane 3, gapped and non-reacted DNA are open circular DNA, while mismatch and non-mismatch DNA are closed circular DNA. In lane 4, nicked DNA and non-mismatch DNA are digested with Spe I-HF, resulting in linear DNA. In lane 5, the subsequent step entails the removal of linear and gapped DNA by T5 exonuclease to isolate mismatch DNA, pBET2 C/A. A single nick site for MMR s introduced. In lane 6, pBET2 C/A is purified using a standard PCR purification kit. In lane 7, the DNA is digested by a nicking endonuclease, forming open circular DNA.

Article Snippet: The plasmid DNA, pBET2 (for sequence, see Addgene ID 240,411), was designed with two nicking sites for the endonuclease Nt.

Techniques: Purification, Agarose Gel Electrophoresis, Staining, Plasmid Preparation