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: A) Direct annealing method. Mismatch substrates are prepared by annealing linearized double‐stranded plasmid DNA ( e.g. replicative form of M13 phage or phagemid) and single‐stranded circular DNA ( e.g. M13 circular ssDNA or phagemid ssDNA) in the presence of high formamide concentration. This reaction mixture is dialyzed while dropping from a high to progressively lower formamide concentrations. Nicked circular molecules are purified via agarose gel electrophoresis and electroelution. B) In vitro mutagenesis method. Mismatch substrates are prepared by annealing synthetic oligonucleotides and single‐stranded circular DNA. After annealing, DNA polymerase and DNA ligase synthesize and seal the new DNA strand, resulting in a circular DNA containing the single mismatch. Closed circular DNA is isolated via CsCl/EtBr density gradient centrifugation. C) Oligo-swapping method. Nicking endonuclease generates a region of ssDNA in plasmid. Annealing with synthetic oligonucleotides, DNA ligase seals it to produce closed circular DNA. Treatment with restriction enzyme and T5 exonuclease isolates the closed circular DNA containing the mismatch.

Article Snippet: 10x restriction endonuclease buffer (10X rCutSmartTM buffer: 500 mM potassium acetate, 200 mM Tris-acetate, 100 mM magnesium acetate, 1000 μg/mL recombinant albumin [pH 7.9 @ 25 °C]) 25 mM ATP: 100 mM Adenosine 5′-Triphosphate (ATP) Disodium Solution (Fuji Wako) is diluted with H2O T4 DNA ligase: 400 units/μL (Takara Bio) SpeI-HF: 20,000 units/ml (NEB) T5 exonuclease: 10,000 units/ml (NEB) PCR purification kit (MACHEREY-NAGEL) Agarose-ME classic type (Nacalai Tesque) Ethidium Bromide Solution (EtBr; 10 mg/mL; Nacalai Tesque) 50x TAE buffer (40 mM Tris, 20 mM acetic acid, and 0.4 mM EDTA; Kanto Chemical) Gel Loading Dye, Purple (6X) (NEB) T4 polynucleotide kinase: 10,000 units/mL (Takara Bio) Nt.BspQI: 10,000 units/mL (NEB) 2.

Techniques: Plasmid Preparation, Concentration Assay, Purification, Agarose Gel Electrophoresis, In Vitro, Mutagenesis, Isolation, Gradient Centrifugation

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: 10x restriction endonuclease buffer (10X rCutSmartTM buffer: 500 mM potassium acetate, 200 mM Tris-acetate, 100 mM magnesium acetate, 1000 μg/mL recombinant albumin [pH 7.9 @ 25 °C]) 25 mM ATP: 100 mM Adenosine 5′-Triphosphate (ATP) Disodium Solution (Fuji Wako) is diluted with H2O T4 DNA ligase: 400 units/μL (Takara Bio) SpeI-HF: 20,000 units/ml (NEB) T5 exonuclease: 10,000 units/ml (NEB) PCR purification kit (MACHEREY-NAGEL) Agarose-ME classic type (Nacalai Tesque) Ethidium Bromide Solution (EtBr; 10 mg/mL; Nacalai Tesque) 50x TAE buffer (40 mM Tris, 20 mM acetic acid, and 0.4 mM EDTA; Kanto Chemical) Gel Loading Dye, Purple (6X) (NEB) T4 polynucleotide kinase: 10,000 units/mL (Takara Bio) Nt.BspQI: 10,000 units/mL (NEB) 2.

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

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: A) Direct annealing method. Mismatch substrates are prepared by annealing linearized double‐stranded plasmid DNA ( e.g. replicative form of M13 phage or phagemid) and single‐stranded circular DNA ( e.g. M13 circular ssDNA or phagemid ssDNA) in the presence of high formamide concentration. This reaction mixture is dialyzed while dropping from a high to progressively lower formamide concentrations. Nicked circular molecules are purified via agarose gel electrophoresis and electroelution. B) In vitro mutagenesis method. Mismatch substrates are prepared by annealing synthetic oligonucleotides and single‐stranded circular DNA. After annealing, DNA polymerase and DNA ligase synthesize and seal the new DNA strand, resulting in a circular DNA containing the single mismatch. Closed circular DNA is isolated via CsCl/EtBr density gradient centrifugation. C) Oligo-swapping method. Nicking endonuclease generates a region of ssDNA in plasmid. Annealing with synthetic oligonucleotides, DNA ligase seals it to produce closed circular DNA. Treatment with restriction enzyme and T5 exonuclease isolates the closed circular DNA containing the mismatch.

Article Snippet: 10x restriction endonuclease buffer (10X rCutSmartTM buffer: 500 mM potassium acetate, 200 mM Tris-acetate, 100 mM magnesium acetate, 1000 μg/mL recombinant albumin [pH 7.9 @ 25 °C]) 25 mM ATP: 100 mM Adenosine 5′-Triphosphate (ATP) Disodium Solution (Fuji Wako) is diluted with H2O T4 DNA ligase: 400 units/μL (Takara Bio) SpeI-HF: 20,000 units/ml (NEB) T5 exonuclease: 10,000 units/ml (NEB) PCR purification kit (MACHEREY-NAGEL) Agarose-ME classic type (Nacalai Tesque) Ethidium Bromide Solution (EtBr; 10 mg/mL; Nacalai Tesque) 50x TAE buffer (40 mM Tris, 20 mM acetic acid, and 0.4 mM EDTA; Kanto Chemical) Gel Loading Dye, Purple (6X) (NEB) T4 polynucleotide kinase: 10,000 units/mL (Takara Bio) Nt.BspQI: 10,000 units/mL (NEB) 2.

Techniques: Plasmid Preparation, Concentration Assay, Purification, Agarose Gel Electrophoresis, In Vitro, Mutagenesis, Isolation, Gradient Centrifugation

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: 10x restriction endonuclease buffer (10X rCutSmartTM buffer: 500 mM potassium acetate, 200 mM Tris-acetate, 100 mM magnesium acetate, 1000 μg/mL recombinant albumin [pH 7.9 @ 25 °C]) 25 mM ATP: 100 mM Adenosine 5′-Triphosphate (ATP) Disodium Solution (Fuji Wako) is diluted with H2O T4 DNA ligase: 400 units/μL (Takara Bio) SpeI-HF: 20,000 units/ml (NEB) T5 exonuclease: 10,000 units/ml (NEB) PCR purification kit (MACHEREY-NAGEL) Agarose-ME classic type (Nacalai Tesque) Ethidium Bromide Solution (EtBr; 10 mg/mL; Nacalai Tesque) 50x TAE buffer (40 mM Tris, 20 mM acetic acid, and 0.4 mM EDTA; Kanto Chemical) Gel Loading Dye, Purple (6X) (NEB) T4 polynucleotide kinase: 10,000 units/mL (Takara Bio) Nt.BspQI: 10,000 units/mL (NEB) 2.

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