Generation of DNA nanocircles containing mismatched bases
The DNA mismatch repair (MMR) system recognizes and repairs errors that escaped the proofreading function of DNA polymerases. To study molecular details of the MMR mechanism, in vitro biochemical assays require specific DNA substrates carrying mismatches and strand discrimination signals. Current ap...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2011-10-01
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| Series: | BioTechniques |
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| Online Access: | https://www.future-science.com/doi/10.2144/000113749 |
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| author | Yu Xiao Caroline Jung Andreas D. Marx Ines Winkler Claire Wyman Joyce H.G. Lebbink Peter Friedhoff Michele Cristovao |
| author_facet | Yu Xiao Caroline Jung Andreas D. Marx Ines Winkler Claire Wyman Joyce H.G. Lebbink Peter Friedhoff Michele Cristovao |
| author_sort | Yu Xiao |
| collection | DOAJ |
| description | The DNA mismatch repair (MMR) system recognizes and repairs errors that escaped the proofreading function of DNA polymerases. To study molecular details of the MMR mechanism, in vitro biochemical assays require specific DNA substrates carrying mismatches and strand discrimination signals. Current approaches used to generate MMR substrates are time-consuming and/or not very flexible with respect to sequence context. Here we report an approach to generate small circular DNA containing a mismatch (nanocircles). Our method is based on the nicking of PCR products resulting in single-stranded 3′ overhangs, which form DNA circles after annealing and ligation. Depending on the DNA template, one can generate mismatched circles containing a single hemimethylated GATC site (for use with the bacterial system) and/or nicking sites to generate DNA circles nicked in the top or bottom strand (for assays with the bacterial or eukaryotic MMR system). The size of the circles varied (323 to 1100 bp), their sequence was determined by the template DNA, and purification of the circles was achieved by ExoI/ExoIII digestion and/or gel extraction. The quality of the nanocircles was assessed by scanning-force microscopy and their suitability for in vitro repair initiation was examined using recombinant Escherichia coli MMR proteins. |
| format | Article |
| id | doaj-art-b93fb702ff03422991d8d748fc709462 |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2011-10-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-b93fb702ff03422991d8d748fc7094622025-08-20T02:25:37ZengTaylor & Francis GroupBioTechniques0736-62051940-98182011-10-0151425926510.2144/000113749Generation of DNA nanocircles containing mismatched basesYu Xiao0Caroline Jung1Andreas D. Marx2Ines Winkler3Claire Wyman4Joyce H.G. Lebbink5Peter Friedhoff6Michele Cristovao71Institute of Biochemistry, Justus-Liebig University, Giessen, Germany1Institute of Biochemistry, Justus-Liebig University, Giessen, Germany1Institute of Biochemistry, Justus-Liebig University, Giessen, Germany1Institute of Biochemistry, Justus-Liebig University, Giessen, Germany2Department of Cell Biology and Genetics, Rotterdam, The Netherlands2Department of Cell Biology and Genetics, Rotterdam, The Netherlands1Institute of Biochemistry, Justus-Liebig University, Giessen, Germany2Department of Cell Biology and Genetics, Rotterdam, The NetherlandsThe DNA mismatch repair (MMR) system recognizes and repairs errors that escaped the proofreading function of DNA polymerases. To study molecular details of the MMR mechanism, in vitro biochemical assays require specific DNA substrates carrying mismatches and strand discrimination signals. Current approaches used to generate MMR substrates are time-consuming and/or not very flexible with respect to sequence context. Here we report an approach to generate small circular DNA containing a mismatch (nanocircles). Our method is based on the nicking of PCR products resulting in single-stranded 3′ overhangs, which form DNA circles after annealing and ligation. Depending on the DNA template, one can generate mismatched circles containing a single hemimethylated GATC site (for use with the bacterial system) and/or nicking sites to generate DNA circles nicked in the top or bottom strand (for assays with the bacterial or eukaryotic MMR system). The size of the circles varied (323 to 1100 bp), their sequence was determined by the template DNA, and purification of the circles was achieved by ExoI/ExoIII digestion and/or gel extraction. The quality of the nanocircles was assessed by scanning-force microscopy and their suitability for in vitro repair initiation was examined using recombinant Escherichia coli MMR proteins.https://www.future-science.com/doi/10.2144/000113749DNA mismatch repairnanocirclesstrand discrimination |
| spellingShingle | Yu Xiao Caroline Jung Andreas D. Marx Ines Winkler Claire Wyman Joyce H.G. Lebbink Peter Friedhoff Michele Cristovao Generation of DNA nanocircles containing mismatched bases BioTechniques DNA mismatch repair nanocircles strand discrimination |
| title | Generation of DNA nanocircles containing mismatched bases |
| title_full | Generation of DNA nanocircles containing mismatched bases |
| title_fullStr | Generation of DNA nanocircles containing mismatched bases |
| title_full_unstemmed | Generation of DNA nanocircles containing mismatched bases |
| title_short | Generation of DNA nanocircles containing mismatched bases |
| title_sort | generation of dna nanocircles containing mismatched bases |
| topic | DNA mismatch repair nanocircles strand discrimination |
| url | https://www.future-science.com/doi/10.2144/000113749 |
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