A rapid, simple DNA mismatch repair substrate construction method
A more flexible and higher yielding in vitro DNA mismatch repair (MMR) substrate construction method, which was developed initially by Wang and Hays, is described for the construction of a nucleotide-based chemical mismatch (G/IU) and a G/T mismatch. Our modifications use the combination of two endo...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2011-06-01
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| Series: | Frontiers in Oncology |
| Subjects: | |
| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fonc.2011.00008/full |
| _version_ | 1818334495071272960 |
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| author | Timothy James Kinsella Timothy James Kinsella Weinan eDu |
| author_facet | Timothy James Kinsella Timothy James Kinsella Weinan eDu |
| author_sort | Timothy James Kinsella |
| collection | DOAJ |
| description | A more flexible and higher yielding in vitro DNA mismatch repair (MMR) substrate construction method, which was developed initially by Wang and Hays, is described for the construction of a nucleotide-based chemical mismatch (G/IU) and a G/T mismatch. Our modifications use the combination of two endonuclease enzymes (NheI and BciVI) and two new redesigned plasmids (pWDAH1A and pWDSH1B). In our modified methodology, plasmids are initially digested with the nicking endonucleases, followed by the streptavidin treatment. The mismatch-containing oligo is then annealed to the gap DNA and finally ligated to produce a mismatch-containing DNA substrate. We report a high ligation efficiency (up to 90%) of these mismatch substrates and confirm recognition using a functional assay. These modifications, coupled with the use of the redesigned plasmids, can be applied for the construction of other types of chemically induced mismatches as well as insertion-deletion loops (IDL) for future in vitro studies of MMR processing by our group and others. |
| first_indexed | 2024-12-13T14:08:26Z |
| format | Article |
| id | doaj.art-928320bbef314f74abce1f49e5fc31a4 |
| institution | Directory Open Access Journal |
| issn | 2234-943X |
| language | English |
| last_indexed | 2024-12-13T14:08:26Z |
| publishDate | 2011-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Oncology |
| spelling | doaj.art-928320bbef314f74abce1f49e5fc31a42022-12-21T23:42:32ZengFrontiers Media S.A.Frontiers in Oncology2234-943X2011-06-01110.3389/fonc.2011.0000811205A rapid, simple DNA mismatch repair substrate construction methodTimothy James Kinsella0Timothy James Kinsella1Weinan eDu2Rhode Island HospitalWarren Alpert Medical School of Brown UniversityCase Western Reserve UniversityA more flexible and higher yielding in vitro DNA mismatch repair (MMR) substrate construction method, which was developed initially by Wang and Hays, is described for the construction of a nucleotide-based chemical mismatch (G/IU) and a G/T mismatch. Our modifications use the combination of two endonuclease enzymes (NheI and BciVI) and two new redesigned plasmids (pWDAH1A and pWDSH1B). In our modified methodology, plasmids are initially digested with the nicking endonucleases, followed by the streptavidin treatment. The mismatch-containing oligo is then annealed to the gap DNA and finally ligated to produce a mismatch-containing DNA substrate. We report a high ligation efficiency (up to 90%) of these mismatch substrates and confirm recognition using a functional assay. These modifications, coupled with the use of the redesigned plasmids, can be applied for the construction of other types of chemically induced mismatches as well as insertion-deletion loops (IDL) for future in vitro studies of MMR processing by our group and others.http://journal.frontiersin.org/Journal/10.3389/fonc.2011.00008/fullmismatch repairiododeoxyuridinemismatch substrates construction |
| spellingShingle | Timothy James Kinsella Timothy James Kinsella Weinan eDu A rapid, simple DNA mismatch repair substrate construction method Frontiers in Oncology mismatch repair iododeoxyuridine mismatch substrates construction |
| title | A rapid, simple DNA mismatch repair substrate construction method |
| title_full | A rapid, simple DNA mismatch repair substrate construction method |
| title_fullStr | A rapid, simple DNA mismatch repair substrate construction method |
| title_full_unstemmed | A rapid, simple DNA mismatch repair substrate construction method |
| title_short | A rapid, simple DNA mismatch repair substrate construction method |
| title_sort | rapid simple dna mismatch repair substrate construction method |
| topic | mismatch repair iododeoxyuridine mismatch substrates construction |
| url | http://journal.frontiersin.org/Journal/10.3389/fonc.2011.00008/full |
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