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Improved methods for creating migratable Holliday junction substrates
Previously, we published a method for creating a novel DNA substrate, the double Holliday junction substrate. This substrate contains two Holliday junctions that are mobile, topologically constrained and separated by a distance comparable with conversion tract lengths. Although useful for studying l...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597647/ https://www.ncbi.nlm.nih.gov/pubmed/23275569 http://dx.doi.org/10.1093/nar/gks1343 |
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author | Chen, Stefanie Hartman Plank, Jody L. Willcox, Smaranda Griffith, Jack D. Hsieh, Tao-shih |
author_facet | Chen, Stefanie Hartman Plank, Jody L. Willcox, Smaranda Griffith, Jack D. Hsieh, Tao-shih |
author_sort | Chen, Stefanie Hartman |
collection | PubMed |
description | Previously, we published a method for creating a novel DNA substrate, the double Holliday junction substrate. This substrate contains two Holliday junctions that are mobile, topologically constrained and separated by a distance comparable with conversion tract lengths. Although useful for studying late stage homologous recombination in vitro, construction of the substrate requires significant effort. In particular, there are three bottlenecks: (i) production of large quantities of single-stranded DNA; (ii) the loss of a significant portion of the DNA following the recombination step; and (iii) the loss of DNA owing to inefficient gel extraction. To address these limitations, we have made the following changes to the protocol: (i) use of a helper plasmid, rather than exogenous helper phage, to produce single-stranded DNA; (ii) use of the unidirectional ϕC31 integrase system in place of the bidirectional Cre recombinase reaction; and (iii) gel extraction by DNA diffusion. Here, we describe the changes made to the materials and methods and characterize the substrates that can be produced, including migratable single Holliday junctions, hemicatenanes and a quadruple Holliday junction substrate. |
format | Online Article Text |
id | pubmed-3597647 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-35976472013-03-15 Improved methods for creating migratable Holliday junction substrates Chen, Stefanie Hartman Plank, Jody L. Willcox, Smaranda Griffith, Jack D. Hsieh, Tao-shih Nucleic Acids Res Methods Online Previously, we published a method for creating a novel DNA substrate, the double Holliday junction substrate. This substrate contains two Holliday junctions that are mobile, topologically constrained and separated by a distance comparable with conversion tract lengths. Although useful for studying late stage homologous recombination in vitro, construction of the substrate requires significant effort. In particular, there are three bottlenecks: (i) production of large quantities of single-stranded DNA; (ii) the loss of a significant portion of the DNA following the recombination step; and (iii) the loss of DNA owing to inefficient gel extraction. To address these limitations, we have made the following changes to the protocol: (i) use of a helper plasmid, rather than exogenous helper phage, to produce single-stranded DNA; (ii) use of the unidirectional ϕC31 integrase system in place of the bidirectional Cre recombinase reaction; and (iii) gel extraction by DNA diffusion. Here, we describe the changes made to the materials and methods and characterize the substrates that can be produced, including migratable single Holliday junctions, hemicatenanes and a quadruple Holliday junction substrate. Oxford University Press 2013-03 2012-12-26 /pmc/articles/PMC3597647/ /pubmed/23275569 http://dx.doi.org/10.1093/nar/gks1343 Text en © The Author(s) 2012. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Online Chen, Stefanie Hartman Plank, Jody L. Willcox, Smaranda Griffith, Jack D. Hsieh, Tao-shih Improved methods for creating migratable Holliday junction substrates |
title | Improved methods for creating migratable Holliday junction substrates |
title_full | Improved methods for creating migratable Holliday junction substrates |
title_fullStr | Improved methods for creating migratable Holliday junction substrates |
title_full_unstemmed | Improved methods for creating migratable Holliday junction substrates |
title_short | Improved methods for creating migratable Holliday junction substrates |
title_sort | improved methods for creating migratable holliday junction substrates |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597647/ https://www.ncbi.nlm.nih.gov/pubmed/23275569 http://dx.doi.org/10.1093/nar/gks1343 |
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