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A 96-well DNase I footprinting screen for drug–DNA interactions
The established protocol for DNase I footprinting has been modified to allow multiple parallel reactions to be rapidly performed in 96-well microtitre plates. By scrutinizing every aspect of the traditional method and making appropriate modifications it has been possible to considerably reduce the t...
Autores principales: | , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2007
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1919508/ https://www.ncbi.nlm.nih.gov/pubmed/17586817 http://dx.doi.org/10.1093/nar/gkm467 |
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author | Ellis, Tom Evans, David A. Martin, Christopher R. H. Hartley, John A. |
author_facet | Ellis, Tom Evans, David A. Martin, Christopher R. H. Hartley, John A. |
author_sort | Ellis, Tom |
collection | PubMed |
description | The established protocol for DNase I footprinting has been modified to allow multiple parallel reactions to be rapidly performed in 96-well microtitre plates. By scrutinizing every aspect of the traditional method and making appropriate modifications it has been possible to considerably reduce the time, risk of sample loss and complexity of footprinting, whilst dramatically increasing the yield of data (30-fold). A semi-automated analysis system has also been developed to present footprinting data as an estimate of the binding affinity of each tested compound to any base pair in the assessed DNA sequence, giving an intuitive ‘one compound–one line’ scheme. Here, we demonstrate the screening capabilities of the 96-well assay and the subsequent data analysis using a series of six pyrrolobenzodiazepine-polypyrrole compounds and human Topoisomerase II alpha promoter DNA. The dramatic increase in throughput, quantified data and decreased handling time allow, for the first time, DNase I footprinting to be used as a screening tool to assess DNA-binding agents. |
format | Text |
id | pubmed-1919508 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2007 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-19195082007-07-24 A 96-well DNase I footprinting screen for drug–DNA interactions Ellis, Tom Evans, David A. Martin, Christopher R. H. Hartley, John A. Nucleic Acids Res Methods Online The established protocol for DNase I footprinting has been modified to allow multiple parallel reactions to be rapidly performed in 96-well microtitre plates. By scrutinizing every aspect of the traditional method and making appropriate modifications it has been possible to considerably reduce the time, risk of sample loss and complexity of footprinting, whilst dramatically increasing the yield of data (30-fold). A semi-automated analysis system has also been developed to present footprinting data as an estimate of the binding affinity of each tested compound to any base pair in the assessed DNA sequence, giving an intuitive ‘one compound–one line’ scheme. Here, we demonstrate the screening capabilities of the 96-well assay and the subsequent data analysis using a series of six pyrrolobenzodiazepine-polypyrrole compounds and human Topoisomerase II alpha promoter DNA. The dramatic increase in throughput, quantified data and decreased handling time allow, for the first time, DNase I footprinting to be used as a screening tool to assess DNA-binding agents. Oxford University Press 2007-06 2007-06-22 /pmc/articles/PMC1919508/ /pubmed/17586817 http://dx.doi.org/10.1093/nar/gkm467 Text en © 2007 The Author(s) http://creativecommons.org/licenses/by-nc/2.0/uk/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Online Ellis, Tom Evans, David A. Martin, Christopher R. H. Hartley, John A. A 96-well DNase I footprinting screen for drug–DNA interactions |
title | A 96-well DNase I footprinting screen for drug–DNA interactions |
title_full | A 96-well DNase I footprinting screen for drug–DNA interactions |
title_fullStr | A 96-well DNase I footprinting screen for drug–DNA interactions |
title_full_unstemmed | A 96-well DNase I footprinting screen for drug–DNA interactions |
title_short | A 96-well DNase I footprinting screen for drug–DNA interactions |
title_sort | 96-well dnase i footprinting screen for drug–dna interactions |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1919508/ https://www.ncbi.nlm.nih.gov/pubmed/17586817 http://dx.doi.org/10.1093/nar/gkm467 |
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