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Nucleic Acids Research 2005 33(6):e60; doi:10.1093/nar/gni060
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Published online 31 March 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Methods Online

An ultrasensitive site-specific DNA recombination assay based on dual-color fluorescence cross-correlation spectroscopy

Michael Jahnz and Petra Schwille*

TU Dresden/BioTec, Institute of Biophysics Tatzberg 47-51, 01307 Dresden, Germany

*To whom correspondence should be addressed. Tel: +49 351 463 40329; Fax: +49 351 463 40342; Email: schwille{at}biotec.tu-dresden.de

Received December 22, 2004. Revised March 9, 2005. Accepted March 9, 2005.

Site-specific exchange of genetic information is mediated by DNA recombinases, such as FLP or Cre, and has become a valuable tool in modern molecular biology. The so far low number of suitable recombinating enzymes has driven current research activities towards alteration of catalytic properties, such as thermostability or recognition sequences. However, identification and analysis of new mutants requires sensitive in vitro activity assays, which traditionally are based on gel electrophoresis. Here, we describe the development of a new sensitive DNA recombination assay based on dual-color fluorescence cross-correlation spectroscopy (DC-FCCS), which works in homogenous solution and does not require any separation step such as electrophoresis. The assay was validated with unlabeled FLP recombinase and different fluorescently labeled DNA substrates containing the FLP recognition target (FRT). This strategy fulfills all requirements for possible application in high throughput screening and engineering of new site-specific DNA recombinases starting from the FLP-FRT system, and is easily adjustable to other systems like Cre/loxP.


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