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Nucleic Acids Research 2005 33(5):e49; doi:10.1093/nar/gni049
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Published online 14 March 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Methods Online

A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting

Juno Choe1,2, Haiwei H. Guo3 and Ger van den Engh1,*

1Institute for Systems Biology 1441 North 34th Street, Seattle, WA 98103-8904, USA 2Department of Genome Sciences, University of Washington Seattle, WA 98195, USA 3Department of Pathology, University of Washington Seattle, WA 98195, USA

*To whom correspondence should be addressed. Tel: +1 206 364 3400; Fax: +1 206 364 346; Email: engh{at}systemsbiology.org

Received November 3, 2004. Revised February 14, 2005. Accepted February 22, 2005.

Molecular biology critically depends upon the isolation of desired DNA sequences. Flow cytometry, with its capacity to interrogate and sort more than 50 000 cells/s, shows great potential to expedite clone characterization and isolation. Intrinsic heterogeneity of protein expression levels in cells limits the utility of single fluorescent reporters for cell-sorting. Here, we report a novel dual-fluorescence strategy that overcomes the inherent limitations of single reporter systems by controlling for expression variability. We demonstrate a dual-reporter system using the green fluorescent protein (GFP) gene fused to the Discosoma red fluorescent protein (DsRed) gene. The system reports the successful insertion of foreign DNA with the loss of DsRed fluorescence and the maintenance of GFP fluorescence. Single cells containing inserts are readily recognized by their altered ratios of green to red fluorescence and separated using a high-speed cell-sorter for further processing. This novel reporter system and vector were successfully validated by shotgun library construction, cloned sequence isolation, PCR amplification and DNA sequencing of cloned inserts from bacteria after cell-sorting. This simple, robust system can also be adapted for diverse biosensor assays and is amenable to miniaturization. We demonstrated that dual-fluorescence reporting coupled with high-speed cell-sorting provides a more efficient alternative to traditional methods of clone isolation.


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