Fluorescence recovery assay for the detection of protein-DNA binding

Xiaoyang Xu, Zhen Zhao, Lidong Qin, Wei Wei, Jon E. Levine, Chad A. Mirkin

Research output: Contribution to journalArticle

48 Scopus citations

Abstract

We report a novel and straightforward fluorescence recovery assay which enables the detection of protein-DNA interactions and simultaneously determines relative binding affinities of sequence-specific DNA-binding proteins for a variety of DNA sequences in a multiplexed format The detection of protein-DNA binding is accomplished by monitoring fluorescence recovery during exonuclease digestion of DNA sequences that are modified with fluorophore-quencher pairs. Retardation of fluorescence recovery occurs with binding of the protein to the putative DNA binding element, which arrests exonuclease digestion. The assay detects protein-DNA binding in a homogeneous solution simply, quickly, and reliably without using radioisotopes. Multiplexing is possible by labeling different DNA sequences with spectrally distinct dyes, allowing simultaneous analysis of experimental and control binding reactions in the same mixture.

Original languageEnglish (US)
Pages (from-to)5616-5621
Number of pages6
JournalAnalytical Chemistry
Volume80
Issue number14
DOIs
StatePublished - Jul 15 2008

ASJC Scopus subject areas

  • Analytical Chemistry

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