Work

Fluorescence Recovery Assay for the Detection of Protein−DNA Binding

Year: 2008

Type: article

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.

Source: Analytical Chemistry

DOI: 10.1021/ac8007016

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

Institutions: Northwestern University, International Institute for Nanotechnology

Language: English

Topic: Advanced biosensing and bioanalysis techniques

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: green

Funders: American Association of University Women, National Institutes of Health, National Cancer Institute, Eunice Kennedy Shriver National Institute of Child Health and Human Development