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Exo-Taq-based detection of DNA-binding protein for homogeneous and microarray format.

Authors
  • Fukumori, Takashi
  • Miyachi, Hirotaka
  • Yokoyama, Kenji
Type
Published Article
Journal
Journal of biochemistry
Publication Date
Oct 01, 2005
Volume
138
Issue
4
Pages
473–478
Identifiers
PMID: 16272142
Source
Medline
License
Unknown

Abstract

The study of DNA-protein interactions is of great importance to understand basic cellular processes such as transcription, replication and recombination. In this research, we developed a novel detection system for DNA-binding proteins (DBPs) involving the exonuclease (Exo) III and Taq DNA polymerase reactions. The system consists of three steps, as follows: the target DBP in the sample solution is incubated with probe DNA, and the probe is digested with Exo III and then extended with Taq using fluorescent dye-labeled dUTP as a substrate. The DBP protects the probe from digestion by Exo III. Therefore, only the DBP-bound probe allows the following extension. We examined this system using the lambda phage Cro repressor in a homogeneous format. The fluorescence image after gel electrophoresis showed a specific band. We also found that this system could be applied to the rapid and efficient detection of DBPs in stem and loop ds-DNA array formats. These results suggest that our method is useful as a new tool for analyzing DNA-protein interactions.

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