© 2005 The Japanese Biochemical Society
Regular Paper |
Exo-Taq-Based Detection of DNA-Binding Protein for Homogeneous and Microarray Format
1 Research Center of Advanced Bionics, National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562; and 2 Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292
* To whom correspondence should be addressed. Tel: +81-29-861-2987, Fax: +81-29-855-3833, E-mail: ke-yokoyama{at}aist.go.jp
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 
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.