Exo-Taq-based detection of DNA-binding protein for homogeneous and microarray format.
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.
Fukumori
Takashi
T
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, Japan.
Miyachi
Hirotaka
H
Yokoyama
Kenji
K
D001483:Base Sequence; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005456:Fluorescent Dyes; D020411:Oligonucleotide Array Sequence Analysis; D012097:Repressor Proteins; D019914:Taq Polymerase / Q000378:metabolism*; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D015342:DNA Probes; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D019914:Taq Polymerase; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III