Simultaneous detection of DNA-binding proteins using exo-Taq-based reaction
Tóm tắt
The DNA-binding protein (DBP) has a wide range of roles such as those in DNA repair, recombination, and gene expression. Recently, a microarray-based method has been developed for the high-throughput analysis of DNA-protein interactions. However, to maximize the advantages of this method, the detection process should be improved so that the method can be applied to many proteins without the use of antibody or sample labeling. Previously, we presented a primary report on the detection of DBP, which is applicable to the microarray format. The system consists of three steps: first, the target DBP in the sample solution is incubated with a probe DNA; second, the probe is digested with Exo (Exonuclease) III; finally, the probe is extended withTaq DNA polymerase using fluorescent dye-labeled dUTP as a substrate. The binding DBP protects the probe from digestion by Exo III. Therefore, only the DBP-bound probe allows the following extension. In this study, the simultaneous detection of multiple DBPs was examined, and then the DBPs were analyzed using a crude extract of the cultured cells to demonstrate the general applicability of the method. Our method can be applied to many DBPs using the same procedure and components, whereas in the antibody-based method, the same number of antibodies as DBPs is needed to detect target DBPs in ELISA (enzyme-linked immunosorbent assay). These results suggest that our method is useful for the high-throughput detection of DBPs in the microarray format.
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