|Budget Amount *help
¥6,300,000 (Direct Cost : ¥6,300,000)
Fiscal Year 1998 : ¥2,800,000 (Direct Cost : ¥2,800,000)
Fiscal Year 1997 : ¥3,500,000 (Direct Cost : ¥3,500,000)
Most of DNA biosensors direct toward the detection of target DNA which has a complementary base sequence of the immobilized DNA on the electrode. On the other hand, DNA molecule can be regarded as ligands to various DNA binding proteins which recognize certain base sequences, mismatch base pair, or characteristic structure of DNA.We investigated the possibility of use of DNA binding proteins to the design of biosensor for gene assay. First, we established the system, in which DNA-protein interaction can be detected, using anti-DNA antibody and DNA modified gold electrode. In this system, ferrocyanide/ferricyanide redox couple was used as anionic redox marker to monitor the protein binding on immobilized DNA.Thus, addition of anti-DNA antibody suppressed the redox currents of marker ions dramatically in concentration dependent manner with the detection limit of 1 nM.In this case, 2-mercaptoethanol treatment of electrode surface was effective to avoid non-specific adsorption of other proteins. Then, DNA detection with the anti-DNA antibody immobilized electrode was also investigated in detail. After the study using the antibody, DNA binding protein was changed to PIT-1, which is a transcriptional factor, as sequence specific DNA binding protein. The redox current of the anionic redox marker was then augmented with the addition of PIT-i in the DNA immobilized electrode system, probably due to the cationic charges of PIT-1 which accelerates the electrochemical reaction of the marker ions. The increase of the redox current was again the concentration dependent of added PIT-1.
In this way, we successfully established the detecting system of sequence specific interaction of DNA-DNA binding protein.