| Budget Amount *help |
¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2003: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2002: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
CO_2 as supercritical fluid (SCF) is non-toxic, non-flammable, and has easily accessible critical conditions, and may be utilized as an environmentally benign solvent substitute. SC-CO2 is used reaction media for enzymatic reaction. The benefits of SC-CO_2 suggest that for some reactions, the yield, the rate of reaction, or selectivity will dramatically be enhanced. In this work, we apply the surfactant-coated polymerase to amplification of DNA with high yields. Despite the fact that the catalytic activities of surfactant-coated enzymes have been extensively explored in routine organic solvents, applications of surfactant-coated enzyme for reaction in SC-CO_2 have been seldom reported. Utilizing the surfactant-coated polymerase with putative proofreading activity in SC-CO_2. it can be expected the DNA amplification with a high fidelity and high yield. This is the first report utilizing surfactant-coated polymerase for amplification of DNA in SC-CO_2.
The surfactant-coated DNA polymerase was prepared by the W/O emulsion method. And we used bis(2-ethylhexyl) sodium sulfosuccinate(Aerosol OT, AOT) sample as surfactant. Aqueous phosphate buffer solution containing polymerase, and isooctane solution containing AOT were mixed, and agitated by a homogenizer. After the mixed solution was dried at room temperature for 4 days, surfactant-coated polymerase was obtained.
In order to identify PCR in SC-C0_2, identification analysis of products were investigated by electrophoresis. The photograph of electrophoresis of the PCR with surfactant-coated KOD polymerase in SC-CO2 shows a similar to the peak that a traditional PCR products. On the other hands, it can not be detected the PCR products in PCR with native polymerase in SC-CO_2. DNA was successfully amplified in SC-CO_2. We found that a surfactant-coated polymerase can be also catalyzed amplification of DNA in SC-CO_2. This enzymatic activity in SC-CO_2 were caused by dispersity of enzyme in reaction media.
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