Development of DNA Amplification Technique by Enzymatic Reaction in Supercritical Fluid

CO_2作为超临界流体（SCF）具有无毒、不可燃、易达到临界条件等特点，可作为一种环保的溶剂替代品。SC-CO2是酶促反应的反应介质。SC-CO_2的优点表明，对于某些反应，产率、反应速率或选择性将显著提高。在这项工作中，我们将表面活性剂包被的聚合酶用于高产量的DNA扩增。尽管表面活性剂包被酶在常规有机溶剂中的催化活性已被广泛研究，但表面活性剂包被酶在SC-CO_2反应中的应用却很少报道。利用表面活性剂包被的聚合酶在SC-CO_2中具有假定的校对活性。可以期望获得高保真度和高产量的DNA扩增。这是第一个利用表面活性剂包被聚合酶在SC-CO_2中扩增DNA的报道。采用W/O乳液法制备表面活性剂包被的DNA聚合酶。并以双（2-乙基己基）琥珀酸钠（气溶胶OT， AOT）样品为表面活性剂。将含聚合酶的磷酸盐缓冲水溶液与含AOT的异辛烷溶液混合，用均质机搅拌。混合溶液室温干燥4天后，得到表面活性剂包被聚合酶。为了对SC-C0_2进行PCR鉴定，对产物进行了电泳鉴定分析。表面活性剂包被的KOD聚合酶在SC-CO2中电泳的照片显示出与传统PCR产物相似的峰。另一方面，在SC-CO_2中，用天然聚合酶无法检测到PCR产物。DNA在SC-CO_2中扩增成功。我们发现表面活性剂包被的聚合酶也可以催化SC-CO_2中DNA的扩增。SC-CO_2酶活性的提高是由酶在反应介质中的分散性引起的。

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.