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Development of DNA Amplification Technique by Enzymatic Reaction in Supercritical Fluid

超临界流体酶促反应 DNA 扩增技术的发展

基本信息

批准号：
14550743
负责人：
MISHIMA Kenji
金额：
$ 2.56万
依托单位：
Fukuoka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-14550743/
关键词：
DNA Amplification Supercritical carbon dioxide Enzyme Surfactant Polymerase chain reaction Polymerase 生態関連物質

项目摘要

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.

CO_2作为超临界流体（SCF）具有无毒、不燃、临界条件容易获得等优点，是一种环境友好的溶剂替代品。SC-CO2是用于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着提高。在这项工作中，我们将表面修饰的聚合酶应用于高产量的DNA扩增。尽管表面修饰酶在常规有机溶剂中的催化活性已被广泛研究，但其在SC-CO_2中的应用却鲜见报道。利用表面活性剂包被的聚合酶在SC-CO_2中具有推定的校对活性。可以期待高保真度和高产率的DNA扩增。本文首次报道了在SC-CO_2中用表面修饰的聚合酶扩增DNA。以双（2-乙基己基）琥珀酸酯磺酸钠（AOT）为表面活性剂。将含有聚合酶的磷酸盐缓冲水溶液和含有AOT的异辛烷溶液混合，并通过均质器搅拌。混合液室温干燥4d后，得到表面包被的聚合酶，通过电泳对产物进行鉴定分析，以鉴定SC-C 0_2中的PCR。表面修饰的KOD聚合酶的PCR产物在SC-CO_2中的电泳图谱与传统PCR产物的电泳图谱相似。而在SC-CO_2中，用天然聚合酶进行PCR反应，则不能检测到PCR产物。在SC-CO_2中成功地扩增了DNA。我们发现，在SC-CO_2中，表面修饰的聚合酶也能催化DNA的扩增。SC-CO_2中的酶活性是由酶在反应介质中的分散性引起的。