Rapid isolation of high quality, multimeric plasmid DNA using zwitterionic detergent
Rapid isolation of high quality, multimeric plasmid DNA using zwitterionic detergent
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DOI：
10.1016/j.jbiotec.2005.05.013
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发表时间：
2005-10-10
影响因子：
4.1
通讯作者：
Akaike, T
中科院分区：
文献类型：
--
作者：
Chowdhury, EH;Akaike, T
从细菌中提纯质粒DNA是重组DNA技术中必不可少的工具，已成为实验室和工业生产中的一项重要任务。此外，“基因治疗”和“基因疫苗”的最新进展也要求生产“千克”级别的药用级质粒DNA。尽管存在许多纯化方案，但大多数都源于一项开创性的工作[Birnboim，H.C.，Doly，J.，1979]。一种筛选重组质粒DNA的快速提取方法。核酸研究报告7,1513-1523]等存在一个或多个缺陷，如纯化时间、纯度或分离的质粒DNA的数量。在这里，我们报道了一种创新的方法，通过在两性离子洗涤剂和碱的帮助下产生‘软蛋白聚集体’来大量分离高纯度和功能性的质粒DNA。用低离子强度的Tris缓冲液进行简单温和的洗涤，可以去除固溶的蛋白质和RNA，并可一步将多聚体DNA从蛋白质聚集体中洗脱出来。此外，分离的质粒DNA易于被限制性内切酶酶切，具有较高的蛋白质表达功能。因此，考虑到它在生产足够纯净的质粒DNA方面的显著简单性和效率，新的策略将在现代重组技术和治疗应用中成为有用的工具。(C)2005年，爱思唯尔出版。
Purification of plasmid DNA from bacteria is an essential tool in recombinant DNA technology and has become an essential task in laboratories to industries. Moreover, the recent progress of 'Gene therapy' and 'Genetic vaccination' also demands production of pharmaceutical grade plasmid DNA in 'kilogram' level. Despite existence of a number of purification protocols, all most all have been originated from a pioneering work [Birnboim, H.C., Doly, J., 1979. A rapid extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513-1523] and so suffer from one or more drawbacks, such as purification time, purity or quantity of isolated plasmid DNA. Here, we have reported an innovative approach for isolation of highly pure and functional plasmid DNA in significant amount, based on generation of 'soft protein aggregate' with the help of zwitterionic detergents and alkali. Solibilized proteins and RNA could be removed by a simple and mild washing with Tris buffer of low ionic strength and multimeric plasmid DNA could be eluted in a single step from the protein aggregate. Additionally, isolated plasmid DNA could easily be digested by restriction enzymes and had high functionality in protein expression. Thus, considering both its remarkable simplicity and efficiency in producing sufficiently pure plasmid DNA, the new strategy would emerge a useful tool in modem recombinant technology and therapeutic applications. (C) 2005 Published by Elsevier B.V.