Rapid isolation of high quality, multimeric plasmid DNA using zwitterionic detergent.
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 modern recombinant technology and therapeutic applications.
Chowdhury
E H
EH
Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
Akaike
Toshihiro
T
D000468:Alkalies / Q000737:chemistry / Q000494:pharmacology; D002458:Cell Fractionation / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry* / Q000302:isolation & purification*; D003902:Detergents / Q000737:chemistry / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D010957:Plasmids / Q000737:chemistry* / Q000302:isolation & purification*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry* / Q000494:pharmacology
D000468:Alkalies; D004269:DNA, Bacterial; D003902:Detergents; D000644:Quaternary Ammonium Compounds; C034730:zwittergent 3-12