One goal of the human genome project is the automated preparation of plasmid DNA from E. coli for subsequent DNA sequencing and restriction mapping. I have developed a radically new procedure for the preparation of plasmid DNA called Sequential Enzymatic Digestion. This method does not employ classical purification steps such as centrifugation, precipitation, extraction with organic solvents, chromatography, or electrophoresis. The method works by the sequential addition of hydrolytic enzymes to an E. coli culture followed by a bead based step for plasmid concentration. The method is readily amenable to automation on commercial liquid handling robots. A proposed small bench top robot employing this method will be capable of producing about one thousand plasmid preparations per day. This purified plasmid DNA will be suitable for applications such as DNA sequencing, restriction endonuclease analysis, PCR. hybridization, cloning, and in vitro transcription.Awardee's statement of the potential commercial applications of the research: The preparation of plasmid DNA from E. coli is a routine procedure performed in all molecular biology labs as a precursor to other procedures such as DNA sequencing, restriction digestion, and cloning. This method will allow complete automation of the procedure, allowing thousands of plasmid preps to be performed daily.National Cancer Institute (NCI)
