An Apparatus for Electrophoretic Capture and Recovery of Circular DNA in Thin Layers

Published

June 1, 2003

Author(s)

Kenneth D. Cole

Abstract

An apparatus was designed for the electrophoretic capture and recovery of circular DNA in thin layers (membranes). Rapid separations were done by the use of a low conductivity buffer and high electric field strengths. Two methods that specifically retain circular DNA in the membranes were demonstrated using the supercoiled and open circular forms of two plasmids with sizes of 4.4 kilobase pairs (kbp) and 13 kbp. Electrophoretic trapping (by an impalement mechanism) in agarose gel-filled membranes used electric field strength to immobilize circular DNA in the membranes. During electrophoretic trapping a critical electric field strength determines the size and physical form of the circular DNA that is retained. The other method of capture utilized the greatly reduced electrophoretic mobility of circular DNA in membranes composed of agarose and the linear polymer hydroxyethyl cellulose. The reduction in electrophoretic mobility was not dependent upon the electric field strength distinguishing it from electrophoretic trapping. The DNA trapped in the membranes was analyzed by placing the membranes into agarose gels and running the DNA out of the membranes at a non-trapping electric field strength. Trapping of circular DNA in the membranes followed by size analysis using agarose gel electrophoresis could be used as a two-dimensional separation for the analysis of complex mixtures. Captured DNA was recovered by two methods. Centrifugation of membranes made with low melting point agarose resulted in a gel slurry that could be heated to release the DNA. The second method of recovery was by electroelution of the membranes. Electroelution was done by using an electrode that was isolated from the DNA by a barrier membrane. A non-trapping electric field was used to reverse the DNA out of the membrane into a small volume of buffer above the membrane. These membranes and methods should be scaleable to process large number of samples or to prepare larger amounts of pure circular DNA by increasing the area of the membranes. The apparatus facilitates the rapid screening of a large number of trapping materials and methods for recovery of DNA.

Citation

Biotechnology and Applied Biochemistry

Volume

37

Pub Type

Journals

Keywords

agarose gel, circular DNA, electroelution, electrophoresis, electrophoretic trapping, membrane, plasmid

Citation

Cole, K. (2003), An Apparatus for Electrophoretic Capture and Recovery of Circular DNA in Thin Layers, Biotechnology and Applied Biochemistry (Accessed December 16, 2024)