Related Experiment Videos
A hybrid mode of rotating gel electrophoresis for separating linear and circular duplex DNA
1Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Abstract:
During agarose gel electrophoresis, alternately applying the electrical potential gradient (E) in two directions (separated by an angle, psi) has been used to enhance the separation by length of linear, double-stranded DNA; the value of psi is usually between 0.5 pi and 0.7 pi radians (Cantor et al. (1988). Ann. Rev. Biophys. Biophys. Chem. 17, 287-304). When the direction of E is changed by rotating the gel (rotating gel electrophoresis, or RGE), open circular DNA longer than 48 Kb is found here to form bands that decrease in either sharpness or migration distance as psi is decreased from 1.1 pi to 0.5 pi radians. A satisfactory band is formed only for psi greater than or equal to 1.1 pi radians. In contrast, the previously-demonstrated separation and banding of linear DNAs (48.5-800 Kb) was observed for psi values between 0.6 pi and 1.4 pi radians. The cause of psi's effect on open circular DNA is electrophoresis during the gel's rotation. RGE by use of psi values above 1.0 pi is a hybrid mode of RGE, incorporating favorable characteristics of both the usual mode and a more recently demonstrated (Serwer, P. and Hayes, S.J. (1989). Applied and Theoretical Electrophoresis, 1, 95-98) mode in which psi is 2 pi radians. To separate linear from open circular DNA and to fractionate by length both of these conformational forms, the following procedure of two-dimensional agarose gel electrophoresis has been developed: (a) a first dimension performed by use of RGE and a psi of 1.4 pi radians, (b) a second dimension performed with an invariant electrical field. Circular DNA has been found among the bacteriophage lambda DNA concatemers (48.5-800 Kb) used as length standards. By use of a psi = 1.4 pi radians, the band of 48.5 Kb monomeric open circular lambda DNA is sometimes accompanied by at least three other bands of open circular DNA; the latter are presumed to be those formed by dimeric, trimeric and tetrameric open circular lambda DNA.