The mobilities of oligomers of phage lambda DNA and of yeast chromosomes in agarose gels during field

The mobilities of oligomers of phage lambda DNA and of yeast chromosomes in agarose gels during field inversion gel electrophoresis (FIGE) were measured at different pulse times and electric fields. Also the ratios between forward and backward pulse times and/or field gradients were varied. The problem of 'band inversion' during FIGE, leading to an ambiguity in the mobility of large DNA fragments, was solved by using two dimensional gel electrophoresis with different parameters in the first and second dimension. The results are compared with those obtained with other pulsed electrophoresis systems and with a theoretical model. INTRODUCTION Linear double stranded DNA fragments with different molecular weight are routinely separated by agarose gel electrophoresis. Under constant field conditions the resolution is lost above a fragment length of about 20 kbp, depending on field strength and agarose concentration. In the last few years, powerful new electrophoretic methods have been developed for the separation of very large DNA molecules, which is important for mapping long parts of genomes. This is possible by periodically changing the direction of the electric field. Different variations in field geometry have been developed, which are distinguished as pulsed field gel electrophoresis (1), orthogonal field alternating gel electrophoresis (2), field inversion gel electrophoresis (3), contour clamped homogeneous field (4), transverse alternating field electrophoresis (5), crossed field electrophoresis (6), rotating field electrophoresis (7), programmable autonomously controlled electrodes (8) and pulsed homogeneous orthogonal gel electrophoresis (9). Some effort has been made to describe the mobility of DNA in agarose gels. The most successful theory, called 'reptation', is based on the assumption, that the molecule moves like a reptile through the tubes formed by the gel (10-16). Recently, numerical simulations of DNA motions have been performed (17,18) and also a logistic function has successfully been used to describe the mobility of linear DNA in agarose gels (19). Several explanations about the behaviour of DNA under pulsed field conditions have been proposed, assuming a relaxation process (20), reorientation of the DNA molecule (1), gel hysteresis (21) or resonance aspects (22). Other investigators have used computer models to simulate the motion of DNA molecules in pulsed field experiments (23 —26), but the physical mechanism of separation is still not clear. Before such a theoretical understanding can be achieved, a systematic study of the mobility of DNA under non-stationary electrophoresis conditions is necessary.