Affinity gel electrophoresis of DNA

The principles of DNA affinity gel electrophoresis are investigated experim entally using velocity and linear dichroism spectroscopy measurements. As a model system we use 1% agarose gels covalently modified with either ethidi um bromide between 0 and 30 mu M or biotin using the same immobilization ch emistry. The method allows the immobilized ethidium bromide to interact wit h the double-stranded DNA by an intercalation type of binding leading to a well-defined DNA-matrix interaction which is reversible, whereas biotin cap tures avidinated DNA irreversibly. At 1 mu M immobilized ethidium bromide T 2 DNA undergoes a weakly perturbed version of the cyclic reptation which is typical of unmodified gels and the velocity is retarded by 35%. At 10 mu M the velocity is retarded by 80% and the mode of migration is strongly pert urbed. In both cases the DNA becomes strongly field-aligned due to the tran sient affinity anchoring to the gel which also causes the velocity retardat ion. Some fundamental aspects of affinity electrophoresis are studied. The affinity effect on the migration disappears when the field force is strong enough to overcome the summed DNA-gel interactions, which indicates that mi gration is slower than in unmodified gels because a fraction of the applied electric force is used to overcome the attraction between DNA and affinity label. Second, DNA migration processes are retarded if they occur on time scales similar to the dissociation time of the DNA-gel affinity complex, wh ereas processes which are much slower are unaffected. Finally, irreversible capture of end-avidinated DNA shows that the long DNA used here encounters the affinity label with high efficiency, perhaps through a directed search by sliding around the labeled gel fibers.