DAPI STAINING OF DNA - EFFECT OF CHANGE IN CHARGE, FLEXIBILITY, AND CONTOUR LENGTH ON ORIENTATIONAL DYNAMICS AND MOBILITY OF THE DNA DURINGAGAROSE-GEL ELECTROPHORESIS

Microscopy studies of the DNA electrophoresis process demands fluoresc ent staining of DNA, and it is important to reveal how the staining af fects the electrophoretic behavior. In this work the influence of the dye DAPI on the behavior of DNA in agarose gel has been studied by mob ility and orientational measurements. From the measurements and compar isons with theories, it is estimated that DAPI, at the binding ratio 0 .2 dye molecule per DNA base, decreases the electrokinetic charge and persistence length of DNA by 13% and at least 40%, respectively, and i ncreaes the contour length by 20%. These changes affect strongly both mobility and orientational dynamics of the DNA, but the mode of motion is not affected for neither short nor long DNA (2-164 kpb). For long DNA that reptates with oscillations between stretched and coiled state s, the steady-state mobility in 1% agarose is reduced by 30% at all st udied fields (5-25 V/cm). In these fields the characteristic times in the buildup of the steady state orientation as well as the reorientati on time and the period time in the oscillatory motion are increased by 60%. For both DAPI-DNA and uncomplexed DNA the field-free relaxation of the major part of the orientation is dominated by two fast processe s with time constants that are similar for the two molecules but with a ratio between the amplitudes that is different. Analysis of this dif ference indicates that the lower mobility of the DAPI-DNA complex is d ue mainly to the strong reduction in the persistence length caused by the dye. However, the difference in the orientational dynamics during migration and in field-free relaxation disappears if comparison is mad e, not at the same field strength, but at field strengths where the tw o molecules show the same mobility.