Study of ethidium bromide effect on dsDNA separation by capillary zone electrophoresis and laser light scattering.

Abstract

The effect of ethidium bromide (EB) on dsDNA separation was studied by using capillary zone electrophoresis (CZE) and laser light scattering (LLS). By increasing the concentration from 1 to 15 microg/mL, EB showed significant influence on the separation of pBR322 HaeIII in terms of resolution, speed, and signal-over-noise ratio. The physicochemical properties of DNA, such as mobility under electric field, charge-over-size ratio, and hydrodynamic radius, showed two declines, a sharp drop followed by a relaxed decrease, with increasing EB concentration. Such observations could be explained by using two modes of the dye binding process: intercalation and outside binding. The combination of results obtained from CZE and LLS also confirmed the existence of outside binding. Compared with intercalation, outside binding showed a weaker dye concentration dependence in neutralizing the negative charges on the DNA fragments. Static light scattering showed that <M(w)> and <R(g)> of DNA in 1xTris-borate-EDTA (TBE) were increased with increasing EB concentration. <R(g)>/<R(h)> of DNA with dye had a value larger than 1.5, suggesting a random coil conformation.