The effect of ethidium bromide on mobility of DNA fragments in agarose gel electrophoresis

Abstract

Ethidium Bromide (EtBr) is sometimes added to running buffer during the separation of DNA fragments by agarose gel electrophoresis. It is used because upon binding of the molecule to the DNA and illumination with a UV light source, the DNA banding pattern can be visualized. The mode of binding of EtBr is intercalation between the base pairs. This binding changes the charge, weight, conformation, and flexibility of the DNA molecule. Since DNA molecules are sized by their relative movement through a gel compared to a molecular weight standard, mobility measurements can be critical to size determinations. After running two identical gels, one without EtBr and one with 0.25, 0.5, 0.75 or 1.0 microgram/mL EtBr in the running buffer, the mobilities of lambda HindIII DNA fragments were compared. The mobility of DNA was always less in the gels with EtBr. Using the reptation theory equation, which describes the mobility of DNA molecules through gel, changes in frictional coefficients were calculated. It was determined that the change in frictional coefficients brought about by the addition of EtBr is directly proportional to the fraction of base pairs of a fragment bound to EtBr. This change in friction is greatest in the largest fragments, which suggests that the stiffening of the molecule by the EtBr binding is the cause for the decreased mobility.

The EB-DNA Liquid Crystalline Complex with High Concentration of Mg2+

“…29,30 Thus, the fraction of liquid crystal phase in the EB-DNA solution is higher than that in the EB-free DNA solution when the concentration is the same. Furthermore, the DNA chains are more rigidity 31,32 in the EB-DNA solution than those in the EB-free DNA solution due to the intercalation of EB, which also results in the decrease of the critical concentration of DNA liquid crystalline phase formation or the increase of the fraction of the liquid crystalline phase in the solution at the same concentration. 30,33 The effect of EB on the ionic environment around DNA chains can be confirmed by 23 Na NMR spectrum.…”

A comparison of DNA stains and staining methods for Agarose Gel Electrophoresis

“…Intercalating dyes change the charge and flexibility of DNA molecules and add weight, altering movement through the gel (Sigmon and Larcom 1996;Miller et al 1999;Huang and Fu 2005;Huang et al 2010).The post-staining method is therefore the most accurate way to size DNA fragments but it is time consuming and costly as more stain is needed.…”

Influence of chloroquine on kinetics of single-cell gel electrophoresis

“…2). Some additional contributions into the hampering of the DNA exit may come from the changes in charge, contour length and stiffness of DNA upon binding of intercalators [15,16].…”