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