PRESENCE OF RNASE-A CAUSES ABERRANT DNA BAND SHIFTS

RNase A, which is routinely added during DNA purification to reduce co ntaminating RNA, causes shifting of DNA bands in agarose gels. DNA ban d sizes on agarose gels increase as much as 10%-20% when RNase A is pr esent. The low concentrations of RNase A typically used to purify DNA cause shifting of select DNA bands, in contrast to higher concentratio ns of RNase A where all bands are shifted and smeared. The binding of RNase A to the DNA is specific and the degree of the shift varies; not all DNA bands are retarded, and the deviation is more pronounced in c ertain buffers. Other proteins, such as bovine serum albumin or protei nase K, do not induce DNA band shift, suggesting the interaction is sp ecific. The binding of RNase A to DNA is reversible. The formation of RNase:DNA complexes may affect experiments involving DNA:protein inter actions such as gel shift, footprinting and filter binding assays. Res earchers performing DNA characterization from miniprep protocols shoul d be aware that RNase may cause the apparent sizes of DNA fragments to be altered and obscure the presence of very small cloned fragments.