Nucleic Acid Blotting: Southern and Northern

Abstract

AbstractE.M. Southern invented blotting of DNA in 1975; the method was extended to RNA in 1977. Standard Southern blotting includes limited depurination, denaturation, and neutralization of the DNA in gels (where they have been separated in size by electrophoresis) and capillary transfer of the DNA onto nitrocellulose or nylon blotting membranes. For northern blotting, RNA is guarded from base and RNase, denatured, separated by electrophoresis, and then blotted to nylon blotting membranes. Both types of blots are then blocked to prevent nonspecific binding, hybridized with probe, and washed. Next the sequences of interest are located by detecting labeled probes. Alternative methods involve dot/slot blotting when the size of the nucleic acid being probed is not of interest. Electrophoretic transfer from polyacrylamide gels can be used when the nucleic acid fragments of interest are too small to be effectively resolved on agarose gels. Artifacts in Southern blot can result from incomplete digestion, overloading the blotting membrane, incomplete blocking, damaged blot media, and air bubbles. In northern blotting, RNA quality must be monitored, and RNA that is degraded or contaminated with excess DNA should be avoided.