Nucleic Acid Blotting: Southern and Northern

E.M. Southern invented blotting of DNA in 1975; the method was extended to RNA and named northern blotting in 1977. 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 basic pH and RNase, denatured, separated by electrophoresis, and then blotted on to nylon blotting membranes. Either type of blot is then blocked to prevent nonspecific binding, hybridized with probe, and washed. Next, the sequences of interest are located by detecting labeled probes. One alternative method involves dot/slot blotting when the size of the nucleic acid being probed is not of interest. Also, electrophoretic transfer from polyacrylamide gels can be used when the nucleic acid fragments of interest are too small to be 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. Curr. Protoc. Essential Lab. Tech. 6:8.2.1-8.2.26. © 2012 by John Wiley & Sons, Inc.