Gene Probes, Principles And Protocols

Methods in Molecular Biology TM VOLUME 179 Gene Probes Principles and Protocols Edited by Marilena Aquino de Muro Ralph Rapley HUMANA PRESS Target Format and Hybridization Conditions 1 1 Target Format and Hybridization Conditions Alex Reid 1. Southern Blotting 1.1. Introduction The isolation of specific regions within the genome of an organism is now normally accomplished by polymerase chain reaction (PCR) amplification using primers specific for the region in question. However, there are occasions where this is not possible (loss of primer sites resulting in no amplification or if there are no primers available). In these cases the detection of the sequence of interest can be achieved by hybridization of a labeled probe to restricted genomic DNA immobilized on a membrane by Southern blotting (1). Genomic DNA is first digested by one or more restriction enzymes and the fragments generated separated by gel electrophoresis. The amount of DNA to be applied to the gel varies from application to application. In general 10 µg of human genomic DNA is needed for the detection of a single copy gene when using radioactively labeled probes and an overnight exposure to X-ray film. This figure can be reduced if the target is either a repetitive element (e.g., ribosomal DNA) or, if plasmid DNA or PCR products are run on the gel. Once the fragments are separated on the gel the DNA is then denatured in situ and transferred by capillary transfer to either a nitrocellulose or nylon membrane. The DNA fragments are then bound to the membrane, which can then be used in