Northern and Southern Blotting and the Polymerase Chain Reaction to Detect Gene Expression
For cancer cells to form a metastasis, cells from the primary tumor must overcome the local adhesive forces, migrate and invade the microcirculation, arrest at a secondary site, and then finally proliferate (1). As implied by its mult]step nature, cancer metastasis is a complex and dynamic process that is likely to be regulated by a series of genes at each step (2). A variety of approaches have been used to discern the molecular events that regulate this process. It is likely that the ability of a cancer cell to form clinically detectable metastases is influenced by a variety of factors, including alt]rations in the pattern of gene expression within the cancer cell. Such changes could be the result]of genetic or epigenetic modifications (3). Alt]ough there has been a growing emphasis on array-based techniques for high-throughput screening of gene expression patterns, there are several well established protocols that can be used to identify such molecular changes. This chapter describes two of these techniques: Northern and Southern blotting.
E. M. Southern first described a method for immobilizing size-fractionated DNA fragments on a nitrocellulose membrane in 1975. Since then, a number of different variations of this blotting method have been developed, as well as a variety of ways by which scientists can generate and hybridize probes to detect specifically the sequences thus immobilized. Southern blotting is now a general term for a number of different methods by which DNA is transferred from a gel to a membrane, and because nitrocellulose is relatively fragile, improved membranes have been developed that are more durable and that have been optimized for allowing binding of nucleic acids.
KeywordsCellulose Formaldehyde Glycerol Phenol EDTA
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