Northern and Southern Blotting and the Polymerase Chain Reaction to Detect Gene Expression
互联网
510
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.
