Basic Techniques Used in the Study of Oncogenes

In addition to resolving DNA fragments of different lengths, gel electrophoresis separates the different molecular configurations of DNA—the covalently closed circular, the nicked circular, and the linear forms. 

Southern s technique could not be applied to the blot transfer of RNA separated by gel electrophoresis. Alwine et al. (A2) therefore devised a procedure in which RNA bands are blot transferred from the gel onto the chemically reactive paper, where they are bound covalently. The reactive paper is prepared by diazotization of aminobenzyl oxymethyl paper, which can be prepared from Whatman paper by a series of uncomplicated reactions. Once covalently bound, the RNA is available for hybridization with radiolabelled DNA probes. Hybridizing bands are detected autoradiographically. This method is termed Northern blotting. 

In the Maxam and Gilbert method (M2) the starting point is a defined DNA restriction fragment. The DNA strand to be sequenced must be radioactively labeled at one end with a 32P-labeled phosphate group. This DNA can be single stranded or of duplex form. The base-specific cleavages depend upon the following points  

Chemical reagents that alter one or two bases in DNA have been characterized (Table 2). The reactions are base specific for example, dimethyl sulfate methylates guanine (at the N7 position). 

An altered base is then removed from the sugar phosphate backbone of DNA. 

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