Technical Considerations

With hundreds of commercially available restriction endonucleases available, selection of appropriate enzyme(s) for use in a particular RFLP analysis depends on the target nucleotide acid sequence of interest (Rogers et al., 1991; Pourzand et al., 1993; Bloch and Grossmann, 1995; Thiers et al., 1997; Buoro et al., 1999; Prix et al., 1999). In many such assays, PCR product can be used directly after amplification, without any intervening manipulation of the sample. No specialized equipment is required for RFLP analysis beyond that normally used for agarose gel electrophoresis (and Southern blot hybridization, if this is used). Only a water bath or incubator is needed for the enzyme digestion process. Time requirements for restriction enzyme digestion depend on the cutting efficiency of the enzyme and the amount of DNA used in the digestion, with incubation times ranging from 1 h to overnight.

Conditions for the cleavage reaction, including concentration of enzyme, buffer, and target nucleic acid, as well as temperature (usually 37°C) and duration of reaction all vary from enzyme to enzyme. Multiple enzymes may be used together if buffer and temperature requirements are similar. If needed, potassium acetate or potassium glutamate buffers may be used, as they tend to accommodate a wide range of restriction enzymes (see more detailed references for specifics on the use of multiple enzymes). Purity of target nucleic acid can be essential for efficient cleavage reactions. Impurities, such as protein, high salt concentrations, anticoagulants, and solvents remaining from DNA extraction or purification processes can all inhibit the function of restriction endonucleases (Bloch et al., 1995). The effects of such contaminants can be mitigated through the use of higher concentrations of enzyme, increased reaction volume, increased time of incubation with the enzyme, or by repurification of the target nucleic acid. Other factors, such as secondary DNA structure (e.g., supercoiling), present in plasmid or some viral preparations, may also inhibit enzyme activity, requiring many times the normal concentration for cleavage.

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