Detection and characterization of molecular amplification products: Agarose gel electrophoresis, southern blot hybridization, restriction enzyme digest analysis, and enzyme-linked immunoassay

The need for accurate detection and characterization of nucleic acid targets has prompted the development of a range of methodologies. Highly complex and often expensive techniques, such as oligonucleotide arrays, are being used increasingly. Such methods can be extremely valuable, but issues such as cost, the need for specialized equipment, and a high level of expertise for both the technical and analytical aspects of implementation may limit their use in a clinical setting (Chee et al., 1996; Cheung et al., 1999). Although such systems are certainly effective for gathering large amounts of information and can be extremely useful in the research arena (Khan et al., 1999), their use may be unnecessary if only single PCR target detection is required. The use of real-time molecular product detection methods, largely relying on the principle of fluorescent resonance energy transfer (Chen et al., 1997) (FRET), has also become quite commonplace. These methods are useful for high-throughput diagnostic assays and are amenable to automation.