Since its development in 1985, polymerase chain reaction (PCR) has revolutionized basic and applied research (1,2). In 1993, Mullis was awarded the Nobel Prize in Chemistry for the development of PCR. With DNA or cDNA as a template, millions of copies of a target sequence are generated during the reaction. Introduction of the thermophilic Thermus aquaticus polymerase increased the specificity of the reaction and made automation and routine use possible (3-5). The ability of PCR to produce multiple copies of a discrete portion of the genome has resulted in its incorporation into techniques used in a wide variety of research and clinical applications. An extraordinary range of clinical applications of PCR have emerged, including diagnosis of inherited disease, human leukocyte antigen (HLA) typing, identity testing, infectious disease diagnosis and management, hematologic disease diagnosis and staging, and susceptibility testing for cancer.

The development of technically simple and reliable methods to detect sequence variations in specific genes is becoming more important as the number of genes associated with specific diseases grows. DNA sequencing is considered the "gold standard" for characterization of specific nucleotide alteration(s) that result in genetic disease. Although sequencing was long considered too cumbersome, expensive, and operator dependent for use in the clinical laboratory, a combination of clinical need and improved technology has brought automated DNA sequencing into routine clinical use. However, even though sequencing technology is now firmly entrenched in the clinical molecular diagnostics laboratory, it is still too expensive and time-consuming for all the laboratory's mutation-detection needs. There are a number of PCR-based mutation-detection strategies that can be used to identify both characterized and uncharacterized mutations and sequence variations.

The degree of allelic heterogeneity, or the number of different mutations in a single gene (each of which cause a specific disorder), influences the method used for mutation detection. For diseases that exhibit no or limited heterogeneity (e.g., sickle cell anemia), assay systems designed to detect specific mutations are appropriate. These types of strategy are also appropriate for disorders in which allelic heterogeneity is high, but only a limited set of mutations are typically analyzed, such as cystic fibrosis. For disorders in which the mutational spectrum is wide (e.g., Duchenne/Becker muscular dystrophy or multiple endocrine neoplasia), a scanning method is needed. A scanning method is also appropriate for analysis of newly identified disease genes, for which there is little or no information regarding the number of disease-causing mutations.

In most applications, PCR is used to amplify specific regions of DNA known to carry or suspected of carrying a mutation. The specific DNA sequence, whether normal or mutated, is then identified by hybridization or electrophoretic separation of the PCR products. In a few techniques, PCR itself is designed to specifically identify the normal and mutant DNA sequence. In this chapter, we will discuss electrophoretic PCR-based techniques for the analysis of DNA and RNA in the clinical laboratory.

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