Polymerase chain reaction (PCR) has revolutionized the field of molecular biology due to its exceptional speed, specificity, and sensitivity as a tool for genetic analysis. Of note also is the exceptional versatility of PCR and the readiness with which users of the technique have learned to adapt and modify its fundamental feature of targeted deoxyribonucleic acid (DNA) amplification to achieve diverse experimental goals.

Figure 1c BRCA1 mutants unanalyzed by SSCP using GeneGel Excel 12.5/24 with the GenePhor electrophoresis unit.

Typical DNA fragment sizes that can be reliably generated after PCR amplification tend to be less than 500 to 600 base pairs in length, although these limitations are rapidly disappearing. There are certainly commercially available polymerases and specialized reaction conditions that allow for much greater processivity, but these are typically reserved for highly specific applications. Thermostable polymerases with fairly moderate processivity are all that is required to perform the majority of widely used PCR applications today. Convenient upstream and downstream analysis of relatively small DNA fragments is important to take full advantage of the convenience, sensitivity, and efficiency of most PCR strategies. This discussion aims to bring together in one place fundamental and applied concepts of how to approach some of these widely used PCR-based assays.