An Introduction to PCR Primer Design and Optimization of Amplification Reactions

PCR optimization is usually performed in order to obtain maximum specificity and yield. In some applications for which the amount of template may be limiting, or when there is a large amount of nontarget sequences, the sensitivity is maximized. Nonoptimized conditions promote artifactual bands resulting from primer dimerization and mispriming, broad bands containing a mixture of correct and incomplete products, and, generally speaking, a poor signal-to-noise ratio. Poorly designed PCR protocols also lack robustness. Such situations can occur in which the PCR is challenged with less than pristine DNAs or when the ingredients of the reaction are not added in the exact amounts as described by the authors of the protocol. Thus, a well-optimized PCR should be reliable in the hands of a coworker or in the laboratory of another institute.