Optimal Design of PCR Assays for Circulating DNA

Carefully considered assay design is essential for PCR-based methods used in circulating DNA (cirDNA) studies. Broadly speaking, there are three main contexts in which cirDNA is subjected to PCR amplification and analyses: (1) in order to quantitate overall cirDNA, or a particular cirDNA sequence; (2) in order to detect mutations or methylation; and (3) as part of sequencing library preparation. In addition to general considerations about primer/probe design, it is equally important to understand the nature and composition of cirDNA in order to optimize analyses. Throughout the research literature, many different approaches have been taken to amplify and quantify cirDNA. Differences in analytical methods influence results, such that studies become incomparable and this hampers the progress of promising clinical biomarkers.