Improving amplification efficiency of qPCR with non-target DNA

Raw dataset for manuscript entitled "Amplification efficiency of quantitative PCR reactions is improved by addition of non-target DNA" submitted to Nucleic Acids Research. Authors: Catherine Reardon, Daniel Manter Abstract: Quantitative PCR (qPCR) is a common, culture-independent method to estimate gene copies at levels as coarse as Kingdom and specific as guild, function or species; however, it is dependent upon several factors that affect the amplification efficiency. Amplification efficiency of an individual qPCR reaction is dependent on template type, qPCR product and assay optimization including primer design. Although qPCR reactions can be optimized, the design of primers to meet guidelines for relatively short amplicon size (75-150 base pair) is often unfeasible especially when targeting a broad phylogeny of organisms or functional genes. The addition of exogenous non-target plasmid DNA (0.05 ng μL-1) significantly increases the amplification efficiency for long target amplicons up to 700 bp. The method shows broad applicability for different template types including circular and linearized plasmid templates (24-30% increase) and cell-free DNA extracts DNA (up to 6%) as well as different thermocyclers and qPCR products. As an exogenous DNA source, the pUC19 plasmid provides flexibility in primer design and reduced chance of non-target amplification due to the relatively simple, short, and known DNA sequence. Overall, this method enhances amplification efficiency and ease of qPCR analyses with reduced template- and well-based variability particularly with long amplicon analyses.