Amplification efficiency of quantitative PCR reactions is improved by addition of non-target DNA

Authors: Reardon, Catherine; Manter, Daniel

Submitted to: Microbial Ecology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/10/2026
Publication Date: 3/31/2012
Citation: Reardon, C.L., Manter, D.K. 2012. Amplification efficiency of quantitative PCR reactions is improved by addition of non-target DNA. Microbial Ecology. 89(84). https://doi.org/10.1007/s00248-026-02719-0.
DOI: https://doi.org/10.1007/s00248-026-02719-0

Interpretive Summary: Quantitative PCR (qPCR) is a common, DNA-based method used to estimate the number of soil microbes, functional groups or species; however, it is dependent upon several factors that affect the amplification efficiency. Amplification efficiency, or reaction efficiency, is dependent on the sample type, qPCR enzymatic product and assay optimization including DNA primer design (sequence and target length). Although qPCR reactions are amiable to optimization, the design of DNA primers to meet the guidelines for relatively short (75-150 bp) amplicon size is often unfeasible especially when targeting broad groups of organisms or functional genes. This report demonstrates that addition of non-target DNA significantly increases the amplification efficiency for long target amplicons therefore pushing the boundaries of target size for this methodology. The method shows promise for different qPCR equipment and products. Plasmid as a non-target DNA provides benefit over more complex sources (i.e., genomic) and flexibility for use with different primers since risk for non-target amplification will be lower. Overall, this method enhances the accuracy and ease of qPCR, particularly when long amplicon analyses are needed.

Technical Abstract: Quantitative PCR (qPCR) is a common, culture-independent method to estimate gene copies at levels as broad 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 length (75–150 base pairs) 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) as a PCR additive significantly increases the amplification efficiency for long target amplicons up to 723 bp. The method is applicable for different template types including circular and linearized plasmids (24–30% increase) and cell-free DNA extracts (up to 26%), as well as different thermocyclers and qPCR products. As an exogenous DNA source, the pUC19 plasmid provides flexibility with a reduced chance of non-target amplification due to the relatively simple, short, and known DNA sequence. Overall, this method enhances amplification efficiency, reduces template bias, and lowers qPCR variability with a strong benefit for long amplicon assays.