# Amplification Efficiency of Quantitative PCR Reactions is Improved by Addition of Non-Target DNA

## TL;DR

Adding non-target DNA improves the efficiency of qPCR reactions, especially for longer DNA sequences.

## Contribution

A new method using non-target plasmid DNA enhances qPCR amplification efficiency for long amplicons.

## Key findings

- Adding 0.05 ng µL-1 of non-target plasmid DNA increases amplification efficiency for long amplicons up to 723 bp.
- The method improves efficiency for various template types including plasmids and cell-free DNA.
- The pUC19 plasmid is a suitable exogenous DNA source with low risk of non-target amplification.

## 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.

The online version contains supplementary material available at 10.1007/s00248-026-02719-0.