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.投稿于《核酸研究》（Nucleic Acids Research）的论文《添加非靶DNA可提升定量PCR反应扩增效率》的原始数据集。作者：凯瑟琳·里尔登（Catherine Reardon）、丹尼尔·曼特（Daniel Manter）。摘要：定量聚合酶链式反应（quantitative PCR，简称qPCR）是一种常用的非培养依赖型技术，可在界级至类群、功能或物种级的宽泛范围内估算基因拷贝数；但其扩增效率受多种因素制约。单个qPCR反应的扩增效率取决于模板类型、qPCR产物及包含引物设计在内的实验优化方案。尽管可对qPCR反应进行优化，但要设计符合较短扩增子长度（75~150碱基对）规范的引物往往难以实现，尤其是当目标覆盖宽泛的系统发育类群或功能基因时。添加外源非靶质粒DNA（0.05 ng μL⁻¹）可显著提升长达700 bp的长靶扩增子的扩增效率。该方法对多种模板类型具有广泛适用性，包括环状与线性化质粒模板（扩增效率提升24%~30%）、无细胞DNA提取物（最高提升6%），同时适配不同的热循环仪与qPCR产物体系。作为外源DNA来源，pUC19质粒因序列相对简单、短小且已知，可为引物设计提供灵活性，并降低非靶扩增的发生概率。总体而言，该方法可提升qPCR分析的扩增效率与操作便捷性，同时降低模板和反应孔间的变异度，尤其适用于长扩增子分析。