The crucial combination of DNA polymerase, qPCR assay and background matrix

Abstract: Quantitative PCR (qPCR) is a commonly used method today in the fields of molecular diagnostics, forensic and food analysis. The real-time measurements of amplification and quantification of specific DNA molecules is one of many reasons why qPCR is the method of choice. However, presence of inhibitory compounds limit this method for example by interacting with either the DNA polymerase or the DNA template, increasing the errors and impairing the detection limit. This is why pre-PCR processing, where the effect of inhibitory compounds are handled prior to qPCR amplification, is of importance. A well-designed robust qPCR assay is essential to obtain reliable results. However, the choice of DNA polymerase also plays a significant role since different DNA polymerases are more or less tolerant to different types of inhibitors. The aims of this project were to study the amplification efficiency and detection limit when combining DNA polymerases with different qPCR assays. Moreover, the performance of the DNA polymerases with qPCR assays was also studied when more or less inhibitory matrices were added. Lastly, due to several parameters affecting the qPCR, a method with the purpose of determining the extension rate assay of the DNA polymerase was set up in the lab. The results showed that the performance of DNA polymerases varied depending on both qPCR assay and matrix. Moreover, the prominence of a robust qPCR assay was clearly observed. The results from the enzymatic rate assay indicated that performance of one DNA polymerase was decreased when inhibitory matrices were added, while the performance of another DNA polymerase on the contrary increased. However, further investigations are needed to elucidate the mechanisms behind this.