Comparison of Six Commercially-Available Dna Polymerases for Direct PCR

Abstract

SUMMARYThe use of a "direct PCR" DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens.

Live‐cell PCR and one‐step purification streamline DNA engineering

“…Another version of the enzyme, KOD Xtrem Hot Start DNA Polymerase was developed for direct PCR of inhibitor-containing blood samples. 21 We found that this polymerase also supported long range PCR of BAC containing cells. However, the polymerase and its proprietary buffer were not equally important to the PCR.…”

“…Although the DNA yield was different depending on the background plasmid structure, 25‐30 µL of PCR mix usually contained a sufficient amount of DNA for screening purposes, that is, elimination of background samples (running 4‐5 µL of PCR mix on gel electrophoresis) and for DNA sequencing (even with additional DNA column purification techniques; Table ). Overall, using live‐cell PCR for the detection of background colonies is a mild improvement in DGR of dsDNA, but this approach has not been used before . Therefore, it is technically novel.…”

“…Overall, using live-cell PCR for the detection of background colonies is a mild improvement in DGR of dsDNA, but this approach has not been used before. 7,[21][22][23] Therefore, it is technically novel.…”

Molecular tools for phytoplankton monitoring samples

“…Previously, other works (Bertozzini et al 2005, Simonelli et al 2009, Eland et al 2012 also compare comercial kits for phytoplankton DNA extraction, or even Taqs for animal samples (Arezi et al 2003, Purzycka et al 2006, Miura et al 2013, Joana & Azevedo 2015, Tahir & Yaqoob 2016. This updated work goes in deep comparing more DNA extraction kits (12) and seven DNA polymerases for phytoplankton samples, to find out the best option for phytoplankton monitoring samples (PMS).…”

A fast and accurate method of detecting Aleutian mink disease virus in blood and tissues of chronically infected mink

“…PCR inhibitors affect the activity of DNA polymerase enzymes (Rådström et al 2004;Eilert and Foran 2009;Monroe et al 2013), and there are differences among enzymes in their ability to directly amplify DNA in blood by PCR (Abu Al-Soud and Rådström 1998;Miura et al 2013). The Omni Klentaq is a Taq DNA polymerase that lacks 278 amino acids in its N-terminal region (Klentaq 1) and contains a glutamic acid to lysine substitution at codon 708, which confers resistance to several PCR inhibitors (Kermekchiev et al 2009) and can directly amplify target DNA from samples containing as much as 25% whole blood, plasma, and serum when used with the inhibitorresistant PCR Enhancer Cocktail (Zhang et al 2010).…”