Plant PCR inhibitor release as a function of sample dissociation method

Abstract

The polymerase chain reaction (PCR) is an invaluable technique used in a wide range of molecular biology applications across multiple disciplines ranging from medical diagnosis and forensic analysis to botanical research. Since PCR is an enzymatic reaction, it is sensitive to inhibitors. PCR inhibitors may interfere with multiple steps in the PCR process leading to reduced sensitivity or even false negatives through inhibition of the amplification of nucleic acids by enzyme binding, nucleic acid cross‐linking/interaction, or through mimicking the nucleic acid structure. Inhibitors result from the original sample or during the extraction process. For plant studies, PCR inhibition may result from contamination by acidic polysaccharides and phenols including xylan, dextran sulfate, and flavonol; among others. While PCR inhibition has been demonstrated for multiple plant‐based compounds and procedural approaches have been recommended for removal of these inhibitors, to our knowledge, there has not been a study to compare the release of these compounds and the subsequent inhibitory effect based on different methods of sample dissociation.Herein, we evaluate three different dissociation techniques, including bead milling, cryo‐grinding and rotor‐stator dissociation for the extraction of genomic DNA and total RNA from sweet basil (Ocimum basilicum) and blueberries (Vaccinium corymbosum) and the release of plant PCR inhibitors. Polysaccharide, phenol, nucleic acid, and protein content was quantified and compared between each approach. qPCR was then employed to monitor the impact of PCR inhibitor content for both classic PCR and RT‐PCRThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.