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|Author(s):||Nathanael D. Olson; Jayne B. Morrow;|
|Title:||Framework for the Evaluation of DNA Extract Suitability for Identification, Quantification and Sequencing of Unknown Microorganisms Microorganisms|
|Published:||December 03, 2012|
|Abstract:||This study provides an evaluation of DNA extraction methods when tasked with identifying unknown microorganisms. Research, public health, and clinical laboratories frequently turn to DNA-based molecular methods to identify and quantify unknown microorganisms for biodetection and metagenomic applications. With over 30 manufacturers of commercial nucleic acid isolation kits, identifying the optimal extraction method is a daunting task when the target organism is known, let alone when the organism is unknown. In this study, five different cells types (Gram positive (2), Gram negative, spore, and yeast) were extracted using six different methods, for a total of 108 extractions. The extracted DNA was evaluated for its suitability for frequently used downstream applications, such as next generation sequencing (NGS) and quantitative polymerase chain reaction (qPCR). Assessment was based on quantity (concentration, yield, and extraction efficiency) and quality (intactness, purity, and the presence of PCR inhibitors). DNA extracts varied significantly in terms of quantity by both cell type and extraction method, with only two extraction methods yielding suitable quantities of DNA for NGS applications. However, DNA was amplified from all extracts using qPCR. Overall, more than 90 percent of the extracted DNA was greater than 1 kb in size and therefore suitable for NGS and qPCR, and none of the cell types or extraction methods had statistically significant inhibition. The purity of the DNA, as assessed by UV spectroscopy, indicated co-extraction of RNA and polysaccharides for a number of samples.|
|Citation:||BMC Research Notes|
|Keywords:||DNA extraction, biodetection, metagenomics|
|Research Areas:||Homeland Security/Forensics/Human Identity, Chemical/Biological/Radiological/Nuclear/Explosives (CBRNE), Biothreat Agent Remediation|