Document details for 'Improved real-time PCR estimation of gene copy number in soil extracts using an artificial reference'

Authors Daniell, T.J., Davidson, J., Alexander, C.J., Caul, S. and Roberts, D.M.
Publication details Journal of Microbiological Methods 91(1), 38-44.
Keywords Gene copy quantification, Internal standard, Quantitative PCR, Relative estimation, Soil ecology
Abstract Application of Polymerase Chain Reaction (PCR) techniques has developed significantly from a qualitative technology to include powerful quantitative technologies, including real-time PCR, which are regularly used for detection and quantification of nucleic acids in many settings, including community analysis where culture-based techniques are not suitable. Many applications of real-time PCR involve absolute quantification which is susceptible to inaccuracies caused by losses during DNA extraction or inhibition caused by co-extracted compounds. We present here an improvement to this approach involving the addition of an artificial internal standard, prior to nucleic acid extraction. By estimating gene target copies by relative abundance, this approach accounts for both loss during extraction and inhibition effects. We present a novel application of relative real time PCR allowing accurate estimation of total bacterial populations both within and across a wide range of soils and demonstrate its improvement over absolute quantification by comparison of both approaches to ELFA analysis of the same soils.
Last updated 2016-05-30
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