Daniell, T.J., Davidson, J., Alexander, C.J., Caul, S. and Roberts, D.M.
Journal of Microbiological Methods 91(1), 38-44.
Gene copy quantification, Internal standard, Quantitative PCR, Relative estimation, Soil ecology
||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.
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