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Digital PCR inhibition mechanisms using standardized inhibitors representing soil and blood matrices
Applied Microbiology, Department of Chemistry at Lund University; Swedish National Forensic Centre (NFC) .
National Institute of Standards and Technology (NIST).
wedish National Forensic Centre (NFC) .
Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering. Swedish National Forensic Centre, Linköping, Sweden.
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2016 (English)Conference paper, Poster (Other academic)
Abstract [en]

Digital PCR (dPCR) enables absolute quantification of nucleic acids by partitioning the sample into hundreds or thousands of minute reactions. By assuming a Poisson distribution for the number of DNA fragments present in each chamber, the DNA concentrationis determined without the need for a standard curve. However, when analyzing nucleic acids from complex matrices such as soil and blood, the dPCR quantification can be biased due to the presence of inhibitory compounds. Here, we present how certain inhibitors disturb dPCR quantification and suggest solutions to these problems. Furthermore, we use real-time PCR, dPCR and isothermal titration calorimetry as tools to elucidate the mechanisms underlying the PCR inhibition. The impact of impurities on dPCR quantification was studied using humic acid as a model inhibitor. We show that the inhibitor-tolerance differs greatly for three different DNA polymerases, illustrating the importance of choosing a DNA polymerase-buffer system that is compatible with the samples to be analysed. Various inhibitory-substances from blood were found to disturb the system in different ways. For example, hemoglobin was found to cause quenching of fluorescence and a dramatic decrease of the number of positive reactions, leading to an underestimation of DNA quantity. IgG caused an increased number of late-starters. The system was more susceptible to inhibition by IgG when single-stranded DNA was used as template, compared with double-stranded DNA. By understanding more about the mechanisms of PCR inhibitors it will be possible to design more optimal PCR chemistries, improving dPCR detection and quantification.

Place, publisher, year, edition, pages
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Natural Sciences Biochemistry and Molecular Biology Law and Society
URN: urn:nbn:se:liu:diva-132620OAI: diva2:1047193
4th qPCR & Digital PCR Congress, London, UK, October 20-21, 2016
Available from: 2016-11-17 Created: 2016-11-17 Last updated: 2016-11-25Bibliographically approved

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