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Limitations of qPCR to estimate DNA quantity: An RFU method to facilitate inter-laboratory comparisons for activity level, and general applicability

Peter Gill, Øyvind Bleka, Ane Elida Fonneløp
doi: https://doi.org/10.1101/2022.05.23.493102
Peter Gill
aForensic Genetics Research Group, Oslo University Hospital, Oslo, Norway
bDepartment of Forensic Medicine, University of Oslo, Oslo, Norway
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  • For correspondence: peterd.gill@gmail.com
Øyvind Bleka
aForensic Genetics Research Group, Oslo University Hospital, Oslo, Norway
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Ane Elida Fonneløp
aForensic Genetics Research Group, Oslo University Hospital, Oslo, Norway
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Abstract

The application of qPCR to estimate the quantity of DNA present is usually based upon a short amplicon (typically c.80bp) and a longer amplicon (typically c.200-300bp) where the latter is used to determine the amount of degradation present in a sample. The data are used to make decisions about a) whether there is sufficient template to amplify? b) how much of the elution volume to forward to PCR? A typical multiplex amplifies template in the region of 100-500bp. Consequently, the results from an 80bp amplicon will tend to overestimate the actual amplifiable quantity that is present in a degraded sample. To compensate, a method is presented that relates the quantity of amplifiable DNA to the average RFU of the amplified fragments. This provides greatly improved accuracy of the estimated quantity of DNA present, which may differ by more than an order of magnitude compared to qPCR. The relative DNA quantities can be apportioned per contributor once mixture proportions are ascertained with probabilistic genotyping software (EuroForMix). The motivation for this work was to provide an improved method to generate data to prepare distributions that are used to inform activity level propositions. However, other applications will benefit, particularly those where extraction and quantification are bypassed: For example direct PCR and Rapid DNA technology. The overall aim of this work was to provide a method of quantification that is standardised and can be used to compare results between different laboratories that use different multiplexes. A software solution “ShinyRFU” is provided to aid calculations.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • https://github.com/peterdgill/ShinyRFU.git

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted May 23, 2022.
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Limitations of qPCR to estimate DNA quantity: An RFU method to facilitate inter-laboratory comparisons for activity level, and general applicability
Peter Gill, Øyvind Bleka, Ane Elida Fonneløp
bioRxiv 2022.05.23.493102; doi: https://doi.org/10.1101/2022.05.23.493102
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Limitations of qPCR to estimate DNA quantity: An RFU method to facilitate inter-laboratory comparisons for activity level, and general applicability
Peter Gill, Øyvind Bleka, Ane Elida Fonneløp
bioRxiv 2022.05.23.493102; doi: https://doi.org/10.1101/2022.05.23.493102

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