Quantitative PCR overestimation of DNA in samples contaminated with tin

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Bonsu, Dan Nana Osei
Higgins, Denice
Simon, Claire
Goodwin, Corey S
Henry, Julianne M
Austin, Jeremy J
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2023
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Abstract

Metals can pose challenges while conducting forensic DNA analysis. The presence of metal ions in evidence-related DNA extracts can degrade DNA or inhibit PCR as applied to DNA quantification (real-time PCR or qPCR) and/or STR amplification, leading to low success in STR profiling. Different metal ions were spiked into 0.2 and 0.5 ng of human genomic DNA in an “inhibition study” and the impact was evaluated by qPCR using the Quantifiler™ Trio DNA Quantification Kit (Thermo Fisher Scientific) and an in-house SYBR Green assay. This study reports on a contradictory finding specific to tin (Sn) ions, which caused at least a 38,000-fold overestimation of DNA concentration when utilizing Quantifiler Trio. This was explained by the raw and multicomponent spectral plots, which indicated that Sn suppresses the Quantifiler Trio passive reference dye (Mustang Purple™, MP) at ion concentrations above 0.1 mM. This effect was not observed when DNA was quantified using SYBR Green with ROX™ as the passive reference, nor when DNA was extracted and purified prior to Quantifiler Trio. The results show that metal contaminants can interfere with qPCR-based DNA quantification in unexpected ways and may be assay dependent. The results also highlight the importance of qPCR as a quality check to determine steps for sample cleanup prior to STR amplification that may be similarly impacted by metal ions. Forensic workflows should recognize the risk of inaccurate DNA quantification of samples that are collected from substrates containing tin.

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Journal of Forensic Sciences

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This is the peer reviewed version of the following article: Bonsu, DNO, Higgins, D, Simon, C, Goodwin, CS, Henry, JM, Austin, JJ. Quantitative PCR overestimation of DNA in samples contaminated with tin. J Forensic Sci. 2023; 00: 1– 8, which has been published in final form at https://doi.org/10.1111/1556-4029.15312. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited.

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This publication has been entered in Griffith Research Online as an advanced online version.

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Other biological sciences

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Criminology

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Bonsu, DNO; Higgins, D; Simon, C; Goodwin, CS; Henry, JM; Austin, JJ, Quantitative PCR overestimation of DNA in samples contaminated with tin, Journal of Forensic Sciences, 2023

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