Quantitative PCR overestimation of DNA in samples contaminated with tin

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 io...

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Bibliographic Details
Published in:Journal of forensic sciences 2023-07, Vol.68 (4), p.1302-1309
Main Authors: Bonsu, Dan Nana Osei, Higgins, Denice, Simon, Claire, Goodwin, Corey S., Henry, Julianne M., Austin, Jeremy J.
Format: Article
Language:English
Subjects:
Amplification
Contaminants
DNA typing
fluorescence quenching
forensic science
Ions
passive reference dye
PCR inhibition
Quantifiler™ trio DNA quantification kit
quantitative PCR
Substrates
Tin
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Summary: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.
ISSN:0022-1198
1556-4029
DOI:10.1111/1556-4029.15312