Quantitative PCR overestimation of DNA in samples contaminated with tin.

Autor: Bonsu DNO; Chemistry and Forensic Sciences, Griffith University, Nathan, Brisbane, Queensland, Australia.; Forensic Research Group, Australian Centre for Ancient DNA (ACAD), School of Biological Sciences, The University of Adelaide, Adelaide, South Australia, Australia., Higgins D; Forensic Research Group, Australian Centre for Ancient DNA (ACAD), School of Biological Sciences, The University of Adelaide, Adelaide, South Australia, Australia.; School of Dentistry, Health and Medical Sciences, The University of Adelaide, Adelaide, South Australia, Australia., Simon C; Forensic Science SA, Attorney-General's Department, Adelaide, South Australia, Australia., Goodwin CS; Thermo Fisher Scientific, Scoresby, Victoria, Australia., Henry JM; Forensic Science SA, Attorney-General's Department, Adelaide, South Australia, Australia., Austin JJ; Forensic Research Group, Australian Centre for Ancient DNA (ACAD), School of Biological Sciences, The University of Adelaide, Adelaide, South Australia, Australia.
Jazyk: angličtina
Zdroj: Journal of forensic sciences [J Forensic Sci] 2023 Jul; Vol. 68 (4), pp. 1302-1309. Date of Electronic Publication: 2023 Jun 16.
DOI: 10.1111/1556-4029.15312
Abstrakt: 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.
(© 2023 American Academy of Forensic Sciences.)
Databáze: MEDLINE
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