An assessment of qPCR assays for DNA concentration and degradation

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2019-05

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Cropper, Emily R.

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Abstract

Forensically challenged samples are often composed of degraded, damaged, or low template mitochondrial DNA (mtDNA). A real-time quantitative polymerase chain reaction (qPCR) assay can help determine if there is sufficient quantity and robust quality of mtDNA to move forward with downstream sequencing and analysis. The fundamental issue with qPCR is that the nominal quantity of the DNA calibrated along the commercial standard used for quantification can vary depending on the supplier and lot numbers. The National Institute of Standards and Technology (NIST) has developed a commercially available human DNA standard, Standard Reference Material (SRM) 2372a, which consists of nuclear DNA (nDNA) and mtDNA data on three wellcharacterized human genomic DNA preparations. The SRM 2372a was used to compare three qPCR assays: a non-commercial triplex assay, for mtDNA quantification, and two commercial assays, Quantifiler Trio (QFTrio) for nDNA quantification, and NovaQUANT for nDNA quantification and determination of the mtDNA/nDNA ratio. Quantification of the SRM uniformly across these three qPCR assays allowed for the conclusion that a robust, reproducible, accurate, and efficient qPCR assay is dependent on (1) the quality and reliability of the DNA standard, (2) the specificity of the qPCR chemistry, and (3) sound primers and probes, to name a few. The findings indicate that commercially available qPCR assays do not necessarily perform as marketed and should be re-verified by a validated DNA SRM.

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