Assessment of human nuclear and mitochondrial DNA qPCR assays for quantification accuracy utilizing NIST SRM 2372a

Journal Article · Mon Apr 18 00:00:00 EDT 2022 · Forensic Science International. Genetics

DOI:https://doi.org/10.1016/j.fsigen.2022.102711· OSTI ID:1981615

Cropper, Emily ^[1]; Coble, Michael D. ^[2]; Kavlick, Mark F. ^[3]

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1. Federal Bureau of Investigation (FBI), Quantico, VA (United States). Research and Support Unit, Visiting Scientist Program; Univ. of North Texas, Ft. Worth, TX (United States). Health Science Center
2. Univ. of North Texas, Ft. Worth, TX (United States). Health Science Center
3. Federal Bureau of Investigation (FBI), Quantico, VA (United States). Research and Support Unit

In forensic DNA casework, a highly accurate real-time quantitative polymerase chain reaction (qPCR) assay is recommended per the Scientific Working Group on DNA Analysis Methods (SWGDAM) (SWGDAM Validation Guidelines for DNA Analysis Methods [1]) to determine whether a DNA sample is of sufficient quantity and robust quality to move forward with downstream short tandem repeats (STR) or sequencing analyses. Most of these assays rely on a standard curve, referred to herein and traditionally as absolute qPCR, in which an unknown is compared, relative to that curve. However, one fundamental issue with absolute qPCR is the quantifiable concentration of commercial assay standards can vary depending on (1) origin, i.e., whether from a cell line or a human subject, (2) supplier, (3) lot number, (4) shipping method, etc. In 2018, the National Institute for Standards and Technology (NIST) released a human DNA standard reference material for evaluating qPCR quantification standards, Standard Reference Material (SRM) 2372a, Romsos et al. (2018) [2] which contains three well-characterized human genomic DNA samples: Component A) a single male1 donor, Component B) a single female₁ donor, and Component C) a 1:3 male₂:female₂ donor, each with certification data for nDNA and informational mitochondrial DNA(mtDNA)/nuclear DNA (nDNA) ratio data. The SRM 2372a was used to assess four qPCR assays: (1) Quantifiler Trio (Thermo Fisher Scientific, Waltham, MA) for nDNA quantification, (2) NovaQUANT (EMD Millipore Corporation, San Diego, CA) for nDNA and mtDNA quantification, (3) a custom duplex mtDNA assay, and (4) a custom triplex mtDNA assay. Additionally, extracts from eighteen (18) skeletal remains were tested with the latter three assays for concordance of DNA concentration and with assays (2) and (3), for the degradation state. Our assessment revealed that an accurate, efficient, and reproducible qPCR assay is dependent on (1) the quality and reliability of the DNA standard, (2) the qPCR chemistry, and (3) the specific primers, and probes (if applicable), used in an assay. Finally, our findings indicate qPCR assays may not always quantify as expected and that performance of each lot should be verified using a well-characterized DNA standard such as the NIST SRM 2372a and adjusted if warranted.

• Research Organization: Oak Ridge Institute for Science and Education (ORISE), Oak Ridge, TN (United States)
• Sponsoring Organization: USDOE Office of Science (SC); Federal Bureau of Investigation (FBI)
• Grant/Contract Number: SC0014664
• OSTI ID: 1981615
• Alternate ID(s): OSTI ID: 1962286
• Journal Information: Forensic Science International. Genetics, Vol. 59, Issue C; ISSN 1872-4973
• Publisher: Elsevier
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Genetics & Heredity
Legal medicine
NIST SRM 2372a
DNA quantification
DNA standard
MIQE guidelines
DNA degradation
mtDNA
nDNA