Highlights
- •MtDNA and nDNA qPCR assays were assessed using the NIST SRM 2372a.
- •Accuracy of DNA standards THP, 143B, and dsT8sig was determined.
- •QPCR efficiency, repeatability, and reproducibility of the assays was determined.
- •Inter-assay reproducibility of DNA concentration and degradation for bone extracts was evaluated.
Abstract
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. 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.SWGDAM Validation Guidelines for DNA Analysis Methods, Approved on 12/05/2016. 〈https://www.swgdam.org/_files/ugd/4344b0_813b241e8944497e99b9c45b163b76bd.pdf〉.
Keywords
To read this article in full you will need to make a payment
Purchase one-time access:
Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online accessOne-time access price info
- For academic or personal research use, select 'Academic and Personal'
- For corporate R&D use, select 'Corporate R&D Professionals'
Subscribe:
Subscribe to Forensic Science International: GeneticsAlready a print subscriber? Claim online access
Already an online subscriber? Sign in
Register: Create an account
Institutional Access: Sign in to ScienceDirect
References
SWGDAM Validation Guidelines for DNA Analysis Methods, Approved on 12/05/2016. 〈https://www.swgdam.org/_files/ugd/4344b0_813b241e8944497e99b9c45b163b76bd.pdf〉.
E. Romsos, et al., Certification of Standard Reference Material 2372a, Human DNA Quantitation Standard, 2018. 〈https://doi.org/10.6028/NIST.SP.260-189〉.
- Forensic aspects of mass disasters: strategic considerations for DNA-based human identification.Leg. Med. 2005; 7: 230-243https://doi.org/10.1016/j.legalmed.2005.01.001
- Evaluating droplet digital PCR for the quantification of human genomic DNA: converting copies per nanoliter to nanograms nuclear DNA per microliter.Anal. Bioanal. Chem. 2018; 410: 2879-2887https://doi.org/10.1007/s00216-018-0982-1
- Production and certification of NIST Standard Reference Material 2372 Human DNA Quantitation Standard.Anal. Bioanal. Chem. 2009; 394: 1183-1192https://doi.org/10.1007/s00216-009-2782-0
- Real-time DNA quantification of nuclear and mitochondrial DNA in forensic analysis.BioTechniques. 2002; 33: 402-411https://doi.org/10.2144/02332rr07
- A duplex real-time qPCR assay for the quantification of human nuclear and mitochondrial DNA in forensic samples: implications for quantifying DNA in degraded samples.J. Forensic Sci. 2005; 50: 1044-1060https://doi.org/10.1520/JFS2004423
FBI Laboratory Quality System Documents, 2020 [cited 2020; Federal Bureau of Investigation, Department of Justice]. Available from: 〈http://fbilabqsd.com〉.
- Quality assurance standards for forensic DNA testing laboratories.Forensic Sci. Commun. 2000; 2: 3
- The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.Clin. Chem. 2009; 55: 611-622https://doi.org/10.1373/clinchem.2008.112797
- Internal validation of human mitochondrial DNA quantification using real-time PCR.J. Forensic Sci. 2014; 59: 1049-1056https://doi.org/10.1111/1556-4029.12477
- Quality assessment of human mitochondrial DNA quantification: MITONAUTS, an international multicentre survey.Mitochondrion. 2011; 11: 520-527https://doi.org/10.1016/j.mito.2011.01.011
- Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number.Anal. Bioanal. Chem. 2009; 394: 457-467https://doi.org/10.1007/s00216-009-2729-5
- DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials.Anal. Bioanal. Chem. 2015; 407: 1831-1840
- Quantification of human mitochondrial DNA using synthesized DNA standards.J. Forensic Sci. 2011; 56: 1457-1463https://doi.org/10.1111/j.1556-4029.2011.01871.x
- Development of a triplex mtDNA qPCR assay to assess quantification, degradation, inhibition, and amplification target copy numbers.Mitochondrion. 2018; https://doi.org/10.1016/j.mito.2018.09.007
FBI DNA Standard Operating Procedures: DNA Procedures for Preparation and Extraction of Calcified Tissue Samples, 2019. 〈http://fbilabqsd.com〉.
Quantifiler Trio Quantification Manual, 2014. [cited 2019 Feb 03]; Available from: 〈http://tools.thermofisher.com/content/sfs/manuals/4485354.pdf〉.
- Sequence and organization of the human mitochondrial genome.Nature. 1981; 290: 457-465https://doi.org/10.1038/290457a0
NovaQUANT™ Human Mitochondrial to Nuclear DNA Ratio Kit, 2011, EMD Millipore Corporation, 〈http://www.emdmillipore.com/US/en/product/NovaQUANT-Human-Mitochondrial-to-Nuclear-DNA-Ratio-Kit,EMD_BIO-72620〉.
- EMPOP—a forensic mtDNA database.Forensic Sci. Int.: Genet. 2007; 1: 88-92https://doi.org/10.1016/j.fsigen.2007.01.018
- Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA.Nature Genetics. 1999; 23: 147https://doi.org/10.1038/13779
- A practical approach to RT-qPCR—Publishing data that conform to the MIQE guidelines.Methods. 2010; 50: S1-S5https://doi.org/10.1016/j.ymeth.2010.01.005
- Guidelines for validation of qualitative real-time PCR methods.Trends in Food Science & Technology. 2014; 37: 115-126https://doi.org/10.1016/j.tifs.2014.03.008
Article info
Publication history
Published online: April 17, 2022
Accepted:
April 12,
2022
Received in revised form:
March 25,
2022
Received:
September 8,
2021
Identification
Copyright
Published by Elsevier B.V.
