Development and validation of a SYBR green-based mitochondrial DNA quantification method by following the MIQE and other guidelines
•We developed a simple SYBR Green-based mtDNA qPCR method.•Experiments were performed following MIQE and other guidelines.•K562 DNA at 100 pg/µL was converted into 16,400 copies/µL mtDNA using plasmid DNA.•NIST SRM 2372a was quantified with differences of up to −35.0% for 1/10 solutions.•Precision,...
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Published in | Legal medicine (Tokyo, Japan) Vol. 58; p. 102096 |
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Main Authors | , , , , , , , |
Format | Journal Article |
Language | English |
Published |
Ireland
Elsevier B.V
01.09.2022
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Subjects | |
Online Access | Get full text |
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Summary: | •We developed a simple SYBR Green-based mtDNA qPCR method.•Experiments were performed following MIQE and other guidelines.•K562 DNA at 100 pg/µL was converted into 16,400 copies/µL mtDNA using plasmid DNA.•NIST SRM 2372a was quantified with differences of up to −35.0% for 1/10 solutions.•Precision, effects of PCR inhibitors, and cross-reactivity were evaluated.
In forensic mitochondrial DNA (mtDNA) analysis, quantitative PCR (qPCR) is usually performed to obtain high-quality sequence data for subsequent Sanger or massively parallel sequencing. Unlike methods for nuclear DNA quantification using qPCR, a calibrator is necessary to obtain mtDNA concentrations (i.e., copies/µL). Herein, we developed and validated a mtDNA quantification method based on a SYBR Green assay by following MIQE [Bustin et al., Clin. Chem. 55 (2009) 611-22] and other guidelines. Primers were designed to amplify nucleotide positions 16,190–16,420 in hypervariable region 1 for qPCR using PowerUp SYBR Green and QuantStudio 5. The optimized conditions were 0.3 µM each primer and an annealing temperature of 60 °C under a 2-step cycling protocol. K562 DNA at 100 pg/µL was converted into a mtDNA concentration of 16,400 copies/µL using linearized plasmid DNA. This mtDNA calibrator was obtained by cloning the synthesized DNA fragments of mtDNA (positions 16,140–16,470) containing a 100-bp inversion. The linear dynamic range of the K562 standard curve was 10,000–0.1 pg/µL (r2 ≥ 0.999). The accuracy was examined using NIST SRM 2372a, and its components A, B, and C were quantified with differences of −29.4%, −35.0%, and −22.0%, respectively, against the mtDNA concentrations calculated from published NIST data. We also examined the specificity of the primers, stability of the reaction mix, precision, tolerance against PCR inhibitors, and cross-reactivity against DNA from various animal taxa. Our newly developed mtDNA quantification method is expected to be useful for forensic mtDNA analysis. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 1344-6223 1873-4162 |
DOI: | 10.1016/j.legalmed.2022.102096 |