Development and validation of a SYBR green-based mitochondrial DNA quantification method by following the MIQE and other guidelines.

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 (r ≥ 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.