Quantification of Human Mitochondrial DNA Using Synthesized DNA Standards*

Abstract

Successful mitochondrial DNA (mtDNA) forensic analysis depends on sufficient quantity and quality of mtDNA. A real-time quantitative PCR assay was developed to assess such characteristics in a DNA sample, which utilizes a duplex, synthetic DNA to ensure optimal quality assurance and quality control. The assay's 105-base pair target sequence facilitates amplification of degraded DNA and is minimally homologous to nonhuman mtDNA. The primers and probe hybridize to a region that has relatively few sequence polymorphisms. The assay can also identify the presence of PCR inhibitors and thus indicate the need for sample repurification. The results show that the assay provides information down to 10 copies and provides a dynamic range spanning seven orders of magnitude. Additional experiments demonstrated that as few as 300 mtDNA copies resulted in successful hypervariable region amplification, information that permits sample conservation and optimized downstream PCR testing. The assay described is rapid, reliable, and robust.

Development of a Universal Internal Positive Control

“…The universal IPC offers the convenience and quality of a fully synthetic DNA template [15,33,34] as well as a potential cost saving relative to commercial IPCs. Another advantage is flexibility of design, for instance, intercalating dye or hydrolysis probe qPCR, including choice of reporter dye and quencher in the latter.…”

“…Examples of commercially available standalone IPCs exist [10,1214]; although cost and/or assay incompatibility, for example, the fluorophore utilized, may exclude their use. As a case in point, a commercial exogenous IPC was successfully utilized in a duplex qPCR assay for quantifying human mtDNA [15,16]; however, subsequent development toward a triplex assay [17] prohibited its use due to dye emission overlap. In addition, custom modifications of a commercial IPC product, for example, an alternative reporter dye and/or alternative quencher, may beavailable at additional cost or not an option.…”

Massively parallel sequencing of complete mitochondrial genomes from hair shaft samples

“…Extract mtDNA yields were assessed using a custom real-time quantitative PCR (qPCR) assay specific for a 105 bp region of the human mtGenome [27] or the Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA) prior to any further processing.…”

Cuantificación en tiempo real de un conjunto de muestras colombianas de relevancia histórica basado en la detección de un fragmento corto de la región hipervariable II del ADN mitocondrial

“…Debido a que las diluciones seriadas se hicieron manualmente, la curva que se presenta carece de cierta linealidad, sin embargo, el coeficiente de correlación lineal obtenido se ajusta a los estándares sugeridos por otros autores (16,17).…”

“…La validez de los resultados obtenidos se estimó verificando su replicación, tal como lo recomiendan otros autores para la detección de ADN de muestras muy degradadas (16,17). Se ha reportado, asimismo, que el caso de las muestras en las que no se obtuvieron resultados replicables coincide con el de aquellas en las que se reportan menos de 100 copias por µl (28), como en este estudio.…”