Base composition profiling of human mitochondrial DNA using polymerase chain reaction and direct automated electrospray ionization mass spectrometry.

We describe an automated system for high-resolution profiling of human mitochondrial DNA (mtDNA) based upon multiplexed polymerase chain reaction (PCR) followed by desolvation and direct analysis using electrospray ionization mass spectrometry (PCR/ESI-MS). The assay utilizes 24 primer pairs that amplify targets in the mtDNA control region, including the hypervariable regions typically sequenced in a forensic analysis. Profiles consisting of product base compositions can be stored in a database, compared to each other, and compared to sequencing results. Approximately 94% of discriminating information obtained by sequencing is retained with this technique. The assay is more discriminating than sequencing minimum HV1 and HV2 regions because it interrogates more of the mitochondrial genome. A profile compared to a population database can be subjected to the same statistics used for assessing the significance of concordant mtDNA sequences. The assay is not hindered by length heteroplasmy, can directly analyze template mixtures, and has a sensitivity of <25 pg of total DNA per reaction. Analysis of 3331 independent trials of the same sample over 28 months produced an average mass measurement uncertainty of 10.1 +/- 8.0 ppm, with >99% of trials producing a full profile with automated analysis. The technique has direct application to analysis of forensic biological evidence.

Evaluation of DNAstable for DNA storage at ambient temperature.

Preserving DNA is important for validation of prospective and retrospective analyses, requiring many expensive types of equipment (e.g., freezers and back-up generators) and energy. While freezing is the most common method for storing extracted DNA evidence or well-characterized DNA samples for validation studies, DNAstable (Biomatrica), a commercially available medium for room temperature storage of DNA extracts was evaluated in this study. Two groups of samples consisting of different DNA quantities were investigated, one ranging from 20 to 400 ng (group 1) and the other one ranging from 1.4 to 20 ng (group 2). The DNA samples with and without DNAstable were stored at four different temperatures [∼25 °C (room temperature), -20 °C, 37 °C or 50 °C]. DNA degradation over several months was monitored by SYBR Green-based qPCR assays and by PCR amplification of the core CODIS STR markers for group 1 and 2 DNA samples, respectively. For the time points tested in this study (up to 365 days), the findings indicate that the -20 °C controls and the DNAstable protected samples at room temperature provided similar DNA recoveries that were higher compared to the unprotected controls kept at RT, 37 °C or 50 °C. These results suggest that DNAstable can protect DNA samples with effectiveness similar to that of the traditional -20 °C freezing method. In addition, extrapolations from accelerated aging experiments conducted at high temperatures support that DNAstable is an effective technology for preserving purified DNA at room temperature with a larger protective impact on DNA samples of low quantity (<20 ng).