Quantitative PCR overestimation of DNA in samples contaminated with tin.

Metals can pose challenges while conducting forensic DNA analysis. The presence of metal ions in evidence-related DNA extracts can degrade DNA or inhibit PCR as applied to DNA quantification (real-time PCR or qPCR) and/or STR amplification, leading to low success in STR profiling. Different metal ions were spiked into 0.2 and 0.5 ng of human genomic DNA in an "inhibition study" and the impact was evaluated by qPCR using the Quantifiler™ Trio DNA Quantification Kit (Thermo Fisher Scientific) and an in-house SYBR Green assay. This study reports on a contradictory finding specific to tin (Sn) ions, which caused at least a 38,000-fold overestimation of DNA concentration when utilizing Quantifiler Trio. This was explained by the raw and multicomponent spectral plots, which indicated that Sn suppresses the Quantifiler Trio passive reference dye (Mustang Purple™, MP) at ion concentrations above 0.1 mM. This effect was not observed when DNA was quantified using SYBR Green with ROX™ as the passive reference, nor when DNA was extracted and purified prior to Quantifiler Trio. The results show that metal contaminants can interfere with qPCR-based DNA quantification in unexpected ways and may be assay dependent. The results also highlight the importance of qPCR as a quality check to determine steps for sample cleanup prior to STR amplification that may be similarly impacted by metal ions. Forensic workflows should recognize the risk of inaccurate DNA quantification of samples that are collected from substrates containing tin.

Degradation of nuclear and mitochondrial DNA after γ-irradiation and its effect on forensic genotyping.

Forensic genotyping can be impeded by γ-irradiation of biological evidence in the event of radiological crime; that is, criminal activity involving radioactive material. Oxidative effects within the mitochondria of living cells elicits greater damage to mitochondrial DNA (mtDNA) than nuclear DNA (nuDNA) at low doses. This study presents a novel approach for the assessment of nuDNA versus mtDNA damage from a comparison of genotype and quantity data, while exploring likely mechanisms for differential damage after high doses of γ-irradiation. Liquid (hydrated) and dried (dehydrated) whole blood samples were exposed to high doses of γ-radiation (1-50 kilogray, kGy). The GlobalFiler PCR Amplification Kit was used to evaluate short tandem repeat (STR) genotyping efficacy and nuDNA degradation; a comparison was made to mtDNA degradation measured using real-time PCR assays. Each assay was normalized before comparison by calculation of integrity indices relative to unirradiated controls. Full STR profiles were attainable up to the highest dose, although DNA degradation was noticeable after 10 and 25 kGy for hydrated and dehydrated blood, respectively. This was manifested by heterozygote imbalance more than allele dropout. Degradation was greater for mtDNA than nuDNA, as well as for hydrated than dehydrated cells, after equivalent doses. Oxidative effects due to water radiolysis and mitochondrial function are dominant mechanisms of differential damage to nuDNA versus mtDNA after high-dose γ-irradiation. While differential DNA damage was reduced by cell desiccation, its persistence after drying indicates innate differences between nuDNA and mtDNA radioresistance and/or continued oxidative effects within the mitochondria.

Singleplex quantitative real-time PCR for the assessment of human mitochondrial DNA quantity and quality.

Mitochondrial DNA (mtDNA) can provide a means for forensic identity testing when genotyping of nuclear DNA (nuDNA) targets is not possible due to degradation or lack of template. For degraded samples, an indication of the quantity and quality of mtDNA is essential to allow selection of appropriately sized targets for hypervariable region (HVR) analysis, which may conserve sample and resources. Three human-specific mtDNA targets of increasing length (86, 190 and 452 base pairs) were amplified by singleplex quantitative real-time PCR (qPCR), capable of providing an index of mtDNA degradation from fragment length information. Quantification was achieved by preparation of a standard curve for each target, using a purified mtDNA standard containing all three targets of interest, which produced a linear, accurate and precise result from 1×108 to 10 copies. These novel assays demonstrated excellent sensitivity, specificity and reproducibility in line with the minimum information for qPCR experiments (MIQE) guidelines. Further, a separate inhibition control reaction was included to guide sample clean-up and ensure the validity of degradation assays. This protocol assists the selection and analysis of appropriately sized targets to maximize the chance of obtaining an informative result in downstream assays like sequencing.

Deep vein thrombosis and stress ulcer prophylaxis in the intensive care unit.

Deep vein thrombosis (DVT) and stress gastric ulcers can be serious complications in patients admitted to the intensive care unit. This review discusses the risk factors associated with the development of DVT and stress-related mucosal disease (SRMD), evaluates the available literature on current options for DVT and stress ulcer prophylaxis, and examines the associated adverse effects and optimal duration of therapy.