Degradation of nuclear and mitochondrial DNA after gamma-irradiation and its effect on forensic genotyping
Forensic genotyping can be impeded by gamma-irradiation of biological evidence in the event of radiological crime. Oxidative effects within the mitochondria elicit 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 gamma-irradiation. Liquid (hydrated) and dried (dehydrated) whole blood samples were exposed to high doses of gamma-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 normalised before comparison by calculation of integrity indices relative to unirradiated controls. For nuDNA, a subset of autosomal STR markers were selected for relative size consistency with three mtDNA targets (86, 190 and 452 bp), including loci of low molecular weight (D2S441, ~75-110 bp), intermediate molecular weight (vWA and D1S1656, ~150-210 bp), and high molecular weight (TPOX and SE33, ~310-450 bp). For STR size groups containing multiple loci, the average peak heights of alleles for each marker were determined. Integrity indices were calculated from the peak height or quantity ratios of increasing amplicon size difference, comprising intermediate/short (Index A), long/intermediate (Index B), and long/short loci (Index C). 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. Findings suggest that oxidative effects due to water radiolysis and mitochondrial function are dominant mechanisms of differential damage to nuDNA versus mtDNA after high-dose gamma-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. Degradation of mtDNA is more severe after gamma-irradiation than nuDNA; this does not adversely impact on genotyping success of blood samples up to 50 kGy.