Post-mortem interval estimation of human skeletal remains by micro-computed tomography, mid-infrared microscopic imaging and energy dispersive X-ray mapping.

In this study different state-of-the-art visualization methods such as micro-computed tomography (micro-CT), mid-infrared (MIR) microscopic imaging and energy dispersive X-ray (EDS) mapping were evaluated to study human skeletal remains for the determination of the post-mortem interval (PMI). PMI specific features were identified and visualized by overlaying molecular imaging data and morphological tissue structures generated by radiological techniques and microscopic images gained from confocal microscopy (Infinite Focus (IFM)). In this way, a more distinct picture concerning processes during the PMI as well as a more realistic approximation of the PMI were achieved. It could be demonstrated that the gained result in combination with multivariate data analysis can be used to predict the Ca/C ratio and bone volume (BV) over total volume (TV) for PMI estimation. Statistical limitation of this study is the small sample size, and future work will be based on more specimens to develop a screening tool for PMI based on the outcome of this multidimensional approach.

Evaluating sequence-derived mtDNA length heteroplasmy by amplicon size analysis.

Length heteroplasmy (LH) in mitochondrial (mt)DNA is usually observed in homopolymeric tracts and manifest as mixture of various length variants. The generally used difference-coded annotation to report mtDNA haplotypes does not express the degree of LH variation present in a sample, even more so, it is sometimes difficult to establish which length variants are present and clearly distinguishable from background noise. It has therefore become routine practice for some researchers to call the dominant type, the "major molecule", which represents the LH variant that is most abundant in a DNA extract. In the majority of cases a clear single dominant variant can be identified. However, in some samples this interpretation is difficult, i.e. when (almost) equally quantitative LH variants are present or when multiple sequencing primers result in the presentation of different dominant types. To better understand those cases we designed amplicon sizing assays for the five most relevant LH regions in the mtDNA control region (around ntps 16,189, 310, 460, 573, and the AC-repeat between 514 and 524) to determine the ratio of the LH variants by fluorescence based amplicon sizing assays. For difficult LH constellations derived by Sanger sequencing (with Big Dye terminators) these assays mostly gave clear and unambiguous results. In the vast majority of cases we found agreement between the results of the sequence and amplicon analyses and propose this alternative method in difficult cases.