Development of a multiplex assay for detection of autosomal and Y-chromosomal STRs, assessment of the degradation state of mitochondrial DNA and presence of mitochondrial length heteroplasmies.

The current focus in most routine forensic casework is detection of autosomal or gonosomal Short Tandem Repeats (STRs). With increasing degradation, STR analysis tends to be less successful up to complete failure. For challenging samples such as telogen hair roots and shafts, touch DNA samples or skeletal remains, mitochondrial DNA (mtDNA) analysis provides a powerful tool. Determination of DNA quantity is an important part in the casework workflow. Several ready-to-use kits are commercially available for nuclear DNA targets. However, quantification of mtDNA targets requires the establishment of an in-house method. Some assays even contain assessment of degradation, which alleviates the choice of target enrichment for sequencing through medium or small amplicons. As Sanger-type Sequencing (STS) still remains the golden standard in many laboratories, identification of heteroplasmies in C-tract regions prior to the sequencing reaction is advantageous. Firstly, primer selection can be expanded with primers binding near the C-tract and secondly, determination of the dominant variant is straightforward. All those quantity (nuclear and mtDNA) and quality (degradation and length heteroplasmies) evaluations usually require at least two separate reactions. Therefore, the aim of this project was the combination of all these targets in one multiplex assay using capillary electrophoresis to spare valuable sample extract. Amplification of representative autosomal and Y-chromosomal STRs allows estimate of success of (Y-)STR analysis. Simultaneously, five length heteroplasmies in the mitochondrial control region are targeted as well as three conservative regions of differing fragment lengths for assessment of the mitochondrial degradation state. Based on the outcome of this assay, forensic examiners can decide if STR analysis may be suitable. In case of absent STR peaks, appropriate proceeding of mtDNA sequencing can be determined.

Best of both: A simultaneous analysis of mRNA and miRNA markers for body fluid identification.

The body fluid identification of traces found at crime scenes is crucial in relation of the circumstance of crime. For this reason, the body fluid identification (BFI) by molecular biological methods has been increasingly investigated in recent decades. Especially the use of messenger RNA (mRNA) has been established and validated by various studies. mRNAs can resist degradation for several decades under dry and dark environmental conditions, but degradation increases greatly e.g., in humid environments and UV radiation. In contrast, the shorter and protein-protected micro RNAs (miRNAs) are less susceptible to degradation, but not all potential markers are tissue-specific. The aim of this study was to develop a simultaneous mRNA/miRNA multiplex assay to take advantage of both types of RNA. The final assay was tested for various body fluids, dilutions, and mixtures. To demonstrate the advantage of a combined mRNA/miRNA assay, older and mostly degraded samples were examined and compared to an established mRNA assay. Initial results from degraded samples show that tissue-specific miRNAs expected could be detected for 93% of the degraded samples compared to mRNA markers with 25% of the mRNA assay. The result is a simultaneous mRNA/miRNA multiplex assay on capillary electrophoresis (CE) for the first time.

The diversity of shedder tests and a novel factor that affects DNA transfer.

Since the first shedder test was formulated almost 20 years ago, a plethora of different test strategies has emerged. The amount of data generated so far is considerable. However, because of the limited reproducibility of its results, the reliability of the shedder concept is frequently questioned. This study provides a literature overview of applied shedder tests that capture the diversity of the concept. It is pointed out to what extent different classification criteria, workflows, and trace evaluation can impair the classification outcome. The robustness of shedder status was assessed by applying a promising approach established by Fonneløp et al. (Forensic Sci Int Genet 29:48-60, 21). Data provide similar results to those in recent studies but also ambiguous shedder classifications. The applied shedder test was adapted based on our own as well as the reviewed data. With novel classification parameters, promising results were achieved. This study reveals uncertainties and inconsistencies of the shedder concept. Recommendations for harmonization and transparency are proposed. Implementation of the recommendations may result in an increased impact on casework and transfer studies, including activity-level assessments. Furthermore, this study shows that moisturizers affect participants' shedder status as well as DNA transfer. The impact appears to remain relevant even 60 min post ointment application but depends greatly on the type of moisturizer applied.