The physical separation and single source DNA profiling of individual mixture components by RNA in situ hybridization-based cell type identification

As submitted by the proposer: Mixtures are frequently encountered in forensic DNA analysis due to the involvement of multiple persons during the commission of certain crimes or due to recovery of mixed specimens due to unavoidable contamination of the crime sample with previously deposited cellular material from other individuals (e.g. from 'touch DNA'). Interpretation of mixture profiles without first separating out the component cell types prior to analysis can be complex and time consuming. Currently, physical separation of mixture components is largely limited to cases of sexual assault in which sperm cells can easily be separated from vaginal epithelia cells due to morphological differences. However, in many cases (e.g. fingernail scrapings, bite-marks, strangulations), non-distinguishable epithelial cell mixtures will be present often with only a minor contribution from the perpetrator. Therefore the proposed work seeks to provide a novel molecular based strategy for the physical separation of epithelial cell types in admixed samples (i.e. mixture de-convolution) using RNA in situ hybridization-based cell type identification. In this work, we will develop RNA in situ hybridization (RNA-ISH) methodologies for the purposes of distinguishing individual cell types {buccal, vaginal or skin) in non-distinguishable epithelial cell mixtures. We will develop highly specific and sensitive epithelial cell probes based on our extensive knowledge and experience with RNA biomarkers for body fluid identification. Optimized strategies for the isolation of cell type populations will be developed using laser capture micro-dissection or micro-manipulation techniques. Robust and sensitive STR amplification strategies will be employed in order to obtain single source profiles from the separated cell types. A full validation, including a demonstration of bona fide casework applications, will be performed in order to demonstrate the reliability and accuracy of the developed methods. The development of an RNA in situ hybridization based cell type identification strategy will permit the physical separation or 'de-convolution' of undistinguishable epithelial cell mixtures prior to DNA profiling. This approach will not only result in single source profiling of each of the individual mixture contributors, but will also provide a prior source attribution {body fluid or tissue of origin) for each of the obtained DNA profiles. This collaborative work (UCF and ESR) brings together world leaders in RNA profiling methods for body fluid identification and the enhanced analysis of low template (single cell) samples. Study results will be presented to the forensic community through comprehensive technical reports, peer-reviewed articles and presentations at domestic and international scientific conferences. ca/ncf