Document Title: Laser Microdissection as a Technique to Resolve Mixtures and Improve the Analysis of Difficult Evidence Samples

Laser microdissection (LM) has proven to be an effective method for cell mixture separations in the forensic laboratory. Its adaptation to the forensics field has provided a means of physically separating the components of assault mixtures as well as improving the collection and DNA analysis associated with low copy number (LCN) samples. The work performed for NIJ Grant # 2006-DN-BX-K032 has focused on the further investigation of the various aspects and benefits of this burgeoning technology. Numerous facets of the processing of evidence containing LCN samples with LM methods were examined including: the best technique to prepare and transfer cells from the evidence to the slide, the enhancement of the extraction process, optimization of the STR amplification procedure, using Fluorescent In Situ Hybridization (FISH) techniques to resolve difficult mixtures, and the effectiveness of laser microdissection across a wide range of evidentiary samples. Findings indicated that the swabbing of substrates followed by a cellulase aided elution did increase the number of cells recovered for LM slide preparation. The QIAamp Micro Extraction kit was found most efficient and reliable for extracting low level samples collected by LM. Amplification results indicate that the Applied Biosystems AmpFlSTR MiniFiler kit is the optimal choice for amplifying LCN samples collected with LM instruments. Additional research was also completed on the cellular separation of problematical sample mixtures. While sperm and epithelial cell sexual assault mixtures can easily be separated based upon morphological differences, combinations of the same cell type are more difficult to separate. For problematic samples like these, a protocol was developed that is capable of successfully separating male/female cellular mixtures of similar morphology using chromosome X/Y The Bode Technology Group, Inc. Page 2 2006-DN-BX-K032 This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.

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