Pre-Extraction Separation of Mixed Source DNA Samples through Micromanipulation and Direct PCR Short Tandem Repeat Typing of Cells

Highly sensitive DNA analysis methods have been in-use for forensic casework at the NYC Office of Chief Medical Examiner since January 2006. Having pioneered these techniques in the United States, we are the first American lab to use these methods to provide accurate and reliable forensic DNA testing results for evidence containing relatively small amounts of DNA. These items are typically touched or grabbed such as the handles of weapons, tools used to gain access to a safe for example, and clothing with no apparent biological staining. This evidence is recovered in the investigation of a wide variety of criminal offenses including homicide, sexual assault, felony assault, and property crime cases. As the evidence is often touched by multiple persons, not just one perpetrator but perhaps multiple perpetrators and/or the victim(s), often the results include DNA contributions from multiple individuals, deconvoluting these mixtures, particularly when dealing with initially small amounts of DNA, 100 pg or less, requires care and may be a complex process. Sometimes, the individual DNA profiles of the contributors cannot be determined, and even when they can, these profiles may be consistent with the victim and thus, may not be informative. In fact, from our extensive experience analyzing touched items, approximately 60% of the time, no foreign DNA profiles are found. Diverse microscopy and micromanipulation techniques are available. These methods may allow the separation of microscopically visible mixture contributions before they inadvertently become pooled in a DNA extraction. Individual contributors are separable on the basis of cell type. Sperm and epithelial cells may be distinguished by morphology. Skin flakes may group by the placement of individual contributors on the object. Specialized staining techniques allow visual discrimination of male from female originating cells of the same morphology. In this study, we propose to provide situational and substrate-specific recommendations for the use of three micromanipulation techniques which have been described in the literature, and to develop a highly efficient method of Direct PCR which will be universal to obtaining the Short Tandem Repeat (STR) profiles of the individual contributors to these substrates. The benefit of Direct PCR is several-fold: streamlines processes, reduces cost, and minimizes DNA loss. The substrates tested will consist of touched items, such as door handles and items of clothing, and mock sexual assault evidence with 2 or 3 volunteers as DNA sources. We will select discrete cellular elements such as individual skin flakes, and cells distinguished by staining or morphology, and place them into individual Direct PCR reactions. We propose that STR profiles originating from touch and trace samples may be able to forgo complex mixture deconvolution and be made progressively more sensitive and probative. Three methods of micromanipulation coupled to Direct PCR will be tested. We will evaluate manual microdissection with and without the use of adhesive microglobes, which involves direct microscopic visualization followed by individual cell plucking. Additionally, Laser Capture Microdissection (LCM), where slide mounted cells can be selected with high resolution will also be assessed. Lastly, robotically assisted Microdissection will be developed and evaluated, where tweezers mounted to a robotic arm pluck the cells with great precision, aided by adhesive microglobes. The OCME currently has LCM and manual microdissection capabilities, which are currently under various stages of preliminary development in our laboratory. Substrates will vary in material and opacity, and preparations will be made and compared from these substrates. Tape lifts, direct visualization of the object, and membrane coated slide preparation will be compared to provide the best process for treating an individual sample type. ca/ncf