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Forensic Stain Identification By RT-PCR Analysis

NCJ Number
Date Published
November 2008
129 pages
The primary aim of this research was to identify mRNA transcripts that would definitively identify the tissue of origin for a DNA sample, to determine if such transcripts survived typical adverse environmental effects that forensic samples might encounter, and to develop rapid multiplex assays for assessing these molecules using small amounts of sample.
Initial work focused on developing a method to co-extract DNA and RNA from the same sample. Several commercial methods were compared for their ability to isolate high-quality nucleic acids. Through these studies, researchers optimized a DNA extraction method that has become the researchers’ standard procedure for extraction of CODIS samples. Researchers assessed the stability of RNA over time using real-time TaqMan-based polymerase chain reaction (PCR) assays for the detection of blood-specific and semen-specific genes. Once RNA was shown to be stable in samples up to 4 years old, researchers attempted to identify two-three tissue-specific transcripts for a variety of stains. In addition to specificity, the sensitivity of the assays was determined by using stains of different sizes. Several technologies were used in multiplexing assays once candidates were shown to be tissue-specific. The Plexor One-Step qRT-PCR System was used to develop three different multiplex assays: blood-semen, semen-sperm, and semen saliva. The semen-saliva assay was designed in collaboration with Promega and is currently in the final stages of development. In cooperation with Promega, a RNA/DNA co-isolation technique was developed that extracts both nucleic acids of sufficient quality and quantity for downstream real-time PCR and single tandem repeat (STR) analyses. Homebrew TaqMan assays were developed for semen-sperm identification as well as a brain screening assay. Luminex-bead technology, in conjunction with the QuantiGene Plex Reagent System, was assessed for its ability to detect tissue-specific markers in forensic stains. 27 tables, 26 figures, 44 references, and a listing of presentations at scientific conferences

Date Published: November 1, 2008