Identification and Separation of Evidence Mixtures Using SNP-Based FISH Techniques and Laser Microdissection.

摘要

Laser microdissection (LM) has proven to be an effective method for cell mixture separations in the forensic laboratory. While sperm and epithelial cell sexual assault mixtures can be easily separated based upon morphological differences, mixtures of the same cell type are more difficult to separate. Past research has demonstrated that male/female cellular mixtures of similar morphology can be successfully separated using X/Y chromosome fluorescence in situ hybridization (FISH) probing. The goal of this research was to separate cellular mixtures of the same morphology and gender by developing FISH probes based on human genetic single nucleotide polymorphisms (SNPs). Screening panels of SNP FISH probes were intended to visually detect the individual contributors of sample mixtures, while laser microdissection would physically separate the cells for further STR processing. Padlock probes paired with tyramide signal amplification and rolling circle amplification were examined as methods of achieving this goal. Findings indicated that the FISH techniques utilized in this research were unsuitable for the detection of single SNP differences between individuals. Differentiation may be achieved in the future through research of other rolling circle amplification methods or by pursuing genetic marker systems that contain larger genetic differences. Multiple improvements in LM sample processing techniques were also examined. The use of cytogenetic on-slide lysis techniques and direct placement of LM collected cells into amplification reactions were evaluated as methods that would allow for the direct collection of nuclei and eliminate the need for DNA extraction prior to amplification.