This report presents the findings and methodology of research that demonstrated genomic DNA can be specifically targeted and covalently attached to a surface, followed by labeling for identification and quantification.
PCR-based DNA profiling technology is the gold standard for human identification technology, being highly discriminatory and requiring only several cells worth of DNA; however, it is still useful to develop alternative technologies that re-imagine how forensic DNA evidence is characterized, which may provide supplemental information under challenging conditions, such as with low copy numbers (LCN) samples, mixtures, and damaged DNA. The current project developed proof-of-concept experiments that demonstrate the potential application of single molecule techniques to forensic samples. The project first demonstrates that the forensic loci in genomic DNA can be specifically targeted for covalent attachment to a surface. This is essential, because the weak signal from a single DNA strand must be recorded for several minutes to determine its identity. This requires that it remain at a fixed position during the measurement. The project then showed that tandem repeat DNA sequences can be annealed to the STR region of the locus; and that by using fluorescent labeled repeats, it is possible to determine the size of the locus by single-step photo- bleaching. In contrast to the surface attached study, freely diffusing DNA samples were also characterized by using fluorescence quenching. Finally, the research examined the effectiveness of a pre-amplification protocol for PCR. The potential demonstrated in this research suggests the development in the near future of reliable techniques for the genotyping of degraded and LCN analysis; however, as a basic research project that explored the potential of various single molecule approaches, there is little immediate impact on the practice and policy of forensic investigations. Still, it encourages the long-term storage of samples that cannot currently be effectively analyzed. 12 figures, 4 tables, and 58 references
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