Rapid forensic DNA typing using microchip electrophoresis
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NO FULL TEXT AVAILABLE. Access is restricted indefinitely. ----- Since first being described in 1985 forensic DNA identification has become one of the most high profile areas of forensic science. DNA analysis is now a routine procedure, with the methods and statistical interpretations widely accepted by both the law enforcement and criminal justice systems. As the field and application of forensic DNA profiling continues to expand, a number of issues have developed. One issue most prominent in the media is the considerable backlog of evidence awaiting analysis. One suggested solution for this problem is the development of faster techniques for screening or triaging of DNA samples. Simultaneously, there is a move towards real-time forensic intelligence gathering requiring methods and technologies that are both rapid and portable. Emerging technologies such as microfluidic devices offer the potential for reduced analysis times, increased sensitivity, and the development of miniaturised, field portable instruments. The Agilent 2100 Bioanalyzer is a commercially available electrophoresis-based microfluidic device. One limitation of the platform is that the current DNA methods are unable to provide the resolution, reproducibility or accuracy required for the analysis of common forensic DNA markers. This project investigates potential improvements to the method that could allow the platform to be used for screening of forensic DNA samples. Optimisation of the instrument parameters and operating script improved the resolution and minimised migration time drift. A Tris-Acetate-EDTA buffer (four times the standard TAE concentration) provided more stable voltages and currents over the course of the assay, improving the reproducibility of the separation. Polymer parameters such as concentration, molecular weight, and polydispersity were investigated and the best separation achieved using a low viscosity/molecular weight poly(N,N-dimethylacrylamide) gel of low-moderate polydispersity at a concentration of 8% (w/v) in the TAE buffer. Method validation results revealed good accuracy (±1.7% RE; ±4 bp) and reproducibility (0.4-1.8% RSD) for DNA fragments across the size range of interest for forensic DNA profiling. 75 DNA samples were obtained that had been typed by the Australian Federal Police using the Promega PowerPlex® 16 System. These samples were amplified using a subset of the PowerPlex® loci (CSF1P0, TPOX, TFI01 and Amelogenin) and analysed in the Bioanalyzer. All Amelogenin profiles were correctly determined. For the three STRs, 90% of alleles were correctly sized, and over 99% of alleles were within the range ±1 repeat from the known profile. The method is thus not yet capable of providing accurate profiles; however it does have the potential to be used for rapid screening or triaging applications. For scenarios such as closed-set disaster victim identification and the re-association of fragmented remains, the advantages of an initial rapid analysis method could outweigh the need for a more discriminating method. To simulate a closed-set victim situation, 22 blind samples (a subset of the original data set) were re-analysed using the Bioanalyzer. The profiles were compared to the PowerPlex® 16 profiles in the original data set and (by way of pattern-comparison) it was possible to correctly identify all 22 samples. Microchip devices such as the Agilent Bioanalyzer are likely to play some role in the future of forensic DNA sciences. The technology could be used for real time field-based intelligence, or to screen through backlogged evidence and identify probative samples. In the future, further improvements to resolution and reproducibility will allow for methods equal to existing capillary electrophoresis methods. Of particular interest will be the next generation of microchip devices, which will undoubtedly be capable of extraction, amplification and separation on a single chip . Analysis times and sample handling will be reduced even further, and could provide investigators with DNA profiles and identifications within minutes in the field.
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