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Multiplex quantitative real-time polymerase chain reaction (qPCR) as a method for detecting PCR amplicon contamination of forensic DNA samples / Tessa Charlotte Dadd
Forensic DNA profiling has become an important tool in the criminal justice system, routinely being used to establish a statistical relationship between DNA evidence and an individual associated with a crime. A major problem facing DNA profiling is laboratory based contamination. This type of contamination typically occurs by the accidental transfer of PCR amplicons produced in previous experiments into fresh samples. Laboratory based contamination by PCR amplicons has been widely researched and many preventative measures are in place to control its occurrence (Hartley and Rashtchian, 1993). Contamination can also occur by the deliberate misuse of PCR amplicons added to DNA evidence at a crime scene. Previous studies by Berryman (2003), Dent (2006) and Frumkin et al., (2009) have demonstrated how PCR amplicons can be re-amplified, and detected in a DNA profile from a sample collected at a contaminated crime scene. Consequently, the resulting DNA profile can appear as a mixture or the original DNA profile can be completely masked by the amplified genomic DNA. In this thesis, a system to detect PCR amplicon contamination was developed. The detection system was designed using the hTERT locus found in the Quantifiler™ Human DNA quantification kit and the D3S1358 locus, which is one of the nine STRs amplified in the AmpF STR® Profiler Plus® PCR Amplification Kit. Ratios of fluorescence from each locus were calculated using the Cycle Threshold (Ct) values obtained from the amplification plots of the hTERT-D3S1358 assay. A narrow range of 0.86-1.04 Ct ( ) was observed for uncontaminated samples in the multiplex reaction. Contamination by PCR amplicons was indicated by an increased Ct ratio value above the established uncontaminated range. The ratio values for contaminated samples were used to determine at which dilution of amplicons the hTERT-D3S1358 assay was able to
detect. Samples contaminated after DNA was extracted could be detected in a 10-7 dilution of amplicons. Further tests using a 10-3 dilution of amplicons to contaminate DNA samples from hair and bloodstains in a simulated crime scene, revealed the amplicons were effectively carried through the standard DNA extraction methods of Chelex® and Qiagen®, and were able to be re-amplified in subsequent PCR reactions. The results presented in this thesis demonstrate the ability to simultaneously quantify the amount of amplifiable DNA in a genomic sample and identify whether the sample has been contaminated with additional alleles in the form of PCR amplicons. Acquiring this information is a necessary step for any forensic DNA analysis, as it can verify that the sample collected is adequate for PCR techniques such as STR analysis and that it is uncontaminated so a genuine DNA profile can be produced.
Thesis (M.For.Sc.)--University of Western Australia, 2010
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