Thesis

Evaluation of direct PCR for forensic DNA profiling and the development of a direct PCR multiplex

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Awarding institution
  • University of Strathclyde
Date of award
  • 2012
Thesis identifier
  • T13202
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Abstract
  • The use of direct PCR with different types of sample was explored in this study. Genomic DNA preparations at various concentrations and buccal cell counts were deposited on commonly encountered substrates, recovered and amplified using direct PCR before subjecting them to capillary electrophoresis. The electropherograms obtained were compared to those obtained using the standard DNA profiling protocol which involves extraction and amplification prior to capillary electrophoresis. Direct PCR was found to be better than the standard DNA profiling protocol in both studies and was further tested with fingerprints, touch DNA on fabric and blood and semen stains on fabrics. All these tests were successful with direct PCR indicating that this technique has the potential to be incorporated into routine forensic DNA testing. Supplementary tests were also carried out to compare the efficiency of the swabbing technique utilised and the effect different substrates had on DNA recovery. Four non-porous substrates, which were glass, stainless steel, plastic and ceramic, and four types of dyed fabrics, which were white cotton, light blue denim, nylon and brown cotton, were used to deposit DNA and the resulting DNA profiles were evaluated. Of the non-porous substrates tested, the highest recovery of DNA was observed with plastic while the lowest was observed with stainless steel. DNA deposited on fabric on the other hand gave variable results which we believe is dependent on the dye used to stain the fabric and the thickness of the fibres used. The results in this experiment indicated that the substrate DNA is deposited on plays an important role in determining the resulting DNA profiles. Finally, a novel multiplex consisting of five autosomal and two Y-chromosomal STRs which also provides the inhibitor status of the sample was developed. This multiplex also addresses the issues concerning sensitivity and robustness that was encountered with other commercially available multiplexes. The multiplex was developed, validated and tested with various mock crime scene samples successfully. Allelic ladder, panels and bins were created to be used with this multiplex to aid in sample designation when subjected to capillary electrophoresis.
Resource Type
DOI
Date Created
  • 2012
Former identifier
  • 948788

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