An investigation into novel DNA manipulation strategies for forensic applications

Abstract

Over recent years there has been considerable interest in the development of faster more robust and sensitive ways of analysing DNA. In partial response to this, the Forensic Science Service® has developed a novel amplification strategy based on single nucleotide polymorphisms. In a continuing effort to speed up the analysis time, the concept of miniaturisation is beginning to receive more attention. Miniaturising the various elements required to complete a DNA analysis is going to require a considerable research effort, specifically when focusing on the areas of DNA extraction and integration as well developing new amplification strategies and their associated detection. This study specifically looked at three areas; microfabrication for DNA extraction, duplex stability assessment with a view to developing more efficient amplification strategies and microarray technology as a replacement for conventional DNA analysis techniques. Microfabricated silicon channels were designed and used in conjunction with a modified Qiagen chemistry. Through optimisation, these channels demonstrated themselves as a robust and efficient platform for DNA extraction. DNA pre-concentration and DNA purification were also demonstrated. In addition, the channels could be used as a means of quantification, by binding a fixed amount of DNA. Their simple design facilitated controllable liquid flow as well as minimising the potential for device blockage. These results demonstrated a proof of principle with regards to efficient miniaturised DNA extraction, providing a feasible first step to a fully integrated DNA analysis system .. Duplex stability is critical to the development of any novel amplification strategy. Future developments require an accurate and efficient assessment of oligonucleotide melting temperature particularly for efficient multiplex development. This investigation determined a new algorithm which calculated duplex melting temperature based on nearest neighbour thermodynamics. This algorithm was incorporated into a Visual Basic programme called MOSAIC. Through a series of validation experiments MOSAIC was found to be more accurate at predicting Tm in lower salt environments than the commercially available software Primer Express. It is expected that the improved algorithm can be used for future primer designs, for any new developing amplification strategy. The use of Microarray technology as an alternative detection strategy for SNP analysis was also investigated. Multiplexes consisting of up to 5 loci were detected by two different methods; direct hybridisation to the SNP site and indirect hybridisation to a universal tail. Both strategies showed equivalent levels of discrimination, reporting correct genotypes in each case. Accurate genotyping from a 10-plex PCR using the indirect approach was also demonstrated.

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