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New Technologies for Genetic Analysis in Research and Diagnostics Settings

By Linda Strömqvist Meuzelaar


This thesis work was designed to provide advanced technologies for the analysis of genetic variation, with emphasis upon complex genome exploration and DNA diagnostics.\ud For DNA sequence analysis, the work was based on Dynamic Allele-Specific Hybridization (DASH). DASH was originally developed for SNP genotyping via melt-curve analysis of allele-specific oligonucleotides. DASH was adapted for more general use in DNA diagnostics, and was demonstrated for mutation scanning of a 97 bp human genomic sequence and parallel detection of multiple mutations in the gyrA gene of Salmonella. Mutation scanning studies utilised a series of partially overlapping probes, where base substitutions were detected by alterations in the melting profile of the probes hybridising to each variant base.\ud Three SNPs in the target sequence were correctly detected in 16 samples. The mutation status of 62 Salmonella strains was accurately determined, using a combination of wildtype and mutation-specific probes. Work was also initiated to convert this concept to a microarray format, and to establish a platform for Thalassaemia diagnostics.\ud To facilitate highly parallel analyses, ‘MegaPlex PCR’ was developed for multiplex amplification of specific DNA fragments. This technology employs solid-phase PCR to capture many different targets and at the same time incorporate common primer sequences. Liquid-phase PCR is then used to amplify all products simultaneously, using a single primer pair. Innovations included the use of partly double-stranded surface-oligonucleotides (‘Barrier Oligos’) to enhance surface reactions, and the use of complex genomic DNA in native format and after pre-amplification. A final proof-of-principle study targeted 50-plex and 75-plex reactions to genotype human SNPs. Next-generation sequencing revealed that up to 86% of the targeted sequences were recovered and less than 10% of the amplified molecules were primer-dimers.\ud With further development, methods such as these should have substantial utility for improved DNA diagnostics and personalised medicine

Publisher: University of Leicester
Year: 2007
OAI identifier: oai:lra.le.ac.uk:2381/9238

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