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Readout Strategies for Biomolecular AnalysesGöransson, Jenny January 2008 (has links)
This thesis describes three readout formats for molecular analyses. A common feature in all works is probing techniques that upon specific target recognition ideally results in equimolar amounts of DNA circles. These are then specifically amplified and detected using any of the techniques presented herein. The first paper presents a method that enables homogeneous digital detection and enumeration of biomolecules, represented as fluorescence-labelled DNA macromolecules. This method offers precise measurements to be performed with a wide linear dynamic range. As an application, two closely related bacterial species were selectively detected. The second paper further investigates and optimizes the properties of the technique presented in paper one. The third paper demonstrates a platform that enables simultaneous quantitative analysis of large numbers of biomolecules. The array format and decoding scheme together propose a digital strategy for decoding of biomolecules. The array and the decoding procedure were characterized and evaluated for gene copy-number measurements. The fourth paper examines a new strategy for non-optical measurements of biomolecules. Characteristics of this technique are investigated, and compared to its optical equivalent, fluorescence polarization.
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Extracting Genomic Variations using Selector TechnologyIsaksson, Magnus January 2010 (has links)
This thesis describes the development and use of a new class of molecular tools called Selector probes, and its potential for investigations of genetic variation. The Selector technology provides multiplex amplification of targeted DNA sequences with a high specificity, and an enrichment factor in the same order of magnitude as PCR. A common feature in this thesis work is to focus the analysis on DNA regions of interest. For example, this technique can be implemented in analysing candidate regions found by whole genome studies that need validation (global to local analysis), and applications requiring detection of rare alleles (common to rare allele), important in for example cancer samples. An assay is presented that allows for fast and simple quantification of relative copy-number variations. The method was proven to be able to detect aneuploidy in chromosome 13, 18, 21 and X, with a resolution enough to distinguish between 4 and 5 copies. The method was successfully applied to solve a biological question regarding a copy-number variation, that explains the Ridge phenotype typical for the dog bread Rhodesian Ridgebacks. The Selector strategy was able to detect and map a tandem duplication with a size of 133 kb, which was characterized with base-pair resolution. A readout platform that facilitates simultaneous digital quantitative analysis of a large numbers of biomolecules is further introduced. The work involves arraying amplified product from successful selection and decoding each molecule by hybridization of fluorophore labeled oligonucleotides. Finally, a genome partitioning method which is applied upstream of next generation sequencing platforms is presented. It is shown that the method provides successful enrichment with 98 % coverage and 94 % specificity and high enrichment uniformity. The technique was applied for mutation analysis of 26 cancer-related genes in tumor cell-lines and tissue.
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