Improved Techniques for High-Throughput Molecular Diagnostics

Detta är en avhandling från Stockholm : Kemi

Författare: Mario Curcio; Kth.; [2002]

Nyckelord: DNA; Molecular Diagnostics;

Sammanfattning: The amount of information derived from sequencing the humangenome is leading to an exponential increase in the rate atwhich genes and genetic disorders are mapped and characterized.As a consequence, the demand for genetic testing is alsodramatically increasing. Screening and assaying methods, otherthan direct sequencing, are gradually becoming available,although with different robustness, sensitivity and throughput.In the work summarized in this thesis, attention was devotedprimarily to the improvement and the development of newtechniques for some of these methods.Considering the role of capillary electrophoresis inmolecular diagnostics and an associated important phenomenon -electroosmotic flow (EOF) - a robust, reproducible procedurefor surface modification of the inner wall of capillaries wasreported (Paper I) and this method was beneficially employed insubsequent projects.The effort to screen and characterize point mutations in theCACNA1A gene, responsible for the Familial Hemiplegic Migraine(FHM) disease, led to the optimization and validation of a verysensitive technique on slab gel called Double Gradient–Denaturing Gradient Gel Electrophoresis (DG-DGGE) (Paper II). A more reliable and robust method forSingle-Strand Conformation Polymorphism analysis (SSCP) bycapillary electrophoresis, making use of neutral pH buffers,was also developed (Paper III), while the next project resulted in thedevelopment of a high-throughput method for assaying knownpolymorphisms by multiplex solid-phase minisequencing inmulti-capillary format using a detection system based on liquidcore waveguiding (Paper IV).As these and other methods, as well as most applications inmolecular diagnostics and molecular biology, depend on thepolymerase chain reaction (PCR), an effort was made to enhancethe throughput of this technology and to minimize reactionvolumes and costs. For this, the concept of a dynamic reactorwas employed, instead of static systems where the reactionmixture is exposed to temperature cycles in a confined space. Acontinuous flow of small-volume reaction mixtures, separated byan immiscible hydrophobic carrier fluid such as aperfluorocarbon, is transported in a hydrophobic tube throughthree zones, which are kept at constant temperatures optimizedfor denaturation, annealing and elongation (Paper V). If combined with a technique for automatedsample loading and collection (Chapter 7), this method should allow veryhigh-throughput miniaturized DNA amplification. A samplehandling concept using hydrophilic anchors is proposed, whichshould also be useful for other miniaturized reactions andchemical processing.Finally, some possible alternative methods are discussed aswell as future trends.Keywords:Amplification, anchor, array, assay,capillary, DGGE, disease, disorder, DNA, droplet, dynamicreactor, electrophoresis, fluorescence, fluorocarbon, gel,genetic alteration, genomics, genotyping, high-throughput,hydrophobic, liquid lid, miniaturized reaction, minisequencing,molecular diagnostics, mutation, PCR, polymerase chainreaction, screening, segmented flow, single nucleotidepolymorphism, SNP, SSCP, test.

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