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Manipulation and extraction of genomic DNA using functionalized magnetic particles for applications in microchip based bioanalysis

Authors Yeung, Siu Wai
Issue Date 2004
Summary A novel approach for extracting living cells' genomic DNA materials utilizing functionalized magnetic particles is developed in this thesis. This strategy is amenable to handle bio-samples in a miniaturized environment and it offers a possibility to separate and purify DNA from other cell lysate mixtures "on-chip", which is known to be a bottle-neck step in an integrated micro-Total-Analysis-System (μTAS). Furthermore, the ability to manipulate and transport magnetic beads linked DNA materials by external magnetic force enable the movement of DNAs to different microreaction chambers for various bioanalysis. Two methods have been studied to capture DNA using functionalized magnetic particles. The first approach involves the immobilization of biotinylated oligo-nucleotides, which are complementary to a selected region of the genomic DNA of interest, to avidin-coated magnetic particles. Through the hybridization between immobilized probes and genome (solid phase hybridization), DNA anchored on the magnetic particles can be separated from the rest of cell lysate mixtures when the magnetic force is applied. The efficiency of genome capturing can be improved by the second method at which the hybridization between biotinylated probes and genome DNAs takes place in the solution phase before the hybrid is linked to the particles via the biotin-avidin linkage (liquid phase hybridization). Conditions such as probe density on particles, probe concentration and the sequence of probe which may affect the capturing efficiency are investigated. A novel solid-phase polymerase chain reaction (PCR) coupled with "sandwiched" hybridization technique are demonstrated to further improve the genome capturing efficiency and the specificity/selectivity of the capturing by magnetic particles. Extraction of genomic DNA from living E.coli is successfully demonstrated in a silicon/glass based microreactor patterned with platinum heaters and sensors, which allow the thermal lysis of cell walls/membranes and the release of genetic materials for the subsequent bioanalytical steps of PCR amplification and hybridization detection. The successful on-chip capturing and decapturing of genomic DNAs illustrated in this thesis is a big step forward toward a total integrated bioanalytical microsystem for crude cells/sample analysis.
Note Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2004
Language English
Format Thesis
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