Invented in the 1950s, gel electrophoresis has now become a routine analytical method to verify the size of nucleic acids and proteins in molecular biology labs. Conventional gel electrophoresis can successfully separate DNA fragments from several base pairs to a few tens of kilo base pairs, beyond which a point is reached that DNA molecules cannot be resolved due to the size independent mobility. In this case, pulsed field gel electrophoresis (PFGE) was introduced to extend the range of DNA fragment sizes that can be effectively separated. But despite the incredible success of PFGE techniques, some important drawbacks remain. First, separation time is extremely long, ranging from several hours to a few days. Second, detection methods still rely on staining the gel after the run. Real time observation and study of band migration behavior is impossible due to the large size of the PFGE device. Finally, many commercial PFGE instruments are relatively expensive, a factor that can limit their accessibility both for routine analytical and preparative use as well as for performing fundamental studies. In this research, a miniaturized PFGE device was constructed with dimension 2cm x 2.6cm, capable of separating DNA fragments ranging from 2.5kb to 32kb within three hours using low voltage. The separation process can be observed in real time under a fluorescence microscope mounted with a cooled CCD camera. Resolution and mobility of the sample were measured to test the efficiency of the device. We also explored manipulating DNA fragments by end labeling DNA molecules with quantum dot nanocrystals. The quantum dot-DNA conjugates can be further modified through binding interactions with biotinylated single-stranded DNA primers. Single molecule visualization was performed during gel electrophoresis and the extension length, entanglement probability and reorientation time of different conjugates were measured to study their effect on DNA migration through the gel. Finally, electrophoresis of DNA conjugates was performed in the miniaturized PFGE device, and shaper bands were observed compared with the non end-labeled sample. Furthermore, by end-labeling DNA with quantum dots, the migration distance of shorter fragments is reduced, providing the possibility of separating a wider range of DNA fragment sizes on the same gel to achieve further device miniaturization.

Thesis (Ph.D.)--York University, 2008. Graduate Programme in Chemistry. / Typescript. Includes bibliographical references (leaves 183-207). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR39001

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

Thesis (M.S.)--Youngstown State University, 2008. / Includes bibliographical references (leaves 50-53). Also available via the World Wide Web in PDF format.

Honors Project--Smith College, Northampton, Mass., 2009. / Includes bibliographical references.

Thesis (M.S.)--Rochester Institute of Technology, 2007. / Typescript. Includes bibliographical references (leaves 91-98).

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第16864号 / 工博第3585号 / 新制||工||1542(附属図書館) / 29539 / 京都大学大学院工学研究科材料化学専攻 / (主査)教授 大塚 浩二, 教授 松原 誠二郎, 教授 田中 勝久 / 学位規則第4条第1項該当

Electrophoresis is a widely used analytical technique in the medical and biotechnology industries. It can provide for the determination of thousands of individual compounds on this small scale. The operating conditions are quite conducive to use with the delicate products of genetic engineering. Due to other complexities associated with scale-up, the process is not widely used on a large scale. A novel recycling electrophoretic instrument was investigated as a preparative protein separation device. The process occurs in a thin film of liquid between two flat plates, in a direction perpendicular to the flow. This device was unique in its use of a relatively high flowrate, and recycling of the process fluid as a method for increasing residence time. The apparatus was operated in three modes, isoelectric focusing (IEF), zone electrophoresis (ZE) and isotachophoresis (ITP). For use in the ITP mode, a computer was used for data acquisition and control functions. Model systems included monoclonal antibodies and lentil lectins.

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