Type of Document Dissertation Author Situma, Catherine N Author's Email Address email@example.com URN etd-04132007-093422 Title Fabrication of DNA Microarrays on Poly(methylmethacrylate) Substrates for Biomolecular Reporting Degree Doctor of Philosophy (Ph.D.) Department Chemistry Advisory Committee
Advisor Name Title Steven A. Soper Committee Chair Isiah Warner Committee Member Robert Cook Committee Member Steven Watkins Committee Member Stephania Cormier Dean's Representative Keywords
- Molecular Beacons
- Fluorescence Resonance Energy Transfer
Date of Defense 2007-03-07 Availability unrestricted AbstractDNA microarrays require the use of substrates with well-established surface modification / probe attachment chemistries. Glass/quartz have been widely adopted as typical support materials since their surface modification chemistries which involve the use of siloxane –based chemistries have been widely studied however; these chemistry is susceptible to hydrolytic cleavage especially at high or low pH values. Recently, polymers have been sought as alternative microarray support materials but their surface modification strategies are not well characterized compared to glass. This report will entail surface photo-modification of PMMA polymer substrates by UV irradiation which produces functional scaffolds of carboxylic groups that allow covalent attachment of amine-terminated oligonucleotide probes onto these surfaces via carbodiimide coupling chemistries. The photo-modification process for microarray fabrication involves only three steps; (1) broadband UV exposure of the polymer surface; (2) carbodiimide coupling of amine-terminated oligonucleotide probes to the surface (via an amide bond) and; (3) washing of the surface.
Since microfluidics offer several advantages such as reduction in reagent cost, reduction in hybridization assay times and parallel processing of samples; we incorporate them in the microarray construction by using poly (dimethylsiloxane) microchannels that are reversibly sealed to the photoactivated PMMA substrates. Parallel sample processing minimizes contamination effects that can give rise to false positives which can be a significant issue especially for diagnostic applications. We demonstrate use of these protocols with linear oligonucleotide probes for screening multiple KRAS 2 mutations possessing high diagnostic value for colorectal cancers whereby a Ligase Detection Reaction/universal zipcode array assays was carried out using parallel detection of two different low abundant DNA point mutations in KRAS 2 oncogenes with allelic composition evaluated at one locus. The same covalent attachment protocols were utilized for immobilizing hairpin probes (molecular beacons) in a microarray format that were used to report on the analysis of complementary DNA (cDNA) specific for fruitless (fru) and ods-site homeobox (OdsH) genes extracted from Drosophila Melanogaster fruit flies. To further improve the analytical sensitivities exhibited by these hairpin probes; we used phthalocyanine dyes for dual labeling of oligonucleotide probes that will be used for reporting on biomolecular association events.
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