Covalent end-immobilization of oligonucleotides onto solid surfaces

With the completion of the Human Genome Project, the focus of genetics research has shifted towards functional genomics, with emphasis on gene expression and polymorphism studies. To this end, there is rapidly increasing interest in solid-phase, high-throughput, combinatorial microarrays for DNA assays. For this purpose I synthesized oligonucleotides (oligos) stepwise onto derivatized SiO2 surfaces. Then double-stranded (ds)DNA molecules with "dangling end" oligo overhangs were immobilized onto the oligo surface by hybridization. Photolysis of psoralen crosslinkers covalently immobilized the dsDNA molecules to the oligo surface. The covalently end-attached dsDNA formed brush-like structures where the dsDNA strand could react under conditions resembling the natural solution-state found in vivo. This method minimized the possibility of nonspecific surface interactions, and could be developed for site-specific segregation of mixed dsDNA sequences from solution onto surface microarrays. Oligo surfaces with different densities were synthesized to determine the conditions that optimize dsDNA hybridization. The oligo surface density was controlled by derivatizing the Si02 surface with mixed compositions of alkylsilane molecules (X-(CH2)11-SiCl3, X= OH or CH3). X-ray photoelectron spectroscopy was used in conjunction with commercially available iodine-labeled nucleotides for quantifying oligo surface densities and stepwise reaction (coupling) efficiencies.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemical Engineering.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2001

Author dc:creator

• Lee, Ivan H. (Ivan Hao), 1967-

Advisor dc:contributor.advisor

• Paul E. Laibinis.

• Chemical Engineering.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/8202

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/8202