HIGH FIDELITY SURFACES AND DNA ARRAYS

University Of Wisconsin Madison, Madison WI

Investigators

Abstract

DESCRIPTION (Investigator's Abstract): The principal goal of this proposal is the development of high quality surfaces and nucleic acid arrays for applications including the analysis of gene expression, of genetic variations, of protein: nuclei acid interactions, and optical mapping. The proposed work falls into two interrelated areas: the development of a variety of surfaces designed and optimized for particular applications; and underlying this, basic research to develop improved understanding and control of the chemistry of the surfaces themselves. The surfaces to be developed fall into three major categories: Uniform (unpatterned) surfaces, surfaces patterned in a square grid pattern, and nanostructured surfaces. The surfaces will be prepared with various chemical functionalities as determined by the application. These functionalities will include hydroxyl groups (substrates for light-directed DNA array fabrication); amino groups (substrates for optical mapping, noncovalent PCR fragment deposition, or covalent attachment of activated oligonucleotides); thiol groups (substrates for covalent attachment of activated oligonucleotides); and thio-reactive or amino-reactive groups (for covalent attachment of oligonucleotides or PCR amplicons). Both vapor-deposited gold films on glass slides, and atomically flat silicon wafers will be employed as substrates. DNA molecules will be attached either by direct chemical coupling or by electrostatic interactions onto vapor-deposited gold thin films that have been chemically modified with densely packed self-assembled monolayers of alkanethiols that are terminated with various functional groups. Attachment to atomically flat hydrogen-terminated silicon surfaces will be accomplished by the UV-mediated reaction of functionalized aliphatic alkenes to form Si-C bonds, also suitable for further modification or direct DNA attachment. The surfaces will be required to meet stringent performance criteria, including physical and chemical stability, thermal stability, amenability to hybridization and enzymatic modification, control of the density of surface-bound DNAs, and reproducibility of preparation.