Novel surface and multicolor charge coupled device-based fluorescent imaging system for DNA microarrays.

Abstract

We report a novel support, concomitant attachment chemistry, and a fluorescent imaging system for DNA microarrays. The support consists of soda lime glass coated with a layer of chromium, which eliminates any autofluorescence from the underlying glass substrate and reduces nonspecific probe binding. Attachment of DNA fragments exceeding 300 nucleotides in length is achieved without chemical modifications of either the chrome surface or the DNA itself. The charge coupled device (CCD)-based imaging system employs a 175 W xenon arc lamp as the light source, allowing the use of many different fluorophors. A 14 mm x 9 mm sample area is imaged with a single exposure, which takes between 5 and 20 s for each color plane in typical genomic comparative genomic hybridization type assays. The spatial resolution is limited only by the pixel size of the CCD chip (9 microm). The oblique illumination geometry combined with effective background reduction afforded by the chromium surface enables the system to achieve a detection limit of <5 x 10(7) fluorophors/cm(2) with 10 s integration. In a model system with arrayed lambda DNA targets a dose response was observed over four orders of magnitude in response to hybridizations with increasing amounts of the fluorescent labeled lambda probe.