Quantitative comparison of protocols for DNA attachment and hybridization to glass

High-density DNA microarrays are a promising technology for high throughput analysis of gene expression. Several DNA attachment and hybridization protocols have been reported using glass substrates. These methods produce inconsistent results and have low signal to noise output. Using fluorescent markers, we quantitatively assessed several published DNA microarray protocols for (1) the efficiency of target DNA attachment to glass, (2) probe-target hybridization efficiency, and (3) the degree of fluorescent labeling of DNA and RNA probes. Protocols tested include DNA attachment via 5' amine mediated covalent bond, and attachment via multiple ionic DNA/glass substrate interactions. Fluorescent labeling of probes by PCR incorporation and Panvera labeling kits were also compared. Ionic substrate-DNA attachment was found to promote the most efficient DNA attachment to glass substrate. 5' covalent attachment methods had less attachment but the attached DNA was more available for probe hybridization and therefore had greater output signal levels.