Fluorescence detection applied to nonelectrophoretic DNA diagnostics on oligonucleotide arrays

DNA analysis based on template hybridization (or hybridization plus enzymatic processing) to an array of surface-bound oligonucleotides is well suited for high density, parallel, low cost and automatable processing. Direct fluorescence detection of labelled DNA provides the benefits of linearity, large dynamic range, multianalyte detection, processing simplicity and safe handling at reasonable cost. Molecular Tool has applied a proprietary enzymatic method of solid phase genotyping (Genetic Bit ^TM Analysis or GBA ^TM) to DNA processing in 96-well plates and glass microscope slides. Glass slides are an inexpensive, convenient format with relatively low fluorescence and the capability for microfabrication of miniature arrays of oligonucleotides. Detecting the fluor-labelled GBA ^TM dideoxynucleotides requires a detection limit of approximately 100 molecules/micrometer ². Commercially available plate readers detect about 1000 molecules/micrometer ², and an experimental setup with an Ar laser and thermoelectrically-cooled CCD can detect approximately 1 order of magnitude less signal. The current limit is due to glass fluorescence, and data is presented describing experimental modifications and analysis to improve the performance. Dideoxynucleotides labelled with fluorescein, eosin, tetramethylrhodamine, Lissamine and Texas Red are being characterized, and photobleaching, quenching and indirect detection with fluorogenic substrates have been investigated.