Waveguide Microarray Scanner

Waveguide Array Readers

Many microarray applications, such as high-throughput SNP detection, require a very high level of specificity. Perfectly matching DNA sequences must be distinguishable from mismatching sequences. The ISB has constructed two microarray readers that improve on the specificity of conventional array hybridization by measuring every probe spot at its optimal stringency during temperature ramping. The designs were primarily driven by the requirement that the array be imaged repeatedly while exposed to a wash solution. Most commercially available scanners move the microarray through a focused laser beam in a confocal-microscope arrangement. While this provides good signal sensitivity, it is rather slow and poorly-suited to use with an array sealed within a hybridization chamber. Both ISB designs can be described as waveguide readers. The "Flying Spot" design uses laser scanning excitation with waveguide collection, while the "Evanescent wave" design uses waveguide excitation and CCD camera imaging.

Non-equilibrium dissociation profiles are used to better discriminate matching and mismatching DNA. After hybridization and washing, arrays may be monitored as temperature is increased.  The resulting dissociation (melting) curves for all matching and mismatching duplexes are used to define dissociation temperatures (T_d). T_d , the point at which 50% of the starting duplexes are still intact, is correlated to melting temperature ( T_m). By comparing Tm and T_d, true hybridization can be distinguished from cross hybridization. This specificity dramatically improves the success of allele-specific hybridization as a genotyping platform