U-STAR: Universal sequence-tag array technology for absolute quantification of per cell transcript profiles

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The importance and tremendous utility of gene expression analysis is now well established and two principle technologies have emerged:  1) DNA microarrays offer a massively parallel and relatively straightforward approach, however they are genome specific and can provide relative, but not absolute, quantification of per cell transcript levels; 2) serial analysis of gene expression (SAGE) is a sequencing-based approach that can provide absolute transcript abundance data, however it is severely disadvantaged by high cost and limited availability of high-throughput DNA sequencing centres, and by costly, labor-intensive sample preparation.  The goal of this project is to develop a universal sequence-tag (micro)array (U-STAR) platform for SAGE-like analyses that combines the advantages, and mitigates the disadvantages, of each of the current technologies.  'Universal' means that a single array can be used to analyze any organism or tissue sample of interest.  The technology exploits short-probe microarrays based on locked nucleic acids (LNA) permitting us to engineer the stability of each probe-target duplex and thereby to equalize the melting temperature across the entire array.  This in turn allows the identification of hybridization conditions that eliminate false signals due to probe-target mismatches.  A new detection system is also proposed that offers potential advantages over conventional fluorescent tags in terms of detection limit and overall precision.  Quantitative reading of hybridized U-STAR arrays will be enhanced by developing a fully integrated instrument employing nanofabrication technologies to provide for highly efficient, stable and precise measurements at each register.  This research will yield a new generation of DNA microarray platform and related instrumentation.  The main advancements that distinguish this new technology are:  i) the universal applicability to any organism of interest; ii) the capability to provide truly quantitative analyses on a per cell basis; iii) the speed, precision, portability and cost saving features of the integrated instrumentation.  The new technology will thus be valuable in a multitude of life science and health research applications, both in research and clinical settings.