Introduction. Microarray Facility

The human genome project has been the catalyst for the development of several high-throughput technologies that have made it possible to map and sequence complex genomes. At this time several bacterial genomes as well as the genome of Sacchromyces cerevisiae have been sequenced. Within the next year the entire human genomic sequence will be completed and this will represent the end of the structural genomics segment of the human genome project. It is clear, however, that the identification of every gene within the genomes of model organisms is only the initial step in our quest to understand what these genes do and how their expression impacts our health. Understanding the functions of the 50,000-100,000 genes comprising mammalian genomes, their implication in disease states, variations within the population and roles in normal development will represent at least if not a more difficult task than the mapping and sequencing efforts currently underway.

At AECOM, we have initiated the Functional Genomics program by designing, constructing and utilizing robotic devices for quantitative parallel assessment of gene expression patterns from thousands of genes in a single experiment. Highly parallel quantitative measurements of gene expression patterns are possible using different methods to produce DNA microarrays. Nucleic acid hybridization to DNA arrays using complex mixtures of probes derived from total cellular mRNA represents a simple method to in theory, measure every different mRNA within the mixture. If detection methods are sensitive enough, quantitation of transcript levels for every gene in an organism is possible if the genome has been sequenced and the identity of the genes are known. One method that is being developed for high-throughput measurement of expression patterns of thousands of genes is the gridded cDNA microarray technology first developed by Pat Brown and colleagues at Stanford University. This method utilizes a high speed, high precision robot to spot thousands of DNA samples onto glass slides. The slides are then simultaneously probed with florescent labeled cDNAs which are generated from mRNA isolated from cells or tissues in two different states that one wishes to compare. A different fluorescent dye is used to make the cDNAs for each physiological state which allows direct comparisons on a single chip. At AECOM, we have built a high speed, ultra high precision robot to make gridded cDNA microarrays on glass slides. Our robot has the precision to spot at least 23,000 PCR products onto a single glass slide. We have obtained from Genome Systems 18,394 unique human cDNAs from the I.M.A.G.E. consortium that represent 15-30% of all human genes. In addition, we have obtained from Genome Systems a similar number of mouse genes. We will produce both human and mouse DNA chips for the quantitative measurement of mRNAs from a variety of model systems. Data from the hybridization reactions are collected using a 2-color laser scanning confocal microscope that is being custom designed and built at AECOM specifically for maximum sensitivity necessary to measure low abundance mRNAs. Finally, informatics for the analysis of databases generated from these experiments will be implimented.

Last updated: Jan. 21, 2003