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Microarrays and molecular biology: beyond gene expression profiling

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This talk will describe the development and application of two novel microarray technologies, focusing on genomic DNA tiling arrays and protein-based arrays. Tiling arrays are designed to represent all of the DNA comprising a chromosome or other genomic locus, without prior consultation of existing gene annotation. Because they are developed for the unbiased interrogation of genomic sequence, tiling arrays enable the discovery of novel transcripts and regulatory elements in eukaryotic genomes. Two examples of microarray-based genome annotation are presented: large-scale mapping of transcribed sequences and identification of transcription factor-binding sites. Chromosome- and genome-wide transcriptional activity is assessed by probing tiling arrays with normal complex tissue RNA ; the locations of cis-regulatory elements are determined via array hybridisation to chromatin-immunoprecipitated DNA , originally bound in vivo by a transcription factor of interest. These approaches are first demonstrated with amplicon arrays representing all of the non-repetitive DNA of human chromosome 22, then extended to the entire genome using maskless photolithographic DNA synthesis technology. A large-scale tiling array survey revealed the presence of thousands of novel transcribed sequences in addition to known and predicted genes, providing the first global transcription map of the human genome. The second part of the talk will discuss the development of protein-based microarrays for the large-scale characterisation of biochemical activity. Full-length, functional proteins are produced from a library of expression clones, purified as GST ::HisX6::fusions and immobilised to various support surfaces at high spatial density. This technology enables high-throughput screening for protein interactions with various analytes and chemical libraries, using procedures analogous to those developed for DNA microarrays. The method was first explored in a pilot study to assay the protein kinases of the budding yeast Saccharomyces cerevisiae using a silicone elastomer microwell array prototype, then extended to the entire yeast proteome using conventional contact-printing microarray technology. This platform is used to assess protein-protein, protein-nucleic acid and small-molecule interactions, enzymatic activity and posttranslational modifications on a proteome-wide scale.

This talk is part of the Computational and Systems Biology series.

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