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• Finish up array applications • Move on to proteomics • Protein microarrays Applications of DNA microarrays • Monitor gene expression – – – – Study regulatory networks Drug discovery - mechanism of action Diagnostics - tumor diagnosis etc. • Genomic DNA hybridizations – – – – • ? Explore microbial diversity Whole genome comparisons - genome evolution Identify DNA binding sites Diagnostics - tumor diagnosis • Identification of DNA regions bound by a protein. • Compare a wild-type strain to a ∆gene (DNA-binding protein). • Do not need any prior knowledge of the sequence the protein binds. Iyer et al. 2001 Nature, 409:533-538 Identifying replication origins in yeast • Only 5% of the genome previously screened for replication origins. • Used known replication initiation factors to perform ChIP/chip analysis • Identified hundreds of additional replication origins in a single experiment. DNA diagnostics • Uses of microarrays is cancer research and diagnosis. – 2733 papers published on microarrays and cancer – 1038 papers published on microarrays, gene expression, cancer diagnosis – 0 since 1997 • Gene expression profiling – Identify genes involved in cancer diagnosis. – Identify gene expression patterns that are associated with disease outcome. • Gene content analysis – Identify genomic regions that are lost or amplified in tumors. Gene expression and cancer • Hierarchical clustering – Method for analyzing microarray data – Gene level analysis – Experiment level analysis Vant Veer et al. 2002 Nature Why study proteins? • They are the machines that make cells function. • RNA levels do not always accurately predict protein levels. – Often processes are regulated at the transcriptional level. – Some processes are controlled posttranscriptionally. • Most often proteins are the targets of drugs. Proteomics -large scale analysis of proteins • Protein levels - Determining the abundance of proteins in a sample. – 2D gel electrophoresis, mass spectrometry, protein microarrays • Interacting proteins - determining which proteins come together to form functional complexes. – Yeast 2-hybrid, affinity purification • Subcellular localization - site of localization can often provide clues to the function of a protein. – GFP tagging, immunofluorescence microscopy. • Protein activity - investigating the biochemical activities of proteins. • Structural genomics - high-throughput analysis of the protein structure From www.probes.com Proteins • Primary structure - sequence – Searching databases – Identifying functional domains • Secondary and tertiary structure - 3D folding of proteins. – Proteins have unique 3D structures – Identify functional domains – VAST - online structural tool from NCBI Western Blot • Determine the presence and level of a protein in a cell lysate. • http://web.mit.edu/esgbio/www/rdna/rdna.ht ml - review of Northern, Western, and Southern blots. Monitoring protein levels - large scale • 2D gel electrophoresis – Old technology - not as useful for lowly expressed proteins. • Mass spectrometry – Many new techniques for protein detection and quantitation being developed. • Protein microarrays • Many developing technologies Protein microarrays • Analysis of thousands of proteins at one time. • Many different types – Antibody arrayed - detect many proteins – Proteins arrayed - detect interacting proteins – Proteins arrayed - detect interacting small molecules – Etc. Templin et al. 2002 Trend in Biotch. Vol 20 Protein:protein interactions Protein activity arrays Small molecule arrays Why bother with DNA microarrays? • Protein microarrays are not as robust – DNA is DNA - all features will behave similarly under single hybridization conditions. – Proteins are unique - will behave differently. • Protein microarrays are costly – $500-1000 per antibody – $10 per oligo • Used for different purposes