finish up array applications move on to proteomics protein microarrays
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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 post-
transcriptionally.
• 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.html - 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
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