• 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

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• 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