Finish up array applications

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• Finish up array applications
• Move on to proteomics
• Protein microarrays

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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, 409533-538
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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.

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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.

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Gene expression and cancer

• Hierarchical clustering
• Method for analyzing microarray data
• Gene level analysis
• Experiment level analysis

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Vant Veer et al. 2002 Nature
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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-transcriptional
  ly.
• Most often proteins are the targets of drugs.

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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

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From www.probes.com
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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

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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.

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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

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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.

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Templin et al. 2002 Trend in Biotch. Vol 20
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Proteinprotein interactions
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Protein activity arrays
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Small molecule arrays
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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