Transcript Lecture_9_2005

• Finish up array applications
• Move on to proteomics
• Protein microarrays
Applications of DNA microarrays
• Monitor gene expression
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Study regulatory networks
Drug discovery - mechanism of action
Diagnostics - tumor diagnosis
etc.
• Genomic DNA hybridizations
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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