Other uses of DNA microarrays

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• Other uses of DNA microarrays
• DNA microarray - image and data analysis
• Proteomics - Chapter 4 p. 184-212.

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Genome of the week - Deinococcus radiodurans

• Highly resistant to DNA damage
• Most radiation resistant organism known
• Multiple genetic elements
• 2 chromosomes, 2 plasmids
• Why call one a chromosome vs. plasmid?

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Why sequence D. radiodurans?

• Learn how this bacterium is so resistant to DNA
  damage
• This bacterium has nearly all known mechanisms
  for repairing DNA damage.
• Redundancy of some DNA damage repair mechanisms.
• Use this organism in bioremediation.
• Sites contaminated with high levels of
  radioactivity
• DOE (Department of Energy) sequences many
  microbial genomes - JGI

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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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Pathogen 1
Pathogen 2
Isolate genomic DNA Label DNA with Cy3 or Cy5
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Genomic DNA hybridizations

• Determine the similarity between two different
  strains.
• Example - E. coli outbreak
• Can only detect presence or loss of genes from
  the sequenced strain. Will NOT detect
  acquisition of new genes into strain being
  tested.

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Image Analysis

• GenePix (Axon)
• Quantitate genes
• Normalize data

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Scanning

• How do we detect the Cy3 and Cy5 labeled cDNA
  hybridized to the slide?
• Cy3 and Cy5 are fluorophores
• Absorb light at one wavelength (excitation) and
  emit light at another wavelength (emission).
• The excitation and emission wavelengths of two
  fluorophores should not overlap
• Laser based scanners - 532nm laser (Cy3) and 635
  nm laser (Cy5) for excitation.
• Also can use microscopes for detection using
  excitation and emission filters.

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Data normalization

• Why do we need to normalize microarray data?
• Correct for experimental errors
• Northern blot example
• Microbial microarrays
• Assume the expression of most genes dont change
• We know every gene - sum the intensity in both
  channels and make the equal.
• Many other ways of normalizing data - not one
  standard way. Area of active research.

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Data analysis

• How do we determine what is a differentially
  expressed gene?
• Fold change?
• What is a significant fold change?
• Statistical analysis of the data
• Multiple repeats of an experiment
• Iterative outlier analysis
• SAM - Significance Analysis of Microarrays -
  applies t-test statistics and false discovery
  rates to analyze microarray data.
• Log2 transformation of data -often used in
  analysis of array data.
• Cy5/Cy3 1 no difference in expression
• Cy5/Cy3 4 or 0.25 4-fold change (4-13 and
  1-0.25 0.75)
• Log2 transformed data - no difference 0
• Log2 of 4 2 Log2 of 0.25 -2.
• Data not bounded by zero