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Other uses of DNA microarrays DNA microarray - image and data analysis

Other uses of DNA microarrays DNA microarray - image and data analysis Proteomics - Chapter 4 p. 184-212. Genome of the week - Deinococcus radiodurans. Highly resistant to DNA damage Most radiation resistant organism known Multiple genetic elements 2 chromosomes, 2 plasmids

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Other uses of DNA microarrays DNA microarray - image and data analysis

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

  2. 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?

  3. 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

  4. 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 • ?

  5. 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

  6. Pathogen 1 Pathogen 2 Isolate genomic DNA Label DNA with Cy3 or Cy5

  7. 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.

  8. Image Analysis • GenePix (Axon) • Quantitate genes • Normalize data

  9. 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.

  10. 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 don’t 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.

  11. 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 (4-1=3 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

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