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

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 ?

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 Nature, 409:

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 = 0.75)
Log2 transformed data - no difference = 0
Log2 of 4 = 2 Log2 of 0.25 = -2.
Data not bounded by zero