1. DNA Microarrays: An Introduction Jochen Mueller mueller@umbi.umd.edu

3. Genome of Halobacterium NRC-1

4. DNA Condition A mRNA Protein DNA Condition B mRNA Protein

5. Microarrays are tools to detect changes in mRNA levels.

6. Flow of information… DNAmRNAProtein …within a cell …in microarray analysis cDNAmRNA tag Probe

7. Whole genome DNA microarrays Construction of the array requires Sequenced genome Identification and annotation of ORFs Design and synthesis of oligonucleotides for amplicon preparation or as probes Printing of amplicons or oligonucleotides

8. Almost every gene is represented on the array Gene B Gene probes Printing or in situ synthesis Gene A

9. Common array formats Probes individually synthesized on array platform Pre-synthesized probes (PCR products or oligonucleotides) are printed onto the array platform photolitho- graphic Ink jet

10. Non-contact Ink Jet printing Ink Jet Pin Spotting Pin Spotting Challenges: Passive volume control Clone contamination Clone storage/handling/tracking Dependent upon physical reagent Labor intensive (PCR etc) Surface artifacts of pins Missing features

11. Analysis of changes in gene expression Sample A RNASample B RNA reverse transcribe incorporating Cy3 or Cy5 dye Sample A cDNA Sample B cDNA remove unincorporated dyes Hybridize

12. Energy metabolism of Halobacterium sp. NRC-1 H+H+ H+H+ H+H+ H+H+ H+H+ Na + AA Cl - ATPADP + P i Arginine Ornithine + CO 2 + NH 4 + Oxidative Phosphorylation (aerobic and anaerobic) Photophosphorylation Substrate-level phosphorylation O2O2 DMSO, TMAO gas vesicles signals

14. Log(rProcessedSignal) Log(gProcessedSignal) selftest anaerobic vs. aerobic respiration

15. Anaerobic respiration vs. aerobic respiration dms bop gvp coxA1/B2 Fermentation vs. aerobic respiration rps/rpl atp arc 0.5 -0.5 0.5 -0.5 log ratio

16. Making discoveries from array data Clustering analysisCo-transcription analysis AB X D P Deciphering regulatory networks Co-regulation of genes