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DNA Microarrays: An Introduction

DNA Microarrays: An Introduction. Jochen Mueller mueller@umbi.umd.edu. Genome of Halobacterium NRC-1. Condition A. DNA. mRNA. Protein. Condition B. DNA. mRNA. Protein. Microarrays are tools to detect changes in mRNA levels. Flow of information…. …within a cell. DNA. mRNA.

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DNA Microarrays: An Introduction

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

  2. Genome of Halobacterium NRC-1

  3. Condition A DNA mRNA Protein Condition B DNA mRNA Protein

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

  5. Flow of information… …within a cell DNA mRNA Protein …in microarray analysis Probe cDNA mRNA tag

  6. 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

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

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

  9. Non-contact Ink Jet printing Pin Spotting Ink Jet • 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

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

  11. Energy metabolism of Halobacterium sp. NRC-1 Photophosphorylation Oxidative Phosphorylation (aerobic and anaerobic) H+ H+ H+ Cl- O2 DMSO, TMAO ATP ADP + Pi H+ gas vesicles Arginine Ornithine + CO2 + NH4+ Substrate-level phosphorylation Na+ AA Na+ H+ signals

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

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

  14. Making discoveries from array data Co-transcription analysis Clustering analysis Co-regulation of genes X A B D P Deciphering regulatory networks

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