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DNA Microarrays:
An Introduction
Jochen Mueller
[email protected]
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
Protein
…in microarray analysis
Probe
cDNA
tag
mRNA
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
Almost every gene is represented on the array
Gene A
Gene B
Gene probes
Printing or
in situ synthesis
Common array formats
Probes individually
synthesized on array
platform
photolithographic
Ink jet
Pre-synthesized probes
(PCR products or oligonucleotides)
are printed onto the
array platform
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
Analysis of changes in gene expression
Sample A RNA
Sample B RNA
reverse transcribe
incorporating Cy3
or Cy5 dye
Sample A cDNA
Sample B cDNA
remove unincorporated dyes
Hybridize
Energy metabolism of Halobacterium sp. NRC-1
Photophosphorylation
Oxidative Phosphorylation
(aerobic and anaerobic)
H+
H+
H+
Cl-
O2
ATP
DMSO, TMAO
ADP + Pi
H+
gas vesicles
Arginine
Ornithine
+ CO2 + NH4+
Substrate-level phosphorylation
Na+
AA Na+
H+
signals
Log(rProcessedSignal)
Log(gProcessedSignal)
selftest
anaerobic vs.
aerobic respiration
Anaerobic respiration vs. aerobic respiration
bop
coxA1/B2
gvp
0.5
-0.5
log ratio
dms
rps/rpl
rps/rpl
atp
Fermentation vs. aerobic respiration
0.5
-0.5
log ratio
arc
Making discoveries from array data
Co-transcription
Clustering analysis
analysis
A
B
X
D
P
Deciphering regulatory networks
Co-regulation of genes