dna microarrays: an introduction
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
DNA Microarrays:An Introduction
Jochen [email protected]
Genome of Halobacterium NRC-1
DNA
Condition A
mRNA
Protein
DNA
Condition B
mRNA
Protein
Microarrays are tools to detect changes
in mRNA levels.
Flow of information…
DNA mRNA Protein
…within a cell
…in microarray analysis
cDNA mRNA
tag
Probe
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 B
Gene probes
Printing or in situ synthesis
Gene A
Common array formats
Probes individuallysynthesized on array
platform
Pre-synthesized probes(PCR products or oligonucleotides)
are printed onto the array platform
photolitho-graphic
Ink jet
Non-contact Ink Jet printing
Ink Jet Pin SpottingPin 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 transcribeincorporating Cy3
or Cy5 dye
Sample A cDNA Sample B cDNA
remove unincorporated dyes
Hybridize
Energy metabolism of Halobacterium sp. NRC-1
H+
H+ H+ H+
H+
Na+
Na+AA
Cl-
ATP ADP + Pi
Arginine Ornithine + CO2 + NH4
+
Oxidative Phosphorylation(aerobic and anaerobic)
Photophosphorylation
Substrate-level phosphorylation
O2 DMSO, TMAO
gas vesicles
signals
Log
(rP
roce
ssed
Sig
nal
)
Log(gProcessedSignal)
selftest anaerobic vs.aerobic respiration
Anaerobic respiration vs. aerobic respiration
dms
bop
gvpcoxA1/B2
Fermentation vs. aerobic respiration
rps/rplrps/rpl atp
arc
0 . 5
- 0. 5
0 . 5
- 0. 5
log
ra
tio
log
ra
tio
Making discoveries from array data
Clustering analysisCo-transcriptionanalysis
A B X D
P
Deciphering regulatory networks
Co-regulation of genes