DNA Microarrays:An Introduction

Jochen Muellermueller@umbi.umd.edu

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