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GeneChip® Probe Arrays
Millions of copies of a specificMillions of copies of a specificoligonucleotide probeoligonucleotide probe
Image of Hybridized Probe ArrayImage of Hybridized Probe Array
>200,000 different>200,000 differentcomplementary probes complementary probes
Single stranded, Single stranded, labeled RNA targetlabeled RNA target
Oligonucleotide probeOligonucleotide probe
**
**
*GeneChipGeneChip Probe ArrayProbe Array
Hybridized Probe CellHybridized Probe Cell
Courtesy of: Mike Lelivelt
Damla D. BilginUniv. of Illinois, Urbana
Photolithographic Synthesis
LampLamp
MaskMask ChipChip
Courtesy of: Mike LeliveltDamla D. BilginUniv. of Illinois, Urbana
Affymetrix Soybean Genome Array
The GeneChip® Soybean Genome Array can be used to study gene expression of over 37,500 soybean (Glycine max) transcripts.
The array includes probe sets to detect approximately 15,800 transcripts for Phytophthora sojae as well as 7,500 Heterodera glycines (cyst nematode pathogen) transcripts.
Damla D. BilginUniv. of Illinois, Urbana
GeneChip® Expression Array Design
GeneGeneSequenceSequence
Probes designed to be Probes designed to be Perfect MatchPerfect Match
Probes designed to be Probes designed to be MismatchMismatch
3’ biased multiple 3’ biased multiple oligo probesoligo probes
UTR
5´5´ 3´3´
Damla D. BilginUniv. of Illinois, Urbana
Synthesis of Ordered Oligonucleotide Arrays
O O O O O
Light(deprotection)
HO HO O O O T T O O O
T T C C O
Light(deprotection)
T T O O O
C A T A TA G C T GT T C C G
MaskMask
SubstrateSubstrate
MaskMask
SubstrateSubstrate
T –T –
C –C –REPEATREPEAT
Courtesy of: Mike LeliveltDamla D. BilginUniv. of Illinois, Urbana
GeneChip Probes (Affymetrix)
• 25-mer oligonucleotides synthesized directly on the glass
• 11-20 probe pairs are selected among all possible 25-mers to represent each transcript
• Perfect Match/Mismatch probe pair strategy
Damla D. BilginUniv. of Illinois, Urbana
Probe Set 1
probe cell
probe pair
Probe Array (chip)
Probe Set 2
Probe Set 3
Damla D. BilginUniv. of Illinois, Urbana
Probe – a single-stranded DNA oligonucleotide complementary to a specific sequence. Each probe cell consists of millions of the same probe molecules.
The intensity of each cell is an average of each of its scanned pixels.
Probe Cell20 - 50 micrometers
Pixel3 – 24 um
Damla D. BilginUniv. of Illinois, Urbana
PM
MM
Probe PairEach pair represents a different subsequence of the gene
PMMM
MM
Probe Cell
Probe Set
Damla D. BilginUniv. of Illinois, Urbana
keck.med.yale.edu Damla D. BilginUniv. of Illinois, Urbana
DefinitionsProbe – a single-stranded DNA oligonucleotide complementary to a specific sequence. Each probe cell consists of millions of probe molecules.Probe Array – a collection of probes sets.Probe Set – a set of probes designed to detect one transcript. 16-20 probe pairs. A 20 probe pair set is made up of 20 PM and 20 MM for a total of 40 probe cells.Probe Pair – Two probe cells, a PM and its corresponding MM.Perfect Match (PM) – probes that are designed to be complementary to the reference sequence.MisMatch (MM) – probes that are designed to be complementary to the reference sequence except for 1 base.Target – sequence from your sample.
Damla D. BilginUniv. of Illinois, Urbana
Procedures for Target Preparation
cDNAcDNAFragmentFragment(heat, Mg(heat, Mg2+2+))
LL LL LL LL
Wash & StainWash & Stain
ScanScan
HybridizeHybridize
(16 hours)(16 hours)
Labeled transcriptLabeled transcript
Poly (A)Poly (A)++//TotalTotal RNARNA
AAAAAAAA
IVTIVT
(Biotin-UTP(Biotin-UTPBiotin-CTP)Biotin-CTP)
Labeled fragmentsLabeled fragments
LL LL
LL
LL
CellsCells
Courtesy of: Mike LeliveltDamla D. BilginUniv. of Illinois, Urbana
Argon laser488nm
570nm
Scanner based on epifluorescence confocal microscopy
Damla D. BilginUniv. of Illinois, Urbana
Damla D. BilginUniv. of Illinois, Urbana
Hybridization oven
Fluid station
Scanner
Damla D. BilginUniv. of Illinois, Urbana
Affymetrix Core Facility at W.M. Keck Center at Univ. of Illinois at Urbana-Champaign
Damla D. BilginUniv. of Illinois, Urbana
Useful web sites
http://www.affymetrix.com/products/arrays/index.affx
http://www.biotech.uiuc.edu/centers/Keck/Functional_genomics/
http://www.iobion.com/support/support_GT_training.html
http://www.cropsci.uiuc.edu/faculty/clough/