1. GeneChip® Probe Arrays*
Hybridized Probe Cell
GeneChip Probe Array
Single stranded,
labeled RNA target
Oligonucleotide probe
Millions of copies of a specific
oligonucleotide probe
>200,000 different
complementary probes
Image of Hybridized Probe Array
Damla D. Bilgin
Univ. of Illinois, Urbana
Courtesy of: Mike Lelivelt

2. Photolithographic Synthesis
Lamp
Mask
Chip
Damla D. Bilgin
Univ. of Illinois, Urbana
Courtesy of: Mike Lelivelt

3. 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. Bilgin
Univ. of Illinois, Urbana

4. GeneChip® Expression Array Design5´ 3´
Gene
Sequence
UTR
3’ biased multiple oligo probes
Probes designed to be Perfect Match
Probes designed to be Mismatch
Damla D. Bilgin
Univ. of Illinois, Urbana

5. 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
C A T A T
A G C T G
T T C C G
Mask
Substrate
T –
C –
REPEAT
Damla D. Bilgin
Univ. of Illinois, Urbana
Courtesy of: Mike Lelivelt

6. 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. Bilgin
Univ. of Illinois, Urbana

7. probe cell probe pair Probe Array (chip) Probe Set 1 Probe Set 2
Damla D. Bilgin
Univ. of Illinois, Urbana
Probe Array (chip)

8. consists of millions of the same probe molecules.
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.
Pixel
3 – 24 um
Probe Cell
micrometers
Damla D. Bilgin
Univ. of Illinois, Urbana

9. Each pair represents a different subsequence of the gene Probe Cell PM MM
Probe Pair
Each pair represents a different subsequence of the gene
Probe Cell
Probe Set
Damla D. Bilgin
Univ. of Illinois, Urbana

10. Damla D. Bilgin
Univ. of Illinois, Urbana
keck.med.yale.edu

11. Definitions
Probe – 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 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. Bilgin
Univ. of Illinois, Urbana

12. Procedures for Target Preparation
Cells
AAAA
Labeled transcript
IVT
(Biotin-UTP
Biotin-CTP)
Poly (A)+/
Total
RNA L L L L
cDNA
Fragment
(heat, Mg2+) L
Wash & Stain L
Hybridize
(16 hours) L L
Scan
Labeled fragments
Damla D. Bilgin
Univ. of Illinois, Urbana
Courtesy of: Mike Lelivelt

13. 570nm
Argon
laser
488nm
Scanner based on epifluorescence confocal microscopy
Damla D. Bilgin
Univ. of Illinois, Urbana

14. Damla D. Bilgin
Univ. of Illinois, Urbana

15. Affymetrix Core Facility at W.M. Keck Center
at Univ. of Illinois at Urbana-Champaign
Hybridization oven
Scanner
Fluid station
Damla D. Bilgin
Univ. of Illinois, Urbana

16. Useful web sites http://www.affymetrix.com/products/arrays/index.affx
Damla D. Bilgin
Univ. of Illinois, Urbana