Overview of Affymetrix GeneChip
Technology
GeneChip Technology
Affymetrix Inc
Miniaturized, high density arrays
Expression arrays 1,300,000 DNA oligos 1-cm by 1-cm
DNA mapping array 7,000,000 DNA oligo 1.3 by 1.3 cm
Manufacturing Process
Solid-phase chemical synthesis and Photolithographic
fabrication techniques employed in semiconductor industry
Photolithographic Synthesis
Manufacturing Process
Probe arrays are manufactured by lightdirected chemical synthesis process which
enables the synthesis of hundreds of thousands
of discrete compounds in precise locations
Lamp
Mask
Chip
Affymetrix Wafer and Chip Format
20 - 50 µm
20 - 50 µm
one
oligonucleotide
sequence per “pixel”
49 - 400
chips/wafer
1.0 cm
up to ~ 1.3 million features/chip
Selection of Expression Probes
Set of oligos to be synthesized is defined from sense sequence
of known genes and EST’s
5’
3’
Sequence
Probes
Perfect Match
Mismatch
Chip
Computer algorithms are used to design photolithographic
masks for use in manufacturing
•
Each gene is represented on the probe array by multiple
probe pairs
•
Each probe pair consists of a perfect match and a
mismatch oligonucleotide
Overview: Creating Targets
mRNA
Reverse Transcriptase
This is what we are
doing in this class
cDNA
in vitro transcription
cRNA
Fragmentation of cRNA
GeneChip Hybridization
mRNA
cRNA
Fragmentation of biotinylated cRNA
Fragmentation -Metal mediated alkaline induced hydrolysis
Hybridization and Staining
Array
RNA:DNA Hybridized Array
Fragmented cRNA Target
Streptavidin phycoerythrin
[Fluorescent dye]
Instrumentation
Affymetrix GeneChip System
3000-7G Scanner
450 Fluidic Station
640 Hybridization Oven
Currently Available GeneChips
Expression Arrays
B. subtilis
Plasmodium Genome Array
Barley Genome Array
Porcine Genome Array
DNA Arrays
10 K DNA mapping
100 K DNA mapping
Bovine Genome Array
Rat Genome Arrays
C. elegans Genome Array
Rice Genome Array
500 K DNA mapping
Canine Genome Array
Soybean Genome Array
Exon Array
Chicken Genome Array
Sugar Cane Genome Array
Resequencing
Drosophila Genome Arrays
Vitis vinifera (Grape) Array
E. coli Genom e Arrays
Wheat Genome Array
Human Genome Arrays
Xenopus laevis Genome Array
Maize Genome Array
Yeast Genome Arrays
Mouse Genome Arrays
Zebrafish Genome Array
P. aeruginosa Genome Array
Arabidopsis Genome Arrays
SNP arrays
Custom GeneChips
Affymetrix offers over 120 prokaryotic arrays that are manufactured by
Nimblegen Inc.
Custom GeneChips are also available for both Eukaryotic and
Prokaryotic systems.
Quality Control Issues
• RNA purity and integrity
• cDNA synthesis efficiency
• Efficient cRNA synthesis, labeling, and
fragmentation
• Target evaluation with Test Chips
Affymetrix GCOS - Data Analysis
Absolute Analysis –whether transcripts are
Present or not (uses data from one probe array
experiment).
Comparison Analysis –determine the relative
change in transcripts (uses data from two probe
array experiments).
Intensities for each experiment are compared to a
baseline/control.