Overview of Affymetrix GeneChip Technology

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Overview of Affymetrix GeneChip Technology
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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
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Photolithographic Synthesis Manufacturing
ProcessProbe arrays are manufactured by
light-directed chemical synthesis process which
enables the synthesis of hundreds of thousands of
discrete compounds in precise locations
Lamp
Mask
Chip
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(No Transcript)
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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
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Selection of Expression ProbesSet of oligos to
be synthesized is defined from sense sequence of
known genes and ESTs
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Sequence
Probes
Perfect Match Mismatch
Chip
Computer algorithms are used to design
photolithographic masks for use in manufacturing
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• Each gene is represented on the probe array by
  multiple probe pairs
• Each probe pair consists of a perfect match and a
  mismatch oligonucleotide

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Overview Creating Targets
mRNA
Reverse Transcriptase
cDNA
This is what we are doing in this class
in vitro transcription
cRNA
Fragmentation of cRNA
GeneChip Hybridization
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mRNA cRNA
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Fragmentation of biotinylated cRNA
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Fragmentation -Metal mediated alkaline induced
hydrolysis
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Hybridization and Staining
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Instrumentation Affymetrix GeneChip
System 3000-7G Scanner 450 Fluidic Station 640
Hybridization Oven
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Currently Available GeneChips
Expression Arrays
DNA Arrays
B. subtilis Barley Genome Array Bovine Genome
Array C. elegans Genome Array Canine Genome
Array Chicken Genome Array Drosophila Genome
Arrays E. coli Genom e Arrays Human Genome
Arrays Maize Genome Array Mouse Genome Arrays P.
aeruginosa Genome Array
Plasmodium Genome Array Porcine Genome Array Rat
Genome Arrays Rice Genome Array Soybean Genome
Array Sugar Cane Genome Array Vitis vinifera
(Grape) Array Wheat Genome Array Xenopus laevis
Genome Array Yeast Genome Arrays Zebrafish Genome
Array Arabidopsis Genome Arrays
10 K DNA mapping 100 K DNA mapping 500 K DNA
mapping Exon Array Resequencing SNP arrays
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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.
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Quality Control Issues

• RNA purity and integrity
• cDNA synthesis efficiency
• Efficient cRNA synthesis, labeling, and
  fragmentation
• Target evaluation with Test Chips

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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.