Overview of Affymetrix GeneChip Technology

1. Overview of Affymetrix GeneChip Technology

2. GeneChip Technology Affymetrix Inc • Miniaturized, high density arrays • Expression arrays 1,300,000 DNA oligos1-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

3. Photolithographic SynthesisManufacturing 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

5. 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

6. Selection of Expression ProbesSet 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

7. Each gene is represented on the probe array by multiple probe pairs • Each probe pair consists of a perfect match and a mismatch oligonucleotide

8. 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

9. mRNA cRNA

10. Fragmentation of biotinylated cRNA

11. Fragmentation -Metal mediated alkaline induced hydrolysis

12. Array RNA:DNA Hybridized Array Fragmented cRNA Target Streptavidin phycoerythrin [Fluorescent dye] Hybridization and Staining

13. Instrumentation Affymetrix GeneChip System 3000-7G Scanner 450 Fluidic Station 640 Hybridization Oven

14. 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

15. 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.

16. Quality Control Issues • RNA purity and integrity • cDNA synthesis efficiency • Efficient cRNA synthesis, labeling, and fragmentation • Target evaluation with Test Chips

17. 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.