Overview of Affymetrix GeneChip Technology

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 DNA Arrays B. subtilis Plasmodium Genome Array Barley Genome Array Porcine Genome Array 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 Drosophila Genome Arrays Resequencing 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 10 K DNA mapping 100 K DNA mapping 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.