The Affymetrix GeneChip system consists of the probe/gene microarray, hybridization oven, fluidics station, computer workstation and a scanner (Figure 1).
Oligonucleotides, usually 25-mers, are directly synthesized onto a glass wafer by a combination of semiconductor-based photolithography and solid phase chemical synthesis technologies. Each array contains up to 900,000 different oligos and each oligo is present in millions of copies. Since oligonucleotide probes are synthesized in known locations on the array, the hybridization patterns and signal intensities can be interpreted in terms of gene identity and relative expression levels by the Affymetrix GeneChip Expression Console.
Each gene is represented on the array by a series of different oligonucleotide probes. Each probe pair consists of a perfect match oligonucleotide and a mismatch oligonucleotide. The perfect match probe has a sequence exactly complimentary to the particular gene and thus measures the expression of the gene. The mismatch probe differs from the perfect match probe by a single base substitution at the center base position, disturbing the binding of the target gene transcript. This helps to determine the background and nonspecific hybridization that contributes to the signal measured for the perfect match oligo. GeneChip Expression Console subtracts the hybridization intensities of the mismatch probes from those of the perfect match probes to determine the absolute or specific intensity value for each probe set. Probes are chosen based on current information from Genbank and other nucleotide repositories. The sequences on the expression arrays are believed to recognize unique regions of the 3’ end of the gene.
GeneChip Hybridization Oven
A “rotisserie” oven is used to carry out the hybridization of up to 64 arrays at one time.
The fluidics station performs washing and staining of the probe arrays. It is completely automated and contains four modules, with each module holding one probe array. Each module is controlled independently through GeneChip Operating software using preprogrammed fluidics protocols.
GeneChip Scanner 3000 with Autoloader
The scanner is a 16-bit sophisticated opto-mechanical confocal scanner which measures fluorescence intensity emitted by the labeled cRNA/cDNA bound to the probe arrays. The scanner uses a high-quality, solid state 532nm Diode-Pumped, Frequency Doubled Nd:YAG (Neodymium-doped Yttrium Aluminum Garnet) Green Laser. The scanner optics are designed to condition the laser beam to deliver an excitation spot size of 3.5 μm and can detect as few as 400 phycoerythrin molecules in a 20 x 20 μm probe site.
The computer workstation with GeneChip Command Console software controls the fluidics station and the scanner. GeneChip Command Console software can control up to twelve fluidics stations using preprogrammed hybridization, wash, and stain protocols for the probe array. The Expression Console software also acquires and converts hybridization intensity data into a presence/absence call for each gene using appropriate algorithms.