Affymetrix GeneChip Technology
Microarray technology is a powerful tool for genetic research that utilizes nucleic acid hybridization techniques and recent advancements in computing technology to evaluate the mRNA expression profile of thousands of genes within a single experiment. It has proven to be an extremely valuable tool to better utilize the enormous amount of information provided by the completion of the Human Genome project.
DNA Microarrays are miniature arrays containing gene fragments that are either synthesized directly onto or spotted onto glass or other substrates. Thousands of genes are usually represented in a single array. A typical microarray experiment involves the following steps:
- Preparation of fluorescently labeled target from RNA isolated from the biological specimens
- Hybridization of the labeled target to the microarray
- Washing, staining, and scanning of the array
- Analysis of the scanned image
- Generation of gene expression profiles
Currently two main types of DNA microarrays are being used: oligonucleotide (usually 25 to 70 mers) arrays and gene expression arrays containing PCR products prepared from cDNAs. In forming an array, oligonucleotides can be either prefabricated and spotted to the surface or directly synthesized on to the surface (in situ). Affymetrix GeneChip system, which will be described in the following pages, uses arrays fabricated by direct synthesis of oligonucleotides on the glass surface.
Affymetrix GeneChip System
The Affymetrix GeneChip system consists of a gene or probe array, hybridization oven, fluidics station, scanner, and a computer workstation (Figure 1).
Figure 1: Affymetrix GeneChip System
Probe/Gene Array: Oligonucleotides, usually 25 mers, are directly synthesized onto a glass wafer by a combination of semiconductor-based photolithography and solid phase chemical synthesis technologies (Figure 2). Each array contains up to 400,000 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 Microarray Suite software.
Figure 2: Manufacturing GeneChip Probe Array
Each gene is represented on the array by a series of different oligonucleotide probes (Figure 3). 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. The Microarray Suite software 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 are believed to recognize unique regions of the 3' end of the gene.
Figure 3: Oligonucleotide Probe Pair
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 Microarray Suite software using preprogrammed fluidics protocols.
The scanner is a sophisticated confocal laser fluorescence scanner which measures fluorescence intensity emitted by the labeled cRNA bound to the probe arrays. The scanner uses an argon-ion laser to excite the fluorophores at 488nm. The scanner can focus the laser to a spot size of less than 4 microns and can detect as few as 400 phycoerythrin molecules in a 20 x 20 .m probe site.
The computer workstation with Microarray Suite software controls the fluidics station and the scanner. Microarray Suite software can control up to eight fluidics stations using preprogrammed hybridization, wash, and stain protocols for the probe array. The software also acquires and converts hybridization intensity data into a presence/absence call for each gene using appropriate algorithms. Finally, the software detects changes in gene expression between experiments by comparison analysis and formats the output into .txt files which can be used with other software programs for further data analysis.