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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. Each array contains up to
900,000 different oligos and each oligo is present
in millions of copies (Figure 2). 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 Operating Software.
Figure 2: Detail of a
GeneChip Tile
Each gene is represented on the array by a series of
different oligonucleotide probes (Figures 3 and 4). 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 GeneChip Operating 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 on the expression arrays are believed to recognize unique regions
of the 3’ end of the gene.
The probe design on the SNP arrays are similar,
however, there are probes for each allele.
Figure 4. Oligonucleotide
Pairs of SNP Arrays
GeneChip Hybridization Oven: A “rotisserie” oven is
used to carry out the hybridization of up to 64 arrays at one time.
Fluidics station: 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.
Affymetrix GeneChip
Scanner 3000: The scanner is a 16-bit sophisticated opto-mechanical confocal scanner which measures fluorescence intensity
emitted by the labeled cRNA 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.
Computer workstation: The computer workstation with
GeneChip Operating software controls the fluidics station and the scanner.
GeneChip Operating 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.
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