Biomedical Engineering Reference
In-Depth Information
In the photolithography process, illustrated in Figure 6-7 , fenestrated masks are placed over the
coated wafer and exposed to UV light. The UV light exposes linkers, which are then available for
nucleotide coupling. A solution containing a single type of deoxynucleotide (A, T, C, or G) is flushed
over the surface, where the nucleotide attaches to the exposed linkers. The process is repeated with
additional masks until the oligonucleotides on the surface of the wafer are 20-25 nucleotides in
length.
Each wafer can produce between about 50 to 400 individual microarrays, each of which can hold up
to 500,000 probes, depending on the yield of the process. In comparison, each glass slide in the
spotting process can hold perhaps 30,000 spots. The quartz wafer is diced and each of the individual
arrays is packaged for use, just as the semiconductor wafers are diced and the individual components
are mounted in a plastic or ceramic housing. A sample of the packaged microarrays is tested by
running control hybridizations. A quantitative test of hybridization is run using standardized control
probes before the microarrays are available for use in competitive hybridization experiments. The
hybridization process is essentially identical to that used in the spotting process outlined in Figure 6-
3 .
A comparison of spotting and the Affymetrix process, summarized in Table 6-3 , reveals that spotting
is associated with quicker setup and modification times. What's more, the spotting process results in
more variability and lower density because it relies on mainly mechanical means. The Affymetrix
process excels at providing absolute, quantitative results instead of qualitative results, in part
because the oligonucleotides are of a fixed length and known quantity. A discussion of the variability
of the spotting technique, in terms of statistical concepts, follows.
Table 6-3. Microarray Fabrication Comparison. Spotting is more variable
than the Affymetrix process.
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