Affymetrix arrays

Spotted arrays

Spotted arrays consist of strands of denatured DNA arranged in grid of spots. DNA can come from expressed mRNA libraries, or manufactured artificially. Common feature is method of DNA attachment: robotic spotting. Developed in research environment and can be used by anyone. A number of alternative proprietary techniques have been developed.

Affymetrix

Sells complete analysis package – arrays, scanner and analysis software. Builds short sequences of oligos directly on the slide surface using photolithography. Must know sequence of interest, but currently not a big problem. Process is expensive and creating one-off custom arrays is not practical. Very precise and allows extremely high density..

Experimental process

• Probe selection. For given target, probe aims to optimise hybridisation while maintain selectivity. 25 bp. 11-20 probes per gene, 1 probe per spot. PM(perfect match) and MM(mismatch) (homomeric base at bp 13), attempt to control for non-specific hybridisation. Probe pairs for particular gene spread across array to control for spatial effects. Each probe represented by cell, 18-20 microns in size. Using 11 PM-MM probes per gene, and 18 micron cells, human genome can be represented on 2 arrays.
• Array fabrication. Multiple identical arrays constructed simultaneously on quartz wafer. Proceeds in stepwise manner, oligos formed one base at a time. At each step photolithographic masks placed over the wafer. UV light through open windows exposes linkers which will bind to a nucleotide. Single wafer yields 50-400 arrays.
• Hybridisation. Similar to spotted arrays, but only one sample per array – makes experimental design much easier! Inter-array variability is very low.
• Data analysis