Oligonucleotide Arrays

This technology, also called by a trade name Genechip® and introduced by Affymetrix (source: www.affymetrix.com), involves in situ synthesis of oligonucleotides of known sequence in a site-specific arrangement on a substrate. This approach can produce hundreds of thousands of different oligonucleotide probes packed at an extremely high density. These oligonucleotides are up to 25 nucleotides (25-mer) long. A schematic of the photolithographic process used is shown in Figure 10.3.

The process of fabrication of a Genechip® combines combinatorial DNA synthesis chemistry with photolithographic techniques adapted from the semiconductor industry (McGall and Fidanza, 2000). The photolithographic process, like for semiconductor chip manufacturing, utilizes ultraviolet light through holes in masks to deprotect photolabile groups and subsequently direct parallel and stepwise synthesis of oligonucleotides with a specific sequence. When using a fused silica or a planar glass substrate, its surface is first covalently modified using a silane reagent to provide hydroxyalkyl groups which serve as the initial synthesis sites. These sites are then extended with linker groups, protected with a photolabile-protecting group, such as 5'-(a-methyl-6-nitropiperonyloxycarbonyl), abbreviated as MeNPOC, which can be activated at specific spatial locations by UV light exposure for addition of nucleoside phosphoramidite monomers, also containing the photolabile group at the 5' (or 3') position. The photodeprotection is induced by the ~350-nm wavelength of the UV light from a commerical photolithographic exposure system.

Repetition of the cycle of changing the mask, deprotecting by photolithography, and adding a nucleotide to ~70 times can allow the synthesis of a com-


Light (deprotection)




GeneChip® Microarray


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Figure 10.3. The use of a unique combination of photolithography and combinatorial chemistry to manufacture Genechip® arrays. (Reproduced with permission from www.affymetrix.com.)

plete array of thousands of 25-mer oligonucleotides in parallel. Generally, 20 pairs of oligonucleotides are arrayed to represent each gene. Each nucleotide matching the gene (perfect match, PM) is paired with a second mismatch oligonucleotide differing only by a central nucleotide (mismatch, MM). MM oligonucleotides serve to detect nonspecific and background hybridization, important for quantifying weakly expressed m-RNAs. The maximum achievable microarray density is determined by the spatial resolution provided by the photolithographic process. Typical dimensions for each array are 24 mm x 24 mm on a 1.6-cm2 chip. This method has been used to display 65,000-400,000 DNA oligonucleotides on a 1.6-cm2 glass surface (Lockhart et al., 1996).

The advantage offered by the Affymetrix Genechip approach is that the microarrays are very uniform. However, as opposed to the spotted array technology discussed earlier where both the sample and the control are hybridized to the same chip using different fluorescent markers, the Genechip approach can handle only one fluorescent marker at a time, thus requiring two chips to compare a sample and a control.

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