GeneChips® from Affymetrix, comprise a variety of microarrays for genome-wide gene expression monitoring studies in mammalian as well as in non vertebrate species. In these arrays, each gene is represented by multiple probe pairs toward the 3’end of the gene. Each probe pair consists of a perfect match and a mismatch oligonucleotide (on average, 20 base pairs in length). The mismatch oligo­nucleotide contains a single base pair mismatch in the center of the probe. The sequence of the probes is chosen to be specific for a particular gene and the probe pairing design facilitates identification and subtraction of non specific hybridization and background signals. The preparation of the mRNA used to query the arrays is fairly straightforward. The mRNA is reverse transcribed into cDNA, which is then used for an in vitro transcription reaction that yields biotin-labeled cRNA. The biotin-labeled cRNA is then hybridized to the arrays. Hybridization signals are developed using immunochemistry and detected using a high-resolution scanner. Also, the match/ mismatch probe system improves the sensitivity of analysis. The presence of reference genes is a useful feature for the normalization and quantitation of the data from different experiments. It is observed that there is up to 3% differences in the performance of chips from different deliveries.
Array Fabrication. In situ synthesis has several advantages over deposition of pre-synthesized oligonucleo­tides. Three approaches have been used to direct oligonu­cleotide synthesis to defined areas of a support for in situ fabrication of arrays: 1) The photochemical deprotection method [2]. 2) Ink-jet delivery of nucleotide precursors to the surface has been developed by a number of companies [3], but is not yet in commercial production. Synthesis can also be localized by confining chemicals physically, for example, using masks or physical barriers; by this means, complex arrays comprising many different, related sequences can be made in a few coupling steps by 3) combinatorial methods. Flooding the precursors through orthogonally intersecting channels has been used to make arrays of all sequences of a chosen length [4,5]: circular or diamond shaped reaction chambers are used to make ‘scanning’ arrays by applying the precursors in a series of overlapping areas on the surface of the support [6].
It is difficult to assess the quality of the oligonucleo­tides made on a surface. The amount of material (approximately 10 pmol per square millimeter at densest packing) is small. However, analysis of oligonucleotides made on cleavable linkers suggests a high quality. Nondestructive measurements can be made by ellipsometry or interferometry, methods recruited from the field of materials science. These techniques can be used for routine quality control, but are not available to most biology laboratories. Pre-synthesized oligonucleotides, on the other hand, can be assessed before they are attached to the surface [7], but it is not presently economical to make large arrays in this way. When large numbers of arrays with the same probes are needed, deposition may be more economical than in situ synthesis. Deposition is also the method of choice for long sequences that are available as polymerase chain reaction (PCR) products. The technology for making spotting arrays is more accessible than that for in situ fabrication.
Polymerase and Ligase Extension. DNA polymerase and ligase enhance the power that can be achieved by hybridization alone. Mismatches close to the center of an oligonucleotide have a strongly destabilizing effect on the duplex. Mismatches at both ends are less destabilizing and thus more difficult to discriminate by hybridization. Poly­merases and ligases, by contrast, are affected more by terminal than internal mismatches. Polymerase uses the tethered oligonucleotide as a primer in an extension reaction in which the subtrate is a dideoxy-nucleotide triphos­phate (ddNTP). The enzyme incorporates and extends only one base, that which is complementary to the next base in the target. This is known as minisequencing [8]. Related methods that use ligase have been developed [9] and are being adapted for use with arrays. Heat-stable enzymes help to overcome the problems that arise from carrying out the reactions at low temperature, conditions which encourage formation of intramolecular folding in the target. Thermostable polymerases and ligases are able to function at high temperatures, where the oligonucleo­tide duplexes have short lifetimes.