Box 1
Methods for Creating a DNA Array
Photolithography: The first step is manufacturing a GeneChip probe array. Affymetrix’s gene chip assembles a series of DNA probe arrays of known sequence on a glass wafer using repeated cycles of photolithography. A combinatorial array of known DNA sequences is thus manufactured at specific sites on the chip. In the next step a fluidics station automates the hybridization of the array with the target DNA marked with a fluorescence tag. The times, temperatures, and stringency of hybridization are controlled in much the same way an experimenter does in a Northern or Southern blot hybridization. Using an argon-ion laser, a scanner excites the fluorescent tag, and the amount of emitted light is proportional to the amount of target DNA at each position of the array generating a quantitative two-dimensional image of hybridization intensity. Image processing software converts the fluorescent intensities from the probe array to generate genetic information.
Fragment-Based DNA Printing: An important difference between this technique and photolithography and the ink-jet methodology is that this method uses DNA fragments, generated, for example, from cDNAs, BACs, and other biological products, and binds them onto an array rather than building synthetic products onto an array. The basic method, currently in use by both the Brown Laboratory and Molecular Dynamics, is essentially a simple chromatographic procedure. A glass slide is coated evenly with a substance that gives the slide an even binding surface that is positively charged, such as amino silane (Molecular Dynamics) or polylysine (Brown Laboratory). DNA fragments are suspended in a denaturing liquid and spotted onto the prepared slide by the arrayer by lowering the printing tips until they just barely touch the surface of the slide. The denatured DNA is favored to react with the positively charged glass slide and binds immediately. These arrays are then hybridized with cDNAs from two separate sources (e.g., similar samples treated under different conditions or samples from different tissue types or organisms). The cDNA from each source is labeled with a different fluorescent marker and hybridized to the array. The scanner, working under similar conditions to those described above, allows one to detect the relative expression of the genes of interest, providing a snapshot of the overall expression levels of genes of interest. [The Brown laboratory website (http://cmgm.stanford.edu/pbrown) provides a complete protocol for their printing methodology.]
Ink-Jet Method: This method, being used for example, by Incyte Pharmaceuticals and Rosetta Biosystems, Inc., is uniquely adapted from the technology currently used in ink-jet printers. In fact, Rosetta Biosystems directly uses only marginally modified ink-jet cartridges supplied by Epson printers. Ink-jet technology works through the piezoelectric effect, whereby a narrow tube containing a liquid of interest (olgonucleotide synthesis reagents for the arrayer) is encircled by an adapter. An electric charge sent across the adapter causes the adapter to expand at a different rate than the tube and forces a small drop of liquid containing phosphoramidite chemistry reagents from the tube onto a coated slide. This methodology allows one, drop by drop, to precisely synthesize an oligonucleotide directly on the slide. Analysis of the array from there proceeds very much along the lines of the previous methods.
