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This slide compares the three methods for preparing microarrays. The top panel illustrates photolithography, the middle panel illustrates mechanical printing, and the bottom panel illustrates ink-jet printing.
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Another method for depositing DNA on glass slides is the use of ink-jet printer technology. Ink-jet printers are designed to deposit very small quantities of ink in precise locations. As adapted for microarray spotting, the printhead draws up a small amount of DNA, moves to a particular point on the slide, and ejects the DNA through a very small hole. This technology has also been adapted to provide for the synthesis of oligonucleotides on slides. After each base is added through the printhead to the growing oligonucleotide, the slide is washed free of excess nucleotides, and the exposed bases are primed for the addition of the next nucleotide. Agilent Technologies has pioneered the use of ink-jet printing for making microarrays. The primary drawback of this approach as compared with photolithography is that the maximum number of spots on a slide is far fewer with the ink-jet printing of DNA.
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Most robotic spotters use pins that act by capillary action similar to that of fountain pens. Multiple pins are mounted together and dipped simultaneously into DNA aliquoted into the different wells of a microtiter plate. The DNA in the wells has usually been amplified from cDNA or genomic DNA, using PCR. Oligonucleotides can also be spotted in this way.
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On Affymetrix GeneChips, there are between 10 and 20 oligonucleotides for each gene. The choice of oligonucleotides is determined using a computer program that searches for nonoverlapping stretches of 20-25 bases that are unique in the genome (in order to prevent cross-hybridization). Furthermore, the computer searches for oligonucleotides that will fit empirically derived design rules that dictate the ratio of G–C pairs vs. A–T pairs and that attempt to reduce the likelihood that the oligonucleotide will hybridize to itself, creating hairpin structures.
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