The Spotted Microarray

Spotted arrays most commonly use coated (or "activated") glass as the solid substrate, and are generally the size and shape of, if not an actual, microscope slide. Using glass as the substrate is beneficial for two reasons: (1) it allows the formation of concentrated spots, thereby increasing the array density, and (2) glass provides a flat surface for grid formation and for the confocal microscope to focus on. The coating chemistry can vary [14], but the common feature of the activated glass is that it allows cross-linking between the positively charged surface and the negatively charged nucleotide probes. The spotted array uses either PCR amplicons or presynthesized oligonucleotides of unique fragments from each of the target genes as probes. The PCR product or oligonucleotide probes are then spotted onto the array surface by an automated robot that uses specially designed pins to pick up the sample from a well and deposit it (spot it) onto the array surface. The probes are spotted in a prearranged grid formation; this allows the analysis software to link each spot with the gene that it represents.

PCR probes are generated in a 96-well or higher format using primers specifically designed to amplify a unique region of the ORF (open reading frame). Primer design is most commonly undertaken using software packages such as Pri-meArray [15, 16], Primer 3 [16], or GenomePRIDE [17] which automatically develop primers for all coding sequences in a genome. The PCR amplicon probes are generally between 200 and 2000 base pairs long. The PCR products must then be purified, and it is often sensible to test a subset to ensure primer and probe specificity.

Long-oligonucleotide spotted arrays were developed as an alternative and beneficial method for the generation of probes, removing the need for time-consuming and error-prone PCR. The probes are single-stranded oligonucleotides synthesized by an external company, so their use makes them inherently faster and cheaper. Also, because they are single-stranded to begin with, they are more sensitive to hybridization with their complementary ORF cDNA (see below). The probes are melting-temperature-normalized and are normally 50-70 nucleotides long. The oligonucleotides are specifically designed to locations in each ORF that increase specificity and reduce cross-reactivity with other probes. As mentioned, the oligonucleotides used for these arrays are purchased in a ready-to-use state (see http:// www.operon.com). The user can choose or design specific probes, or alternatively it is possible to purchase a predesigned set of probes that cover every ORF from one of many genomes. Operon currently sells complete genome array ready sets of oligonucleotide probes for many bacterial pathogens including E. coli, M. tuberculosis, Bacillus anthracis, Neisseria meningitides, and Listeria monocytogenes. It is also possible to buy a set of approximately 35 000 probes that match the human genome, allowing host studies with this method too. However, this system is relatively new, and it is currently unknown whether there are problems with the storage of single-stranded nucleotides that are inherently more susceptible to degradation than double-stranded probes generated by PCR.

0 0

Post a comment