Table 1 Construction of DNA microarray

Microarrays are constructed by synthesizing oligonucleotides on glass slides, by spotting PCR-amplified cDNA clones from EST data (eukaryotes), or by spotting PCR products from genomic DNA template (prokaryotes) using a set of gene-specific primers. The method described in this paper has the advantage of being both more cost effective and less labor intensive for the construction of microarrays from prokaryotic organisms. The procedure is most useful for those laboratories working with organisms for which commercial microarrays are unavailable. The salient features of our approach include:

   • Bipartite Primer: The use of a bipartite primer for the amplification of genes has several advantages (see text). Importantly, it permits the use of a common primer for the amplification of all genes in the second and subsequent amplification steps. This simple modification in primers alleviates the problem of primer-limitation and therefore, is particularly valuable in constructing many arrays for a multi-investigative, collaborative effort.

   • Two stage amplification: The use of the two-stage amplification process increases the signal-to-noise ratio by diminishing the hybridization signal resulting from contaminating genomic DNA used for amplification of PCR fragments. Additionally, it also yields relatively uniform quantities of PCR products.

   • Purification and Quantitation: For efficient binding of the amplified products and to reduce the background signal, it is essential to purify the products. We found ethanol precipitation unsatisfactory, whereas the use of Multiscreen filter plates gave excellent results. One drawback with this technique is the relatively lower percentage recovery of smaller fragments. Therefore, following purification, fragments were quantified and normalized to an equal concentration.

   • Printing: We checked the suitability of various slide surfaces (poly-Lysine and amine) and the spotting buffers from a number of commercial vendors to ensure reproducible and uniform spot morphology and maximal retention of DNA. We found that Superamine slides and MSP spotting buffer from Telechem gave satisfactory results.