DNA Arrays Platforms Techniques and Targets

DNA array technology is based on the well-established and long-exploited principle of nucleic acid hybridization. However, now for the first time it offers the possibility of simultaneously conducting tens or hundreds of thousands of simultaneous hybridizations. Structurally different platforms are currently used for DNA arrays: Macroarrays consisting of dot blots on nitrocellulose or nylon membranes have the disadvantage of moderate throughput and uncontrolled binding of oligonucleotides [40]. A modification to this technique, reverse line blot hybridization [41], enables reasonable throughput if the number of probes is limited to about 10-20. With the widespread availability of microarray core technologies, planar glass microarrays have become the most widely used type of array, owing to their general utility and moderate cost. DNA microarrays consist of microscopic checkerboards of hundreds to thousands of different DNA sequences produced by several methods, of which the two most commonly used techniques are spotted glass slide microarray and high-density oligonucleotide array technology. In the spotted microarray, presynthesized single-stranded or double-stranded DNA is bound or "printed" onto glass slides. The DNA can be generated from cloned, synthesized, or PCR-amplified material. Because of the technical simplicity of this approach, spotted microarrays can be produced in-house as well as purchased from commercial providers. High-density oligonucleotide arrays are constructed by synthesizing short (< 25-mer) oligonucleotides in situ on glass wafers using a photolithographic manufacturing process and are thus only available commercially.

Regarding the structure of microarrays, those with a selected panel of targets can be distinguished from whole-genome DNA microarrays. The former may consist of particular virulence genes or virulence-associated DNA fragments and are applied in virulence typing of microbial strains, whereas the whole-genome DNA microarrays are more designed to perform comprehensive analyses of single isolates, e.g., microbial transcriptome studies. This whole-genome approach has been used to construct a 4290 open reading frame (ORF) E. coli microarray [42, 43] and a 3834-ORF M. tuberculosis microarray [44] as well as an array containing 1660 unique sequences for Helicobacter pylori [45]. Each of the ORFs can be obtained by PCR amplification that uses ORF-specific oligonucleotides, but photolithographic synthesis of oligonucleotides in situ has also been successfully employed for the production of complete ORF chips.

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