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By Springer-Verlag, Jorg Hoheisel

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To a protein-encoding mRNA a 5′ tag sequence and a 3′ ribosome-blocking sequence are attached (A). In a pool of such molecules, such as a randomly mutated gene library, each mRNA is paired with a unique tag and all have the same 3′ sequence. Following in-vitro translation either on a microarray or in a test tube, the nascent protein remains attached to the mRNA (B). During hybridization the tag directs each mRNA-protein to a particular address on the Tag probe array (C), where all the proteins are screened simultaneously for activity (D).

Up to 50 kb of sequence can thus be determined with 200,000 probes, or 400,000 probes if both DNA strands are interrogated. Impending decreases in array feature size (see Sect. 4) will extend this capacity further. 2 kb coding sequence of the ATM gene [33], BRCA1 coding mutations [34, 35], p53 mutations [36, 37], cytochrome P450 variants involved in drug metabolism [38], and HIV sequence variants [39, 40], among others. The last three arrays are commercial products currently available from Affymetrix.

A A known biallelic polymorphism at position 0 is interrogated by a block of four or five probe sets (five in this example). Each probe set consists of four probes, a perfect match and a mismatch to allele A, and a perfect match and a mismatch to allele B. One probe set in a block is centered directly over the polymorphism “0”), and others are centered upstream (–4, –1) and downstream (+1, +4). B The sequences of the probe set centered over the polymorphism is shown. C Sample images of blocks showing homozygous A, heterozygous A/B, or homozygous B at the same SNP site.

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