Chip arrays consist of oligonucleotide probes that are synthesized in situ on silica wafers under the control of photolithographic chemistry (11). The lengths of both the probes and targets are critical to the sensitivity and specificity of the hybridization reactions. Probe oligomers between 20 and 60 bases in length provide the best balance between discriminant and sensitive hybridization. As well as the correct oligonucleotides, "missense" oligonucleotides are created in parallel for each probe, which allow the specificity of the hybridization reaction to be determined by comparison of the extent of hybridization to the missense and sense probes, as well as background noise reduction by subtraction of missense from sense signal. In the Affymetrix system (see www.affymetrix.com/), the sense probes are 25 mers (high specificity but reduced sensitivity compared with longer oligomers) and are referred to as "perfect matches" (PM), whereas the missense probes are termed "mismatches" (MM), differing from their sense probe by just their middle, 13th base. The lower the value of the missense:sense signal ratio, the better. For each gene, a set of 11-20 PM and MM probe pairs is laid down, thereby increasing the sensitivity for detecting a gene transcript to, at best, 1 in 300,000 transcripts. Through the reduction in feature size, as many as 54,000 gene probes can now be included on one chip. As with dot-blot slides, the target for oligonucleotide chips is created from test sample RNA. The RNA is reverse transcribed into cDNA followed by transcription back to RNA, as cRNA. Because this two-step procedure results in amplification of the mRNA, less RNA is required for chip arrays compared with dot-blot arrays (5-10 |g vs 30 |g). To produce the labeled target, biotin-conjugated bases are used in the synthesis of the cRNA. The labeled cRNA requires fragmentation into lengths of 20 to 200 base pairs, followed by denaturing before application to the array. Unlike dot-blot arrays, competitive hybridization with a reference RNA is not required for interchip array expression profile comparison because the oligonucleotide synthesis of each feature is so precisely controlled that interchip variation is minimal. The cost of commercially available generic chips has reduced substantially since their introduction, making the technology affordable to an increasing number of research facilities. Furthermore, customized chips can be ordered that contain only the gene features of interest from previous experiments or known to be critical to a particular illness, tissue, or set of experimental conditions.
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