Gene chips

Although sequence data provide a profile of all the genes present in a genome, they give no information as to which genes are switched on (transcribed) and, hence, which are functionally active at any given time/under any given circumstances. Gene transcription results in the production of RNA, either mRNA (usually subsequently translated into a polypeptide) or rRNA or tRNA (which have catalytic or structural functions; Chapter 3). The study of under which circumstances an RNA species is expressed/not expressed in the cell/organism can provide clues as to the biological function of the RNA (or, in the case of mRNA, the function of the final polypeptide product). Furthermore, in the context of drug lead/target discovery, the conditions under which a specific mRNA is produced can also point to putative biopharmaceuticals/drug targets. For example, if a particular mRNA is only produced by a cancer cell, that mRNA (or, more commonly, its polypep-tide product) may represent a good target for a novel anti-cancer drug.

Levels of RNA (usually specific mRNAs) in a cell can be measured by well-established techniques such as northern blot analysis or by PCR analysis. However, the recent advent of DNA microarray technology has converted the identification and measurement of specific mRNAs (or

Figure 4.2 Generalized outline of a gene chip. In this example, short oligonucleotide sequences are attached to the anchoring surface (only the outer rows are shown). Each probe displays a different nucleotide sequence, and the sequences used are usually based upon genome sequence information. The sequence of one such probe is shown as AGGCA. By incubating the chip with, for example, total cellular mRNA under appropriate conditions, any mRNA with a complementary sequence (UCCGU in the case of the probe sequence shown) will hybridize with the probes. In reality, probes will have longer sequences than the one shown above

Figure 4.2 Generalized outline of a gene chip. In this example, short oligonucleotide sequences are attached to the anchoring surface (only the outer rows are shown). Each probe displays a different nucleotide sequence, and the sequences used are usually based upon genome sequence information. The sequence of one such probe is shown as AGGCA. By incubating the chip with, for example, total cellular mRNA under appropriate conditions, any mRNA with a complementary sequence (UCCGU in the case of the probe sequence shown) will hybridize with the probes. In reality, probes will have longer sequences than the one shown above other RNAs if required) into a 'high-throughput' process. DNA arrays are also termed oligonucleotide arrays, gene chip arrays or, simply, chips.

The technique is based upon the ability to anchor nucleic acid sequences (usually DNA based) on plastic/glass surfaces at very high density. Standard gridding robots can put on up to 250 000 different short oligonucleotide probes or 10 000 full-length cDNA sequences per square centimetre of surface. Probe sequences are generally produced/designed from genome sequence data; hence, chip production is often referred to as 'downloading the genome on a chip'. RNA can be extracted from a cell and probed with the chip. Any complementary RNA sequences present will hybridize with the appropriate immobilized chip sequence (Figure 4.2). Hybridization is detectable as the RNA species are first labelled. Hybridization patterns obviously yield critical information regarding gene expression.

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