Innovative and powerful hybridization technologies developed in the early 1990s permit numerous hybridization assays to be simultaneously conducted on a common sample under the same conditions. A DNA or oligonucleotide microarray consists of many thousands or millions of different unlabeled DNA or oligonucleotide probe populations that have been fixed to glass or another suitable surface within a high-density grid format. Within each grid square are large numbers of identical copies of just one probe (a grid square with its probe population is called a feature). For example, oligonucleotide microarrays often have a 1.28 cm × 1.28 cm surface that contains very many different features that each occupy about 5 or 10 μm2 (Figure1).
Fig1. Principle of microarray hybridization. A microarray is a solid surface on which molecules can be fixed at specific co-ordinates in a high-density grid format. Oligonucleotide or DNA microarrays have thousands to millions of different synthetic, single-stranded oligonucleotide or DNA probes fixed at specific pre-determined positions in the grid. As shown by the expanded item enclosed within the circle (left), each grid square will have many identical copies of a single type of oligonucleotide or DNA probe (a feature). An aqueous test sample containing a heterogeneous collection of labeled DNA fragments or RNA transcripts is denatured and allowed to hybridize with the probes on the array. Some probes (e.g., the A1 feature) may find numerous complementary sequences in the test population, resulting in a strong hybridization signal; for other probes (e.g., the B1 feature) there may be few complementary sequences in the test sample, resulting in a weak hybridization signal. After washing and drying of the microarray, the hybridization signals for the numerous different probes are detected by laser scanning, giving huge amounts of data from a single experiment. (For ease of illustration, we show test-sample nucleic acids with end labels, but normally they would contain labels on internal nucleotides.)
A test sample—an aqueous solution containing a complex population of fluorescently labeled denatured DNA or RNA—is hybridized to the different probe populations on the microarray. After a washing step to remove nonspecific binding of labeled test-sample molecules to the array, the remaining bound fluorescent label is detected using a high resolution laser scanner. The signal emitted from each feature on the array is analyzed using digital imaging software that converts the fluorescent hybridization signal into one of a palette of colors according to its intensity (see Figure 1).
Because the intensity of each hybridization signal reflects the amount of labeled molecules that have bound to a feature, microarray hybridization is used to quantitate different sequences in complex test-sample populations such as different samples of genomic DNA or total cellular RNA (or cDNA). As described in later chapters, frequent applications include quantifying different transcripts (expression profiling) and also scanning genomes to look for large-scale deletions and duplications.
