Dna Microarrays
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By examining where and when genes are expressed in a cell or organism, one can acquire valuable hints to its function, for genes compile the genetic make-up of an organism and exploring the function of genes is helping to uncover the complexity of ourselves and of other forms of life (1). Significant developments have been made in gene monitoring techniques specifically in DNA microarrays which only very recently revolutionized genome expression analysis (1).
Despite continuous improvements and modification to the technique, DNA microarrays are still no more than a glass microscope slide studded with individual immobile nucleotide fragments (1, 2). The fundamentals of DNA microarrays are set on complementary base-pairing (3), and because the exact sequence and position of every segment on the slide is known, each serves as a probe for a specific gene (1).
The two main microarray systems are spotted DNA and oligonucleotide arrays (4); there are others with various difference but all are essentially derived from the same simple design (3). Messenger RNA from the cells of interest is first converted into cDNA which is then labeled with a fluorescent probe and incubated with the microarray where hybridization occurs; positions of hybridization are detected with a scanning-laser microscope (1). For comparison studies, typically two differently labeled samples (a test and a control) are mixed and the scanned intensity of each dye is proportional to the amount of hybridization by the respective cDNA (4).
Regardless of simplicity, what makes microarrays truly ground-breaking is its ability to analyze thousands of nucleotides in a single assay as opposed to blotting techniques (5). This high capacity in turn saves both time and resources (4). It should also be noted that the technology allows for the interaction of different molecular pathways to be studied simultaneously and the degree of specificity is so fine-tuned that subtle differences are more easily detected (4). Nonetheless, DNA microarrays are limited to sufficient amounts of RNA that will allot a proper study (5). With microarrays, because of the large quantities of DNA it is easy to be distracted by substantial changes while possibly bypassing expression changes of smaller magnitude in other genes which may hold greater significance (4). However, the greatest challenge is ironically developing a systematic way to properly organize and interpret the large volumes of data (2).
Information gathering from microarrays constitutes two main areas of study (4). Firstly is genotyping in which DNA microarrays aid in the identification and characterization of organisms (4). Microarrays produced from a known sequenced genome, a pathogen for example, can be used to compare genes that are expressed and conserved with other pathogens genes that are un-sequenced. In addition, microarrays can also be specially prepared to detect polymorphisms by using multiple variant sequences of each gene (4). The second area of study is gene expression which, mentioned earlier, provides clues to gene function, the basis of cellular physiology (1). This study has been applied variously from the examination of genes that ripen strawberries to the expression of cancerous cells (1).
An in-depth study of gene expression was conducted by a team from Stanford University to explore the metabolic properties of Saccharomyces cerevisiae, yeast. The main focus of the investigation was conducted during the metabolic shift from anaerobic (fermentation) to aerobic (respiration) (2). The change from fermentation to respiration, or the diauxic shift, is the result of widespread changes in gene expression of fundamental cellular processes. DNA microarrays containing probes of nearly the entire yeast genome were used to monitor the changes and extrapolate patterns in expression (2). DeRisi et. al. observed that as yeast progressively consumed their carbon source (glucose), messenger RNA levels for approximately 900 genes were induced while another 1200 declined in activity. Close to half