In this article we will discuss about the uses of DNA microarrays.
The DNA Microarray technology is used to determine the level of expression of many thousands of genes simultaneously. This new approach is used not for individual genetic loci, rather, for the analysis of genome-wide patterns of gene expression. Using DNA microarrays, it is possible to estimate the relative level of gene expression of each gene in the genome.
The DNA microarray or chip is a high density grid system, consisting of a flat solid substrate about the size of a postage stamp that can be used to detect hybridisation of target DNA under appropriate conditions. The chip contains 10,000 to 100,000 distinct spots, from 75 to 150 µm in diameter.
The spacing between spots on an array is usually 100 to 200 µm. Each spot contains a different immobilised DNA sequence that can be hybridised with DNA (or RNA) from a large number of different cells. Two types of chips are currently available: one, in which oligonucleotides have been synthesised directly on the chip, one nucleotide at a time, by automated procedures.
These chips have hundreds of thousands of spots per array; second, chips in which double-stranded DNA sequences of 500 to 5000 base pairs have been deposited through drops by capillary action from miniaturized devices mounted on the movable head of a robotic workstation.
These chips have tens of thousands of spots per array. The surface onto which DNA is spotted is critically important. The ideal surface immobilizes the target DNAs, and is compatible with stringent probe hybridisation conditions.
The procedure shown (Fig. 24.1) depicts only 6 spots in a chip, each of which contains a DNA sequence that serves as a probe for a different gene. Experimental cells are used for the extraction of cellular mRNA, and a control sample of mRNA from another source. The samples are subjected to reverse transcription to obtain DNA strands.
In the experimental material, the primer for reverse transcription is tagged with a green fluorescent label, while primers of the control material receive red fluorescent label. After the DNA strands have been obtained in sufficient quantity, the fluorescent samples are mixed and hybridised with the DNA in the spots in the chip. The hybridisation is competitive because the two samples were mixed.
Therefore, the density of red and green strands bound to the chip is proportional to the concentration of red or green molecules in the mixture. Genes that are over-expressed in the experimental sample relative to the control will have more green strands hybridised to the spot, whereas those that are under-expressed in the experimental sample relative to the control will have more of red strands hybridised to the spot.
The intensity of fluorescence is viewed by placing the chip under a laser scanning microscope or a fluorescence microscope that scans each pixel, which is the smallest discrete unit in a visual image. The intensity of fluorescent label is recorded. The signals are synthesised to produce a signal value for each spot in the microarray.
The signals indicate the relative levels of gene expression through colour. Green or yellow green indicate over-expression in experimental sample, while red or orange indicates under-expression in experimental sample. Yellow indicates equal expression in both experimental and control samples.
DNA microarray technology is useful for study of large number of cells growing under different conditions, at different developmental stages, or at different stages of a disease. Besides detection of gene expression, this technology can be used to detect mutations and polymorphisms, to map genomic DNA clones, and to compare the gene expression pattern in normal and diseased tissues.