Molecular Basis of Heredity: Part 4. Gene Identification and Tests
For the microarray image shown on this slide, let us assume that each spot on the array contains the DNA of a different form of a polymorphic marker. For every form or variation of the marker, there is a separate spot on the array. DNA from the test specimen is applied to the array and allowed to hybridize (bind specifically) to the DNA on the microarray. This hybridization is based on the complementarity of the DNA double helix. Under the proper conditions, the DNA from the specimen will find its complementary counterpart on the array and hybridize (bind) to it. The DNA from the specimen is labeled with a fluorescent tag so that it can be seen with a color camera. In this way, the specimen can be genotyped for each marker represented on the array, based on the pattern of spots to which it hybridizes. For example, if a dimorphic marker (marker with two forms– A and B) is used, test samples can be genotyped as AA, BB or AB based on the pattern of fluorescence on the array. Using this technology, the entire genome of a specimen can be investigated for a large number of markers simultaneously, significantly reducing the labor, time and cost involved in the analysis. Imagine that for a particular marker a preponderance of the B allele is found in the case group but not in the control group: one might suspect that there is a susceptibility gene somewhere in the region of that particular marker. The genomic region surrounding the marker could then be further investigated to attempt to identify disease-associated genes and mutations.
Microarrays also can be used for other purposes as well, such as investigating gene expression in cells. In this application of microarray technology, DNA sequences representing all (or many) of the genes in the genome are immobilized on the array. Fluorescently labeled probes derived from the RNA of a particular cell type are applied to the array to see which genes are being expressed in the cells (and which are not). The spots corresponding to expressed genes will light up because a fluorescently labeled specimen hybridizes there. Spots corresponding to unexpressed genes will remain unlabelled. Under the proper experimental conditions, quantitation of the amount of gene expression also may be possible. This is a useful approach for comparing gene expression between normal and disease tissue. Any differences found could indicate candidate genes associated with susceptibility to or progression of the disease.
To interpret the microarray shown in this slide in a slightly different way, let us now assume that this is a gene expression microarray and that there is a spot on the microarray bearing DNA from every gene in the human genome. Let us also assume that control cDNA, cDNA derived from normal tissue has been tagged with GREEN fluorescent dye and hybridized to the target DNA on the microarray, and that sample cDNA, cDNA derived from diseased tissue is tagged with RED fluorescent dye and hybridized to the target DNA on the microarray. The spots that show green suggest that there is no expression of that particular gene in the disease tissue. The spots that show red suggest that there is no expression of that particular gene in normal tissue. The spots that show yellow would suggest that there is expression of that particular gene in both normal and disease tissue. The spots that show no fluorescence suggest that there is no expression of that particular gene in either normal or disease tissue. Discovery of the differences in gene expression between normal and disease tissue allows researchers to focus their studies on the genes whose expression differs between the normal and disease state and accelerate their understanding of the biochemical pathways that might be activated, or inactivated, in association with a particular disease.
Another use of microarray technology is to search for small gains or losses in genomic DNA in patients with particular disorders. For this application, DNA representing a large percentage of the DNA in the human genome is ordered on an array. A specimen from a patient with a disorder of unknown etiology is labeled with one fluorescent dye and mixed with equal quantities of control DNA from a person without the disorder that has been labeled with a different fluorescent dye. These two DNA samples are then allowed to hybridize to the DNA on the array. By analyzing the fluorescence bound to each of the spots on the array, scientists can determine if portions of the genome are missing from the patient DNA, or occur in more copies than usual in the patient DNA. In this way, researchers can identify regions of the genome where losses or gains of genetic information might be associated with the symptoms in the patient. This is called comparative genomic hybridization.
Keywords: gene identification | genetic disorders | genetic marker | genetics | techniques | microarray | gene expression | human genome | genomic hybridization
- National Center for Biotechnology Information, National Library of Medicine. (2003). A Science Primer: microarrays: chipping away at the mysteries of science and medicine. Retrieved 12-08-2005 from http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html
- Nussbaum, R. L., McInnes, R. R., & Willard, H. F. (2004). Thompson & Thompson: Genetics in Medicine (6th ed.). Saunders, an imprint of Elsevier.
Visiscience Corporation. (2005). Microarray. ScienceSlides. All rights reserved.
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