Gene Mapping by In Situ Hybridization
The previous mapping methods are indirect in that they provide information on the physical location of a gene on a particular chromosome but without actually visualizing the gene's map position. A more direct approach is in situ hybridization, which involves hybridizing DNA (or RNA) probes directly to metaphase chromosomes spread on a slide and visualizing the hybridization signal (and thus the location of the gene to which the probe hybridizes) under a microscope.
The DNA in metaphase chromosomes is denatured in place (hence, in situ) on the slide, and hybridization of a labeled probe is allowed to proceed. Methods for mapping single-copy gene sequences by in situ hybridization originally were laborious and slow, requiring long exposures of the slides under photographic emulsion to detect the location of hybridized probe that had been labeled with low-level isotopes, such as tritium. Mapping with confidence required analysis of many metaphase spreads to distinguish the real hybridization signal from background radioactivity. However, more sensitive techniques have now been developed that enable rapid detection of hybridized probes labeled non radioactively with compounds that can be visualized by fluorescence microscopy (Fig). Even in a single metaphase spread, one can easily see the position of the gene being mapped.
In combination with banding methods for chromosome identification, fluorescence in situ hybridization can be used to map genes to within 1 to 2 million base pairs (1000 to 2000 kb) along a metaphase chromosome. Although this degree of resolution is a considerable improvement over other methods, it is still substantially larger than the size of most individual genes.
Figure: Gene mapping by in situ hybridization of a biotin-labeled DNA probe for the human muscle glycogen phosphorylase gene (MGP) to a spread of human metaphase chromosomes. Location of the MGP gene is indicated by the bright spots seen over each chromatid at the site of the gene in band q13 of chromosome 11. The mapping of MGP to 11q13 also assigns the locus for McArdle disease, an autosomal recessive myoglobinuria caused by deficiency of MGP. (Photograph courtesy of Peter Lichter, Yale University)
The previous mapping methods are indirect in that they provide information on the physical location of a gene on a particular chromosome but without actually visualizing the gene's map position. A more direct approach is in situ hybridization, which involves hybridizing DNA (or RNA) probes directly to metaphase chromosomes spread on a slide and visualizing the hybridization signal (and thus the location of the gene to which the probe hybridizes) under a microscope.
The DNA in metaphase chromosomes is denatured in place (hence, in situ) on the slide, and hybridization of a labeled probe is allowed to proceed. Methods for mapping single-copy gene sequences by in situ hybridization originally were laborious and slow, requiring long exposures of the slides under photographic emulsion to detect the location of hybridized probe that had been labeled with low-level isotopes, such as tritium. Mapping with confidence required analysis of many metaphase spreads to distinguish the real hybridization signal from background radioactivity. However, more sensitive techniques have now been developed that enable rapid detection of hybridized probes labeled non radioactively with compounds that can be visualized by fluorescence microscopy (Fig). Even in a single metaphase spread, one can easily see the position of the gene being mapped.
In combination with banding methods for chromosome identification, fluorescence in situ hybridization can be used to map genes to within 1 to 2 million base pairs (1000 to 2000 kb) along a metaphase chromosome. Although this degree of resolution is a considerable improvement over other methods, it is still substantially larger than the size of most individual genes.
Figure: Gene mapping by in situ hybridization of a biotin-labeled DNA probe for the human muscle glycogen phosphorylase gene (MGP) to a spread of human metaphase chromosomes. Location of the MGP gene is indicated by the bright spots seen over each chromatid at the site of the gene in band q13 of chromosome 11. The mapping of MGP to 11q13 also assigns the locus for McArdle disease, an autosomal recessive myoglobinuria caused by deficiency of MGP. (Photograph courtesy of Peter Lichter, Yale University)
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