Restriction landmark genomic scanning
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Restriction landmark genomic scanning (RLGS) is a genome analysis method for rapid simultaneous visualization of thousands of landmarks, or
restriction sites Restriction sites, or restriction recognition sites, are located on a DNA molecule containing specific (4-8 base pairs in length) sequences of nucleotides, which are recognized by restriction enzymes. These are generally palindromic sequences (beca ...
. Using a combination of restriction enzymes some of which are specific to DNA modifications, the technique can be used to visualize differences in
methylation In the chemical sciences, methylation denotes the addition of a methyl group on a substrate, or the substitution of an atom (or group) by a methyl group. Methylation is a form of alkylation, with a methyl group replacing a hydrogen atom. These t ...
levels across the genome of a given organism. RLGS employs direct labeling of DNA, which is first cut by a specific series of restriction enzymes, and then labeled by a radioactive isotope (usually phosphorus-32). A two-dimensional
electrophoresis Electrophoresis, from Ancient Greek ἤλεκτρον (ḗlektron, "amber") and φόρησις (phórēsis, "the act of bearing"), is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric fie ...
process is then employed, yielding high-resolution results. The radioactive second-dimension gel is then allowed to expose a large sheet of
film A film also called a movie, motion picture, moving picture, picture, photoplay or (slang) flick is a work of visual art that simulates experiences and otherwise communicates ideas, stories, perceptions, feelings, beauty, or atmosphere ...
. The radiation produced by the radioactive labeling will cause the film to be exposed wherever the restriction fragments have migrated during electrophoresis. The film is then developed, yielding a visual representation of the results in the form of an autoradiograph. The same combination of restriction enzymes will produce the same pattern of 'spots' from samples from the same organisms, but different patterns for different types of organism. For example, human and mouse DNA will produce distinctly different patterns when treated with the same combination of enzymes. These finished auto-rads can be examined against each other, revealing any changes in gene expression that lead to visual differences in the film. Each autoradiograph contains thousands of spots, each corresponding to a labeled DNA restriction landmark. RLGS becomes useful when doing whole-genome scans, and can effectively do the work of thousands of polymerase chain reactions at once. It readily detects alterations deviating from normal, and thus is exceptionally effective in identifying hyper/hypomethylation in tumors, deletions or amplifications of genes, or simply changes in
gene expression Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. The ...
throughout the development of an organism.


Sources

* *http://www.informatics.jax.org/searches/accession_report.cgi?id=MGI:71287
a picture of an auto-rad from Roswell Park Comprehensive Cancer Center


References

{{reflist Molecular biology Laboratory techniques Genomics