Restriction landmark genomic scanning
Encyclopedia
Restriction Landmark Genomic Scanning (or RLGS) is a genome
analysis method that allows for rapid simultaneous visualization of thousands of landmarks, or restriction sites
. Using a combination of restriction enzyme
s some of which are specific to DNA modifications
, the technique can be used to visualize differences in methylation
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
process is then employed, yielding high-resolution results. The radioactive second-dimension gel is then allowed to expose a large sheet of film
. 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 very useful when doing whole-genome
scans, and can effectively do the work of thousands of polymerase chain reaction
s at once. It readily detects alterations deviating from normal, and thus is exceptionally effective in identifying hyper/hypomethylation
in tumor
s, deletions or amplifications of genes, or simply changes in gene expression
throughout the development of an organism.
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
analysis method that allows for rapid simultaneous visualization of thousands of landmarks, or restriction sites
Restriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
. Using a combination of restriction enzyme
Restriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
s some of which are specific to DNA modifications
Epigenetics
In biology, and specifically genetics, epigenetics is the study of heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence – hence the name epi- -genetics...
, the technique can be used to visualize differences in methylation
DNA methylation
DNA methylation is a biochemical process that is important for normal development in higher organisms. It involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring...
levels across the genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
of a given organism. RLGS employs direct labeling of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
, which is first cut by a specific series of restriction enzymes, and then labeled by a radioactive isotope
Isotope
Isotopes are variants of atoms of a particular chemical element, which have differing numbers of neutrons. Atoms of a particular element by definition must contain the same number of protons but may have a distinct number of neutrons which differs from atom to atom, without changing the designation...
(usually phosphorus-32
Phosphorus
Phosphorus is the chemical element that has the symbol P and atomic number 15. A multivalent nonmetal of the nitrogen group, phosphorus as a mineral is almost always present in its maximally oxidized state, as inorganic phosphate rocks...
). A two-dimensional
Two-dimensional gel electrophoresis
Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins...
electrophoresis
Electrophoresis
Electrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...
process is then employed, yielding high-resolution results. The radioactive second-dimension gel is then allowed to expose a large sheet of film
Photographic film
Photographic film is a sheet of plastic coated with an emulsion containing light-sensitive silver halide salts with variable crystal sizes that determine the sensitivity, contrast and resolution of the film...
. 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
Autoradiograph
An autoradiograph is an image on an x-ray film or nuclear emulsion produced by the pattern of decay emissions from a distribution of a radioactive substance...
. 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 very useful when doing whole-genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
scans, and can effectively do the work of thousands of polymerase chain reaction
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
s at once. It readily detects alterations deviating from normal, and thus is exceptionally effective in identifying hyper/hypomethylation
DNA methylation
DNA methylation is a biochemical process that is important for normal development in higher organisms. It involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring...
in tumor
Tumor
A tumor or tumour is commonly used as a synonym for a neoplasm that appears enlarged in size. Tumor is not synonymous with cancer...
s, deletions or amplifications of genes, or simply changes in gene expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
throughout the development of an organism.
Sources
- http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=8388788
- http://www.informatics.jax.org/searches/accession_report.cgi?id=MGI:71287
- a picture of an auto-rad from Roswell Park Cancer Institute
