Long-Range Restriction Mapping
Restriction mapping is a technique that utilizes the ability of restriction enzymes to selectively cleave DNA to obtain a map of the order of the restriction sites. The pattern of the sites, and so the sequences of DNA they have recognized, can be used to clarify the arrangement of bases comprising the DNA region under investigation.
Long-range restriction mapping allows the study of large regions of DNA, particularly mammalian DNA, which can encompass regions that have not yet been cloned-- not yet obtained in any tangible form. The main utility of long-range restriction maps is to place upper and lower limits on the physical distance that separates two or more DNA markers known to be linked together. This information then guides the investigator in deciding whether or not to investigate the particular DNA region in more detail, or whether to investigate other DNA regions. Long-range restriction mapping thus speeds up the restriction analysis of DNA.
The technique requires two tools—the first is a method for separating very large DNA fragments based on their differing sizes, and the second is a set of reagents for cutting DNA at relatively rare restriction sites. Use of restriction enzymes that recognized relatively common DNA sequences would generate many short fragments of DNA, instead of the long pieces necessary for the technique's success.
The required methodology was invented by Schwartz and Cantor and is known as Pulsed Field Gel Electrophoresis (PFGE). The technique permits the physical separation of DNA molecules that vary in size up to nine megabases. PFGE became practically useful for mapping mammalian chromosomes with the discovery of a special class of rare-cutting restriction enzymes. Such enzymes recognize a sequence of eight bases, instead of the regular six bases recognized by other restriction enzymes, and also recognize a particular dinucleotide, CpG, that is uncommon in mammalian DNA.
To perform the mapping, genomic DNA of very high molecular weight is simultaneously treated with several rare-cutting restriction enzymes to generate several large fragments. These fragments are then treated with individual rare-cutting enzymes. All the samples are loaded onto an electrophoretic gel and the fragments separated into visibly distinct bands based on their size. The pattern of banding provides a map of the DNA region under study.
Prior to the advent of cloning technology, long-range restriction mapping provided the most feasible means for estimating physical distances between areas of DNA. Such physical mapping is now often more easily accomplished by cloning. Nevertheless, there are still many situations where a region of interest is flanked by the markers (known regions of the DNA) that are too distant from each other to allow rapid cloning.
One area where long-range restriction mapping has been, and continues to be useful is in generating the large defined stretches of DNA used to construct the Yeast Artificial Chromosome cloning system. This cloning system remains important for mouse genetic studies.
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