Restriction digest
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A restriction digest is a procedure used in molecular biology to prepare DNA for analysis or other processing. Also known as DNA fragmentation, it uses a restriction enzyme to selectively cleave strands of DNA into shorter segments, which are more suitable for analytical techniques such as chromatography. Restriction digests are performed in Genetic fingerprinting techniques using restriction fragment length polymorphisms, and are used in many other DNA manipulations.
A given restriction enzyme cuts DNA segments within a specific nucleotide sequence, and always makes its incisions in the same way. These recognition sequences are typically only four to twelve nucleotides long. Because there are only so many ways to arrange the four nucleotides--A,C,G and T--into a four or eight or twelve nucleotide sequence, recognition sequences tend to "crop up" by chance in any long sequence. Furthermore, restriction enzymes specific to hundreds of distinct sequences have been identified and synthesized for sale to laboratories. As a result, potential "restriction sites" appear in almost any gene or chromosome. Meanwhile, the sequences of some artificial plasmids include a "linker" that contains dozens of restriction enzyme recognition sequences within a very short segment of DNA. So no matter the context in which a gene naturally appears, there is probably a pair of restriction enzymes that can snip it out, and which will produce ends that enable the gene to be spliced into a plasmid (i.e. which will enable what molecular biologists call "cloning" of the gene).
Once the cuts are made with the restriction enzymes, the plasmid can then be loaded into a gel for electrophoresis. In electrophoresis, the negatively charged DNA is pulled through an agarose gel, causing the shortest fragments of the plasmid to extend farther into the gel, and separating each cut segment from the other. A marker is also loaded with the gel, indicating the amount of base pairs in each segment of the plasmid. The new DNA fragment may then be extracted from the gel by cutting it, and doing a gel purification. Through ligation of a vector which has also been cut by a restriction enzyme, the fragment of interest can then be inserted into this vector, and a new synthetic plasmid is formed. The new plasmid is then transformed into a biological system, and replication occurs.
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[edit] Possible Uses
Restriction digests are necessary for performing any of the following analytical techniques:
- RFLP - Restriction Fragment Length Polymorphism
- AFLP - Amplified Fragment Length Polymorphism
- RAPD - Randomly Amplified Polymorphic DNA
- STRP - Simple Tandem Repeat Polymorphism
[edit] Various Restriction Enzymes
There are numerous types of restriction enzymes, each of which will cut DNA differently. (See article on Restriction enzymes for examples). There are some that cut a three base pair sequence while others can cut four, six, and even eight. Each enzyme has distinct properties that determine how efficiently it can cut and under what conditions. Most manufacturers that produces such enzymes will often provide a specific buffer solution that contains the unique mix of cations and other components that aid the enzyme in cutting as efficiently as possible. Different restriction enzymes also have different optimal temperatures under which they function.
[edit] External Links
- New England Biolabs - Manufacturer of restriction enzymes. This site contains highly detailed information on numerous enzymes, their optimal temperatures, and recognition sequences.
[edit] See also
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