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Restriction Digestion of DNA

BY: Lakshmi K Sugavanam | Category: Biotech-Research | Submitted: 2011-04-09 19:54:48
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Article Summary: "Importance of restriction digestion as a tool in DNA analysis. It is sometimes called as DNA fragmentation..."

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A restriction digestion is a procedure used in molecular biology to digest DNA into smaller fragments for further analysis. It is sometimes called as DNA fragmentation. It was described by Hartl and Jones as 'The enzymatic technique that can be used for cleaving DNA molecules at specific sites, ensuring that all DNA fragments that contain a particular sequence have the same size; furthermore, each fragment that contains the desired sequence has the sequence located at exactly the same position within the fragment. The cleavage method makes use of an important class of DNA-cleaving enzymes isolated primarily from bacteria. These enzymes are called restriction endonucleases or restriction enzymes, and they are able to cleave DNA molecules at the positions at which particular short sequences of bases are present.'

Restriction enzymes are enzymes that are isolated from bacteria that recognize specific sequences in DNA and then cut the DNA to produce fragments, called restriction fragments. These restriction endonucleases make a cut wherever that sequence occurs in the DNA.

The process of restriction digestion is carried out by mixing the DNA and the restriction enzymes together. Salts like NaCl or MgCl2 have to be added to serve as a buffer and maintain the pH. The buffer is specific for a specific enzyme. Then, the whole mixture has to be incubated at a specific temperature for an hour or longer as per the specialized requirement, for the reaction to occur optimally. Since the enzymes are proteins, they can get denatured easily. To prevent this, they have to be carefully stored in ice or in the freezer till they have to be taken out to be used. After the specified reaction time is complete, the mixture is put back on ice to arrest the restriction process. If allowed to continue, non-specific degradation of DNA might occur. Then, the digested sample is run on agarose gel to view the various digests and these can be purified for further experiments. The digested DNA obtained after this process is very often amplified using PCR which makes it more suitable for analysis using agarose gel electrophoresis or chromatography techniques.

One specific restriction enzyme cleaves DNA at a specific sequence of nucleotides, which is known as a restriction site. These are recognition sequences that may be four to twelve bases long. Most long sequences will have the presence of at least a few of these restriction sites. Many restriction enzymes have been recognized for hundreds of such restriction sites. Each restriction enzyme will cleave the DNA differently. The efficiency and the conditions at which they can digest DNA differ from enzyme to enzyme. Most manufacturers that produce such enzymes provide the buffer solution containing the unique mix of cations and other components, specific to that enzyme that is required by it to digest the DNA as efficiently as possible. Different restriction enzymes also have different optimal temperatures under which they function.

DNA can be cut with two or more enzymes. But, in such cases, the buffer solution should be compatible with all of them. Some special enzymes may require some specific conditions like the addition of Bovine Serum Albumin (BSA) into the reaction mixture, or detergents like Triton X100 to reduce the surface tension.

Some restriction enzymes have been known to exhibit unusual behavior like cutting at sites other than its known restriction sites, at extreme conditions. This is known as star activity. Star activity is mostly presented at sites that are similar in base composition to the restriction site. For example, EcoRI has a restriction site GAATTC. Under unusual conditions, it may restrict a DNA having a restriction site CAATTC.

When artificial plasmids are constructed, they are typically designed to be carrying at least a few of such restriction sites so that a gene or DNA fragment of interest can be inserted into the vector for cloning purposes.

Restriction digestion is a necessary process to be able to perform analysis of the DNA by RFLP, AFLP or STRP techniques. Restriction enzymes play a very important role in the construction of recombinant DNA molecules, as is done in gene cloning experiments. Another application of restriction enzymes is to map the locations of restriction sites in DNA. It is used in gene fingerprinting and in RFLP experiments.

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