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Concept
Restriction digest
Summary
A restriction digest is a procedure used in molecular biology to prepare DNA for analysis or other processing. It is sometimes termed DNA fragmentation, though this term is used for other procedures as well. In a restriction digest, DNA molecules are cleaved at specific restriction sites of 4-12 nucleotides in length by use of restriction enzymes which recognize these sequences.
The resulting digested DNA is very often selectively amplified using polymerase chain reaction (PCR), making it more suitable for analytical techniques such as agarose gel electrophoresis, and chromatography. It is used in genetic fingerprinting, plasmid subcloning, and RFLP analysis.
A given restriction enzyme cuts DNA segments within a specific nucleotide sequence, at what is called a restriction site. These recognition sequences are typically four, six, eight, ten, or twelve nucleotides long and generally palindromic (i.e. the same nucleotide sequence in the 5' – 3' direction). Because there are only so many ways to arrange the four nucleotides that compose DNA (Adenine, Thymine, Guanine and Cytosine) into a four- to twelve-nucleotide sequence, recognition sequences tend to occur by chance in any long sequence. Restriction enzymes specific to hundreds of distinct sequences have been identified and synthesized for sale to laboratories, and as a result, several potential "restriction sites" appear in almost any gene or locus of interest on any chromosome. Furthermore, almost all artificial plasmids include a (often entirely synthetic) polylinker (also called "multiple cloning site") that contains dozens of restriction enzyme recognition sequences within a very short segment of DNA. This allows the insertion of almost any specific fragment of DNA into plasmid vectors, which can be efficiently "cloned" by insertion into replicating bacterial cells.
After restriction digest, DNA can then be analysed using agarose gel electrophoresis. In gel electrophoresis, a sample of DNA is first "loaded" onto a slab of agarose gel (literally pipetted into small wells at one end of the slab).
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