STRP – Short tandem repeat polymorphism.AFLP – Amplified fragment length polymorphism.RFLP – Restriction fragment length polymorphism.Restriction digests are also necessary for performing any of the following analytical techniques: The vector typically contains a multiple cloning site where many restriction site may be found, and a foreign piece of DNA may be inserted into the vector by first cutting the restriction sites in the vector as well the DNA fragment, followed by ligation of the DNA fragment into the vector. Restriction digest is most commonly used as part of the process of the molecular cloning of DNA fragment into a vector (such as a cloning vector or an expression vector). This is in contrast to proteins, in which there is no 'backbone', and net charge is generated by different combinations and numbers of charged amino acids. Net charge in DNA is produced by the sugar-phosphate backbone. Since none of the four nucleotide bases carry any charge, net charge becomes insignificant and size is the main factor affecting rate of diffusion through the gel. The molecules travel at different rates (and therefore end up at different distances) depending on their net charge (more highly charged particles travel further), and size (smaller particles travel further). The gel is then subjected to an electric field, which draws the negatively charged DNA across it. 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). 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.Īfter restriction digest, DNA can then be analysed using agarose gel electrophoresis. 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. 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. 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. the same nucleotide sequence in the 5' – 3' direction). These recognition sequences are typically four, six, eight, ten, or twelve nucleotides long and generally palindromic (i.e. It is used in genetic fingerprinting, plasmid subcloning, and RFLP analysis.Ī given restriction enzyme cuts DNA segments within a specific nucleotide sequence, at what is called a restriction site. 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. 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. The cleavage method makes use of an important class of DNA-cleaving enzymes isolated primarily from bacteria. This enzymatic technique can be used for cleaving DNA molecules at specific sites, ensuring that all DNA fragments that contain a particular sequence at a particular location have the same size furthermore, each fragment that contains the desired sequence has the sequence located at exactly the same position within the fragment. It is sometimes termed DNA fragmentation (this term is used for other procedures as well). JSTOR ( May 2009) ( Learn how and when to remove this template message)Ī restriction digest is a procedure used in molecular biology to prepare DNA for analysis or other processing.Unsourced material may be challenged and removed.įind sources: 'Restriction digest' – news Please help improve this article by adding citations to reliable sources. This article needs additional citations for verification.

Restriction fragments