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 overhanging nucleotide strand is called a sticky end because it can easily bond with complementary DNA fragments.This article needs additional citations for verification. This creates DNA fragments with one nucleotide strand that overhangs at the end. Other restriction enzymes, like Eco RI, cut through the DNA strands at nucleotides that are not exactly opposite each other. SmaI is an example of a restriction enzyme that cuts straight through the DNA strands, creating DNA fragments with a flat or blunt end. Restriction enzymes cut through both nucleotide strands, breaking the DNA into fragments, but they don’t always do this in the same way. DNA fragments: Blunt or sticky ends?ĭNA consists of two complementary strands of nucleotides that spiral around each other in a double helix. For example, EcoRI was the first restriction enzyme isolated from Escherichia coli strain RY13, whereas HindIII was the third enzyme isolated from Haemophilus influenzae strain R d. They are named after the genus and species of the organism they were isolated from and are given a number to indicate the order in which they were found. Scientists have identified and purified hundreds of different types of restriction enzymes. They are used for DNA cloning and DNA fingerprinting. Restriction enzymes are a basic tool for biotechnology research. Recognition sites are usually only short - 4-8 nucleotides. Each restriction enzyme recognises a different and specific recognition site, or DNA sequence.
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