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Encyclopedia > Restriction digest

A restriction digest is a molecular biology procedure used 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. Molecular biology is the study of biology at a molecular level. ... DNA replication Deoxyribonucleic acid (DNA) is a nucleic acid which carries genetic instructions for the biological development of all cellular forms of life and many viruses. ... It has been suggested that Endonuclease be merged into this article or section. ... Chromatography is a family of analytical chemistry techniques for the separation of mixtures. ... Genetic fingerprinting, DNA testing, DNA typing, and DNA profiling are techniques used to distinguish between individuals of the same species using only samples of their DNA. Its invention by Sir Alec Jeffreys at the University of Leicester was announced in 1985. ... In molecular biology, the term restriction fragment length polymorphism (or RFLP) is used in two related contexts: as a characteristic of DNA molecules (arising from their differing nucleotide sequences) by which they may be distinguished, and as the laboratory technique which uses this characteristic to compare DNA molecules. ...

A given restiction 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). part of a DNA sequence A DNA sequence (sometimes genetic sequence) is a succession of letters representing the primary structure of a real or hypothetical DNA molecule or strand, The possible letters are A, C, G, and T, representing the four nucleotide subunits of a DNA strand (adenine, cytosine, guanine...

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. See also: DNA sequencing DNA sequencing is the process of determining the nucleotide order of a given DNA fragment, called the DNA sequence. ...



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