![]() They are covalently ligated together through the standard 3' to 5' phosphodiester bonds of DNA. For the purposes of DNA cloning, purified DNA ligase is used to covalently join the ends of a restriction fragment and vector DNA that have complementary ends. DNA ligase During normal DNA replication, DNA ligase catalyzes end-to-end joining (ligation) of short fragments of DNA, called Okazaki fragments. Restriction enzymes can also make straight cuts in the two DNA strands at their recognition site, which generates blunt ends. Cut that creates a sticky end Cut that creates a blunt end Many restriction enzymes make staggered cuts in the two DNA strands at their recognition site, which generates fragments with a single stranded "tail" that overhangs at both ends, called a sticky end. The modification enzyme adds a methyl group to one or two bases, and the presence of this methyl group prevents the restriction endonuclease from cutting the DNA. This is done by modifying the host DNA at or near each potential cleavage site. A restriction site is typically a palindromic sequence, which means that the restriction-site sequence is the same on each strand of DNA when read in the 5' to 3' direction.įor each restriction enzyme, bacteria also produce a modification enzyme so that a host bacterium's own DNA is protected from cleavage. Restriction Enzymes Restriction enzymes are endonucleases produced by bacteria that typically recognize small base pair sequences (called restriction sites) and then cleave both strands of DNA at this site. ![]() Two enzymes facilitate the production of such recombinant DNA molecules:ġ. Therefore, the long DNA molecules that compose an organism's genome must be cleaved into fragments that can be inserted into the vector DNA. Only relatively small DNA molecules can be cloned in any available vector. In order for DNA cloning to be completed, it is necessary to obtain discrete, small regions of an organism's DNA that constitute specific genes. Here, DNA fragmentation is a molecular genetic technique that permits researchers to use recombinant DNA technology to prepare large numbers of identical DNA molecules. This is a form of asexual reproduction where an organism splits into fragments and then each of these fragments develop into mature, fully grown individuals that are clones of the original organism (See reproductive fragmentation).ĭNA cloning can also be performed intentionally by laboratory researchers. However, problems within a cell can sometimes cause fragmentation that results in irregularities such as red blood cell fragmentation and sperm cell DNA fragmentation.ĭNA cloning can be performed spontaneously by the cell for reproductive purposes. These two ways in which fragmentation is used in cellular processes describe normal cellular functions and common laboratory procedures performed with cells. Apoptosis is the programmed destruction of cells, and the DNA molecules within them, and is a highly regulated process. DNA cloning is important in asexual reproduction or creation of identical DNA molecules, and can be performed spontaneously by the cell or intentionally by laboratory researchers. In cell biology, fragmentation is useful for a cell during both DNA cloning and apoptosis. For other uses, see Fragmentation (disambiguation).įragmentation describes the process of splitting into several pieces or fragments.
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