Plasmid

From DrugPedia: A Wikipedia for Drug discovery

A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae).Plasmid size varies from 1 to over 200 kilobase pairs (kbp).Plasmids used in genetic engineering are called vectors. Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to multiply (make many copies of) or express particular genes. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Next, the plasmids are inserted into bacteria by a process called transformation. Then, the bacteria are exposed to the particular antibiotics. Only bacteria which take up copies of the plasmid survive the antibiotic, since the plasmid makes them resistant. In particular, the protecting genes are expressed and the expressed protein breaks down the antibiotics. In this way the antibiotics act as a filter to select only the modified bacteria. Now these bacteria can be grown in large amounts, harvested and lysed to isolate the plasmid of interest.Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest,it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for,However, a plasmid can only contain inserts of about 1-10 kbp. To clone longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes or yeast artificial chromosomes could be used.There are several methods to isolate plasmid DNA from bacteria, Alkline lysis method and imdiperep kits are there.Plasmid DNA may appear in one of five conformations, which (for a given size) run at different speeds in a gel during electrophoresis. "Nicked Open-Circular" DNA has one strand cut. "Relaxed Circular" DNA is fully intact with both strands uncut, but has been enzymatically "relaxed" (supercoils removed). "Linear" DNA has free ends, either because both strands have been cut, or because the DNA was linear in vivo. "Supercoiled" DNA is fully intact with both strands uncut, and with a twist built in, resulting in a compact form. "Supercoiled Denatured" DNA is like supercoiled DNA, but has unpaired regions that make it slightly less compact; this can result from excessive alkalinity during plasmid preparation. The rate of migration for small linear fragments is directly proportional to the voltage applied at low voltages. At higher voltages, larger fragments migrate at continually increasing yet different rates. Therefore the resolution of a gel decreases with increased voltage. Because of its tight conformation, supercoiled DNA migrates faster through a gel than linear or open-circular DNA.