Cloning Vectors
Cloning vectors are DNA molecules used to insert fragments of foreign DNA into a host cell. The vector still has the properties of multiplying and self-replication. These vectors introduce foreign DNA into the host cells, which then may divide in large quantities. The major cloning vectors are plasmids, cosmids, yeast artificial chromosomes (YACs), and recombinant molecules.
Plasmids are replications of DNA that appear as a circular piece outside of the chromosome. The replicon or replicating piece is stable and inherited in an extrachromosomal state, distinct from the normal bacterial genome. Under normal conditions, the plasmid is not essential for cell survival. Small pieces of DNA (from 50 to 5,000 base pairs) may be inserted. This DNA is referred to as recombinant DNA.
When the recombinant DNA is ligated or put into the plasmid vector, it then is introduced into a system, such as bacteria, that will amplify it. Under normal conditions bacteria do not take up the free plasmids, so they must be made "competent" to take them in. Bacteria may be treated with a substance like calcium chloride and then heat shocked. This procedure is called transformation. For example, the Amp gene codes for resistance to ampicillin, an antiobiotic. By using a plasmid-containing Amp, it can be determined which bacteria have taken up the plasmid by their resistance to ampicillin. A number of artificially constructed plasmids are now being used as cloning vectors.
Cosmids, another kind of vector, contains the cos gene of phage lambda. Phages are small particles that prey upon and destroy bacteria. Lambda is a specific type of phage, and the cos gene is a specific fragment of DNA from the phage. Cosmids are artificially constructed and can be packaged in lambda phage particles for insertion into the bacterium Escherishia coli. Cosmids may be used in cloning larger fragments of up to 45 kilobases (kb). Cosmids allow for insertion of up to 45,000 base pairs.
Another cloning vector is the yeast artificial chromosome or YAC. This artificially constructed cloning vector is made from telomeric, centromeric, and replication origin sequences (phases of cell division). The fragments can be up to 400 kb and are much larger than those of plasmids and cosmids.
Viruses are used as vectors. The virus is a non-living biological entity that reproduces only with a host cell. Viruses consist of nucleic acid covered with a protein. Inside the cell, the virus bonds with the host cell to produce copies.
Vectors are often recombinant molecules using DNA sequencing from many sources. Recombinant DNA techniques put together these DNA molecules to serve various purposes. For example, inserting a gene coding for human insulin into bacteria will create a powerful drug for those with diabetes. To combat cystic fibrosis, a virus has been genetically manipulated to deliver a working gene to the patient's lungs. The gene, after being inderted by a vector, then begins to code for the protein that is not produced normally produced by the patient, or that is produced in less than sufficient quantities.
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