Multiple cloning site

A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. The purpose of an MCS in a plasmid is to allow a piece of DNA to be inserted into that region. An MCS is found in a variety of vectors, including cloning vectors to increase the number of copies of target DNA, and in expression vectors to create a protein product. In expression vectors, the MCS is located downstream of the promoter. In some instances, a vector may not contain an MCS. Rather, an MCS can be added to a vector. The first step is designing complementary oligonucleotide sequences that contain restriction enzyme sites along with additional bases on the end that are complementary to the vector after digesting. Then the oligonucleotide sequences can be annealed and ligated into the digested and purified vector. The digested vector is cut with a restriction enzyme that complements the oligonucleotide insert overhangs. After ligation, transform the vector into bacteria and verify the insert by sequencing. This method can also be used to add new restriction sites to a multiple cloning site. Multiple cloning sites are a feature that allows for the insertion of foreign DNA without disrupting the rest of the plasmid which makes it extremely useful in biotechnology, bioengineering, and molecular genetics. MCS can aid in making transgenic organisms, more commonly known as a genetically modified organism (GMO) using genetic engineering. To take advantage of the MCS in genetic engineering, a gene of interest has to be added to the vector during production when the MCS is cut open. After the MCS is made and ligated it will include the gene of interest and can be amplified to increase gene copy number in a bacterium-host. After the bacterium replicates, the gene of interest can be extracted out of the bacterium.