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 A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...

. 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 vector A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, ...

s to increase the number of copies of target DNA, and in

expression vector An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for ...

s to create a protein product. In expression vectors, the MCS is located downstream of the promoter.

Creating a multiple cloning site

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 A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class ...

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. MCS_creation

Uses

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 Biotechnology is the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services. The term ''biotechnology'' was first used ...

bioengineering Biological engineering or bioengineering is the application of principles of biology and the tools of engineering to create usable, tangible, economically-viable products. Biological engineering employs knowledge and expertise from a number o ...

, and

molecular genetics Molecular genetics is a sub-field of biology that addresses how differences in the structures or expression of DNA molecules manifests as variation among organisms. Molecular genetics often applies an "investigative approach" to determine the ...

. MCS can aid in making transgenic organisms, more commonly known as a

genetically modified organism A genetically modified organism (GMO) is any organism whose genetic material has been altered using genetic engineering techniques. The exact definition of a genetically modified organism and what constitutes genetic engineering varies, wit ...

(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. In some instances, an expression vector can be used to create a protein product. After the products are isolated, they have a wide variety of uses such as the production of

insulin Insulin (, from Latin ''insula'', 'island') is a peptide hormone produced by beta cells of the pancreatic islets encoded in humans by the ''INS'' gene. It is considered to be the main anabolic hormone of the body. It regulates the metabolism ...

, the creation of

vaccine A vaccine is a biological preparation that provides active acquired immunity to a particular infectious or malignant disease. The safety and effectiveness of vaccines has been widely studied and verified.

s, production of

antibiotics An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting bacterial infections, and antibiotic medications are widely used in the treatment and preventio ...

, and creation of gene therapies.

Example

One bacterial plasmid used in genetic engineering as a plasmid cloning vector is pUC18. Its polylinker region is composed of several restriction enzyme recognition sites, that have been engineered into a single cluster (the polylinker). It has restriction sites for various restriction enzymes, including

EcoRI ''Eco''RI (pronounced "eco R one") is a restriction endonuclease enzyme isolated from species ''E. coli.'' It is a restriction enzyme that cleaves DNA double helices into fragments at specific sites, and is also a part of the restriction modifica ...

, BamHI, and PstI. Another vector used in genetic engineering is pUC19, which is similar to pUC18, but its polylinker region is reversed. ''E.coli'' is also commonly used as the bacterial host because of the availability, quick growth rate, and versatility. An example of a plasmid cloning vector which modifies the inserted protein is pFUSE-Fc plasmid. In order to genetically engineer insulin, the first step is to cut the MCS in the plasmid being used. Once the MCS is cut, the gene for human insulin can be added making the plasmid genetically modified. After that, the genetically modified plasmid is put into the bacterial host and allowed to divide. To make the large supply that is demanded, the host cells are put into a large fermentation tank that is an optimal environment for the host. The process is finished by filtering out the insulin from the host. Purification can then take place so the insulin can be packaged and distributed to individuals with diabetes.

References

{{reflist Genetics techniques