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{{Short description|Small piece of maintainable DNA}}
[[File:PBR322 color.svg|thumb|Schematic representation of the [[pBR322]] plasmid, one of the first plasmids widely used as a 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 [[molecular cloning|cloning]] purposes.<ref>{{cite web |url=http://www.theodora.com/genetics/#cloningvector |title=Definition of cloning vector |work=Genome Dictionary |access-date=2012-10-18}}</ref> The cloning vector may be DNA taken from a [[virus]], the [[Cell (biology)|cell]] of a higher organism, or it may be the [[plasmid]] of a bacterium. The [[Vector (molecular biology)|vector]]
There are many types of cloning vectors, but the most commonly used ones are genetically engineered [[plasmid]]s. Cloning is generally first performed using ''[[Escherichia coli]]'', and cloning vectors in ''E. coli'' include plasmids, [[bacteriophage]]s (such as [[Lambda phage|phage λ]]), [[cosmids]], and [[bacterial artificial chromosome]]s (BACs). Some DNA, however, cannot be stably maintained in ''E. coli'', for example very large DNA fragments, and other organisms such as yeast may be used. Cloning vectors in yeast include [[yeast artificial chromosome]]s (YACs).
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===Cloning site===
All cloning vectors have features that allow a gene to be conveniently inserted into the vector or removed from it. This may be a [[multiple cloning site]] (MCS) or polylinker, which contains many unique [[restriction enzyme|restriction sites]]. The restriction sites in the MCS are first cleaved by restriction enzymes, then a [[Polymerase chain reaction|PCR]]-amplified target gene also digested with the same enzymes is ligated into the vectors using [[DNA ligase]]. The target DNA sequence can be inserted into the vector in a specific direction if so desired. The restriction sites may be further used for [[sub-cloning]] into another vector if necessary.
Other cloning vectors may use [[topoisomerase]] instead of ligase and cloning may be done more rapidly without the need for restriction digest of the vector or insert. In this [[TOPO cloning]] method a linearized vector is activated by attaching topoisomerase I to its ends, and this "TOPO-activated" vector may then accept a PCR product by ligating both the 5' ends of the PCR product, releasing the topoisomerase and forming a circular vector in the process.<ref>{{cite web |url=http://www.invitrogen.com/site/us/en/home/brands/Product-Brand/topo/The-Technology-Behind-TOPO-Cloning.html |title=The Technology Behind TOPO® Cloning |work=Invitrogen }}</ref> Another method of cloning without the use of DNA digest and ligase is by [[Site-specific recombination|DNA recombination]], for example as used in the [[Gateway Technology|Gateway cloning system]].<ref>{{Cite book |chapter=Gateway cloning for protein expression |vauthors=Esposito D, Garvey LA, Chakiath CS |title=High Throughput Protein Expression and Purification |series=Methods in Molecular Biology |year=2009 |volume=498 |pages=[https://archive.org/details/highthroughputpr00shar/page/31 31–54] |doi=10.1007/978-1-59745-196-3_3 |pmid=18988017 |isbn=978-1-58829-879-9 |chapter-url-access=registration |chapter-url=https://archive.org/details/highthroughputpr00shar/page/31 }}</ref><ref>{{cite web |url=http://www.embl.de/pepcore/pepcore_services/cloning/cloning_methods/recombination/gateway/ |title=Cloning Methods - Recombination cloning systems |work=EMBL }}</ref> The gene, once cloned into the cloning vector (called entry clone in this method), may be conveniently introduced into a variety of expression vectors by recombination.<ref>{{cite web |url=http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning/Gateway-Technology.html |title=Gateway® Recombination Cloning Technology |work=Invitrogen}}</ref>
===Selectable marker===
A [[selectable marker]] is carried by the vector to allow the selection of positively [[Transformation (genetics)|transformed]] cells. [[Antibiotic]] resistance is often used as marker, an example being the [[beta-lactamase]] gene, which confers resistance to the [[penicillin]] group of [[beta-lactam antibiotics]] like [[ampicillin]]. Some vectors contain two selectable markers, for example the plasmid pACYC177 has both ampicillin and [[kanamycin]] resistance gene.<ref name = "Casali_2003">{{cite book |
Another kind of selectable marker allows for the positive selection of plasmid with cloned gene. This may involve the use of a gene lethal to the host cells, such as [[barnase]],<ref>{{cite journal
===Reporter gene===
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==Types of cloning vectors==
A large number of cloning vectors are available, and choosing the vector may depend upon a number of factors, such as the size of the insert, copy number and cloning method. Large insert may not be stably maintained in a general cloning vector, especially for those with a high copy number, therefore cloning large fragments may require more specialised cloning vector.<ref name =
[[File:PUC19.svg|thumb|The pUC plasmid has a high copy number, contains a multiple cloning site (polylinker), a gene for ampicillin antibiotic selection, and can be used for blue-white screen.]]
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===Plasmid===
{{main|Plasmid vector}}
Plasmids are autonomously replicating circular extra-chromosomal DNA. They are the standard cloning vectors and the ones most commonly used. Most general plasmids may be used to clone DNA
Some plasmids contain an [[M13 bacteriophage]] origin of replication and may be used to generate single-stranded DNA. These are called [[phagemid]]s, and examples are the [[pBluescript]] series of cloning vectors.
===Bacteriophage===
The bacteriophages used for cloning are the [[Bacteriophage lambda|λ phage
There is also a lower size limit for DNA that can be packed into a phage, and vector DNA that is too small cannot be properly packaged into the phage. This property can be used for selection - vector without insert may be too small, therefore only vectors with insert may be selected for propagation.<ref>{{cite book |title=Gene Cloning and DNA Analysis: An Introduction|author= TA Brown|publisher=Wiley-Blackwell |page=100 |isbn= 978-1444334074 |url= https://books.google.com/books?id=Ju8XeJL9Fc4C&pg=PA100 |date= 2010-04-19}}</ref>
===Cosmid===
[[Cosmids]] are plasmids that incorporate a segment of bacteriophage λ DNA that has the cohesive end site (''cos'') which contains elements required for packaging DNA into λ particles. Under apt origin of replication (ori), it can replicate as a plasmid It is normally used to clone large DNA fragments between 28 and 45 Kb.<ref name =
===Bacterial artificial chromosome===
Insert size of up to 350 kb can be cloned in [[bacterial artificial chromosome]] (BAC). BACs are maintained in ''E. coli'' with a copy number of only 1 per cell.<ref name = "
===Yeast artificial chromosome===
[[Yeast artificial chromosome]] are used as vectors to clone DNA fragments of more than 1 mega base (1Mb=1000kb) in size. They are useful in cloning larger DNA fragments as required in mapping genomes such as in
===Human artificial chromosome===
[[Human artificial chromosome]] may be potentially useful as a gene transfer vectors for gene delivery into human cells, and a tool for expression studies and determining human chromosome function. It can carry very large DNA fragment (there is no upper limit on size for practical purposes), therefore it does not have the problem of limited cloning capacity of other vectors, and it also avoids possible insertional mutagenesis caused by integration into host chromosomes by viral vector.<ref>{{cite journal |
===Animal and plant viral vectors===
Viruses that infect plant and animal cells have also been manipulated to introduce foreign genes into plant and animal cells. The natural ability of viruses to adsorb to cells
[[File:Blue-white test.jpg|thumb|An LB agar plate showing the result of a blue white screen. White colonies may contain an insert in the plasmid it carries, while the blue ones are unsuccessful clones.]]
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Many general purpose vectors such as [[pUC19]] usually include a system for detecting the presence of a cloned DNA fragment, based on the loss of an easily scored phenotype. The most widely used is the gene coding for ''E. coli'' [[beta-galactosidase|β-galactosidase]], whose activity can easily be detected by the ability of the enzyme it encodes to hydrolyze the soluble, colourless substrate [[X-gal]] (5-bromo-4-chloro-3-indolyl-beta-d-galactoside) into an insoluble, blue product (5,5'-dibromo-4,4'-dichloro indigo). Cloning a fragment of DNA within the vector-based ''lacZα'' sequence of the β-galactosidase prevents the production of an active enzyme. If X-gal is included in the selective agar plates, transformant colonies are generally blue in the case of a vector with no inserted DNA and white in the case of a vector containing a fragment of cloned DNA.
== See also ==
* [[Vector (molecular biology)]]
* [[Plant transformation vector]]
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* [[Golden Gate Cloning]]
== References ==
{{Reflist|2}}
[[Category:Genetics techniques]]
[[Category:Molecular biology]]
[[Category:Cloning]]
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