Editing Cloning vector (section)

==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 = "Casali_2003" />

[[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.]]

===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 inserts of up to 15 kb in size.  One of the earliest commonly used cloning vectors is the [[pBR322]] plasmid.  Other cloning vectors include the [[pUC19|pUC]] series of plasmids, and a large number of different cloning plasmid vectors are available.  Many plasmids have high copy numbers, for example, [[pUC19]] has a copy number of 500-700 copies per cell,<ref name = "Casali_2003" /> and high copy number is useful as it produces greater yield of recombinant plasmid for subsequent manipulation. However low-copy-number plasmids may be preferably used in certain circumstances, for example, when the protein from the cloned gene is toxic to the cells.<ref>{{cite web |url= http://www.mfa.od.ua/page23.htm |title= Copy number |work= Genetics Institute, Inc. |access-date= 2013-03-06 |archive-url= https://archive.today/20130419075932/http://www.mfa.od.ua/page23.htm |archive-date= 2013-04-19 |url-status= dead }}</ref>

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]] and [[M13 phage]].<ref>{{cite journal | vauthors = Chauthaiwale VM, Therwath A, Deshpande VV | title = Bacteriophage lambda as a cloning vector | journal = Microbiological Reviews | volume = 56 | issue = 4 | pages = 577–591 | date = December 1992 | pmid = 1480110 | pmc = 372889 | doi = 10.1128/mr.56.4.577-591.1992 }}</ref>  There is an upper limit on the amount of DNA that can be packed into a phage (a maximum of 53 kb), therefore to allow foreign DNA to be inserted into phage DNA, phage cloning vectors may need to have some non-essential genes deleted, for example the genes for [[lysogeny]] since using phage λ as a cloning vector involves only the lytic cycle.<ref>{{cite book | vauthors = Glick BR, Pasternak JJ |year=2005 |title=Molecular Biotechnology Principles and Applications of Recombinant DNA |edition=3rd |publisher=ASM Press |url= https://books.google.com/books?id=Wz3CtTBe9aUC&pg=PA86 |isbn=9781555816124 }}</ref>  There are two kinds of λ phage vectors - insertion vector and replacement vector.  Insertion vectors contain a unique cleavage site whereby foreign DNA with size of 5–11 kb may be inserted.   In replacement vectors, the cleavage sites flank a region containing genes not essential for the lytic cycle, and this region may be deleted and replaced by the DNA insert in the cloning process, and a larger sized DNA of 8–24 kb may be inserted.<ref name = "Casali_2003" />

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 = "Casali_2003" />

===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 = "Casali_2003" />  BACs are based on [[Fertility factor (bacteria)|F plasmid]], another artificial chromosome called the [[P1-derived artificial chromosome|PAC]] is based on the [[P1 phage]].

===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 the [[Human Genome Project]]. It contains a telomeric sequence, an autonomously replicating sequence (features required to replicate linear chromosomes in yeast cells). These vectors also contain suitable restriction sites to clone foreign DNA as well as genes to be used as selectable markers.

===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 | vauthors = Kim JH, Kononenko A, Erliandri I, Kim TA, Nakano M, Iida Y, Barrett JC, Oshimura M, Masumoto H, Earnshaw WC, Larionov V, Kouprina N | display-authors = 6 | title = Human artificial chromosome (HAC) vector with a conditional centromere for correction of genetic deficiencies in human cells | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 108 | issue = 50 | pages = 20048–20053 | date = December 2011 | pmid = 22123967 | pmc = 3250132 | doi = 10.1073/pnas.1114483108 | doi-access = free | bibcode = 2011PNAS..10820048K }}</ref><ref>{{cite journal | vauthors = Kouprina N, Earnshaw WC, Masumoto H, Larionov V | title = A new generation of human artificial chromosomes for functional genomics and gene therapy | journal = Cellular and Molecular Life Sciences | volume = 70 | issue = 7 | pages = 1135–1148 | date = April 2013 | pmid = 22907415 | pmc = 3522797 | doi = 10.1007/s00018-012-1113-3 }}</ref>

===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, introduce their DNA and replicate have made them ideal vehicles to transfer foreign DNA into eukaryotic cells in culture. A vector based on [[SV40|Simian virus 40]] (SV40) was used in first cloning experiment involving mammalian cells. A number of vectors based on other type of viruses like [[Adenoviridae|Adenoviruses]] and [[Papillomaviridae|Papilloma virus]] have been used to clone genes in mammals. At present, retroviral vectors are popular for cloning genes in mammalian cells. In case of plants like [[Cauliflower mosaic virus]], [[Tobacco mosaic virus]] and [[Geminiviridae|Gemini viruses]] have been used with limited success.
[[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.]]