A cosmid is a type of hybrid plasmid that contains a Lambda phagecos sequence. Cosmids DNA sequences are originally from the lambda phage. They are often used as a cloning vector in genetic engineering. Cosmids can be used to build genomic libraries. They were first described by Collins and Hohn in 1978. Cosmids can contain 37 to 52 kb of DNA, limits based on the normal bacteriophage packaging size. They can replicate as plasmids if they have a suitableorigin of replication : for example SV40 ori in mammalian cells, ColE1 ori for double-strandedDNA replication, or f1 ori for single-stranded DNA replication in prokaryotes. They frequently also contain a gene for selection such as antibiotic resistance, so that the transformed cells can be identified by plating on a medium containing the antibiotic. Those cells which did not take up the cosmid would be unable to grow. Unlike plasmids, they can also be packagedin vitro into phage capsids, a step which requires cohesive ends, also known as cos sites also used in cloning with a lambda phage as a vector, however nearly all the lambda genes have been deleted with the exception of the cos sequence. The hybrid cosmid DNA in the capsids can then be transferred into bacterial cells by transduction. Since there is a requirement for in vitro packaging whereby at least 38 kb of DNA is required between the cos sites, the vector without insert DNA will not be packaged DNA. This instability can largely be counteracted by using a host bacterium with specific mutations affecting DNA recombination.
Cos sequences are ~200 base pairs long and essential for packaging. They contain a cosN site where DNA is nicked at each strand, 12 bp apart, by terminase. This causes linearization of the circular cosmid with two "cohesive" or "sticky ends" of 12bp. The cosB site holds the terminase while it is nicking and separating the strands. The cosQ site of next cosmid is held by the terminase after the previous cosmid has been packaged, to prevent degradation by cellular DNases.
Cosmid features and uses
Cosmids are predominantly plasmids with a bacterial oriV, an antibiotic selection marker and a cloning site, but they carry one, or more recently two, cos sites derived from bacteriophage lambda. Depending on the particular aim of the experiment, broad host range cosmids, shuttle cosmids or 'mammalian' cosmids are available. The loading capacity of cosmids varies depending on the size of the vector itself but usually lies around 40–45 kb. The cloning procedure involves the generation of two vector arms which are then joined to the foreign DNA. Selection against wild type cosmid DNA is simply done via size exclusion. Cosmids, therefore, always form colonies and not plaques. Also the clone density is much lower with around 105 – 106CFU per µg of ligated DNA. After the construction of recombinant lambda or cosmid libraries the total DNA is transferred into an appropriate E. coli host via a technique called in vitro packaging. The necessary packaging extracts are derived from E. coli cI857 lysogens and Eam. These extracts will recognize and package the recombinant molecules in vitro, generating either mature phage particles or recombinant plasmids contained in phage shells. These differences are reflected in the different infection frequencies seen in favour of lambda-replacement vectors. This compensates for their slightly lower loading capacity. Phage libraries are also stored and screened more easily than cosmid libraries. Target DNA: the genomic DNA to be cloned has to be cut into the appropriate size range of restriction fragments. This is usually done by partial restriction followed by either size fractionation or dephosphorylation to avoid chromosome scrambling, i.e. the ligation of physically unlinked fragments.
