Cosmid
Encyclopedia
A cosmid, first described by Collins and Hohn in 1978, is a type of hybrid plasmid
(often used as a cloning vector
) that contains cos sequences, DNA sequences originally from the Lambda phage
. Cosmids can be used to build genomic libraries
.
Cosmids are able to contain 37 to 52 kb
of DNA, while normal plasmids are able to carry only 1–20 kb. They can replicate
as plasmids if they have a suitable origin of replication: for example SV40 ori in mammalian cells, ColE1 ori for double-stranded DNA replication or f1 ori for single-stranded DNA replication in prokaryote
s. They frequently also contain a gene for selection such as antibiotic resistance, so that the transfected 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 packaged in phage capsid
s, which allows the foreign genes to be transferred into or between cells by transduction
. Plasmids become unstable after a certain amount of DNA has been inserted into them, because their increased size is more conducive to recombination
. To circumvent this, phage transduction is used instead. This is made possible by the cohesive ends, also known as cos sites. In this way, they are similar to using the lambda phage as a vector, but only that all the lambda genes have been deleted with the exception of the cos sequence.
Cos sequences are ~200 base pairs long and essential for packaging. They contain a cosN site where DNA is nicked at each strand, 12bp apart, by terminase. This causes linearization of the circular cosmid with two "cohesive" or "sticky ends" of 12bp. (The DNA must be linear to fit into a phage head.) The cosB site holds the terminase while it is nicking and separating the strands. The cosQ site of next cosmid (as rolling circle replication
often results in linear concatemers) is held by the terminase after the previous cosmid has been packaged, to prevent degradation by cellular DNases.Picture
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 (red- gam- Sam and Dam (head assembly) and Eam (tail assembly) respectively). These extracts will recognize and package the recombinant molecules in vitro, generating either mature phage particles (lambda-based vectors) or recombinant plasmids contained in phage shells (cosmids). 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 library are also stored and screened easier than cosmid (colonies!) 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 (using calf-intestine phosphatase) to avoid chromosome scrambling, i.e. the ligation of physically unlinked fragments.
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
(often used as a cloning vector
Cloning vector
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted. The insertion of the fragment into the cloning vector is carried out by treating the vehicle and the foreign DNA with a restriction enzyme that creates the same overhang, then ligating the fragments...
) that contains cos sequences, DNA sequences originally from the Lambda phage
Lambda phage
Enterobacteria phage λ is a temperate bacteriophage that infects Escherichia coli.Lambda phage is a virus particle consisting of a head, containing double-stranded linear DNA as its genetic material, and a tail that can have tail fibers. The phage particle recognizes and binds to its host, E...
. Cosmids can be used to build genomic libraries
Library (biology)
In molecular biology, a library is a collection of DNA fragments that is stored and propagated in a population of micro-organisms through the process of molecular cloning...
.
Cosmids are able to contain 37 to 52 kb
Base pair
In molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called a base pair...
of DNA, while normal plasmids are able to carry only 1–20 kb. They can replicate
DNA replication
DNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for biological inheritance. The process starts with one double-stranded DNA molecule and produces two identical copies of the molecule...
as plasmids if they have a suitable origin of replication: for example SV40 ori in mammalian cells, ColE1 ori for double-stranded DNA replication or f1 ori for single-stranded DNA replication in prokaryote
Prokaryote
The prokaryotes are a group of organisms that lack a cell nucleus , or any other membrane-bound organelles. The organisms that have a cell nucleus are called eukaryotes. Most prokaryotes are unicellular, but a few such as myxobacteria have multicellular stages in their life cycles...
s. They frequently also contain a gene for selection such as antibiotic resistance, so that the transfected 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 packaged in phage capsid
Capsid
A capsid is the protein shell of a virus. It consists of several oligomeric structural subunits made of protein called protomers. The observable 3-dimensional morphological subunits, which may or may not correspond to individual proteins, are called capsomeres. The capsid encloses the genetic...
s, which allows the foreign genes to be transferred into or between cells by transduction
Transduction (genetics)
Transduction is the process by which DNA is transferred from one bacterium to another by a virus. It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector. Transduction does not require cell-to-cell contact , and it is DNAase resistant...
. Plasmids become unstable after a certain amount of DNA has been inserted into them, because their increased size is more conducive to recombination
Genetic recombination
Genetic recombination is a process by which a molecule of nucleic acid is broken and then joined to a different one. Recombination can occur between similar molecules of DNA, as in homologous recombination, or dissimilar molecules, as in non-homologous end joining. Recombination is a common method...
. To circumvent this, phage transduction is used instead. This is made possible by the cohesive ends, also known as cos sites. In this way, they are similar to using the lambda phage as a vector, but only that all the lambda genes have been deleted with the exception of the cos sequence.
Cos sequences are ~200 base pairs long and essential for packaging. They contain a cosN site where DNA is nicked at each strand, 12bp apart, by terminase. This causes linearization of the circular cosmid with two "cohesive" or "sticky ends" of 12bp. (The DNA must be linear to fit into a phage head.) The cosB site holds the terminase while it is nicking and separating the strands. The cosQ site of next cosmid (as rolling circle replication
Rolling circle replication
Rolling circle replication describes a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids, the genomes of bacteriophages, and the circular RNA genome of viroids...
often results in linear concatemers) is held by the terminase after the previous cosmid has been packaged, to prevent degradation by cellular DNases.Picture
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 (linked to SV40 oriV and mammalian selection markers) 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 wildtype cosmid DNA is simply done via size exclusion. Cosmids, however, always form colonies and not plaques. Also the clone density is much lower with around 105 - 106 CFUCFU
CFU may refer to:* Cambridge Fans United, fans of the English football team Cambridge United F.C.* Canadian Freelance Union, a Canadian trade union for freelance media contributors* Caribbean Football Union, the nominal governing body for Caribbean football...
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 (red- gam- Sam and Dam (head assembly) and Eam (tail assembly) respectively). These extracts will recognize and package the recombinant molecules in vitro, generating either mature phage particles (lambda-based vectors) or recombinant plasmids contained in phage shells (cosmids). 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 library are also stored and screened easier than cosmid (colonies!) 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 (using calf-intestine phosphatase) to avoid chromosome scrambling, i.e. the ligation of physically unlinked fragments.