Cosmid Cloning Vectors:
Fragments from30to46kb can be accommodated by a cosmid vector.Cosmids combine essential elements of a plasmid and Lambda systems.
Cosmids are extracted from bacteria and mixed with restriction endonucleases.
Cleaved cosmids are mixed with foreign DNA that has been cleaved with the same endonuclease.
Recombinant cosmids are packaged into lambda caspids
Recombinant cosmid is injected into the bacterial cell where the rcosmid arranges into a circle and replicates as a plasmid. It can be maintained and recovered just as plasmids.
Bacterial Artificial Chromosomes(BACs) and Yeast Artificial Chromosomes(YACs)
BACs can hold up to 300 kbs.
The F factor of E.coli is capable of handling large segments of DNA.
Recombinant BACs are introduced into E.coli by electroportation ( a brief high-voltage current). Once in the cell, the rBAC replicates like an F factor.
Example: pBAC108L
Has a set of regulatory genes, OriS, and repE which control F-factor replication, and parA and parB which limit the number of copies to one or two.
A chloramphenicol resistance gene, and a cloning segment.
YACs can hold up to 500 kbs.
YACs are designed to replicate as plasmids in bacteria when no foreign DNA is present. Once a fragment is inserted, YACs are transferred to cells, they then replicate as eukaryotic chromosomes.
YACs contain: a yeast centromere, two yeast telomeres, a bacterial origin of replication, and bacterial selectable markers.
YAC plasmid?Yeast chromosome
DNA is inserted to a unique restriction site, and cleaves the plasmid with another restriction endonuclease that removes a fragment of DNA and causes the YAC to become linear. Once in the cell, the rYAC replicates as a chromosome, also replicating the foreign DNA.
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