Introduction
- Cosmid vectors are hybrid plasmid with a bacterial “ori” sequence and a “cos” sequences derived from the lambda phage first described by Collins and Hohn in 1978.
- The cosmid contains: an origin of replication (ori) that allows it to replicate as a bacterial plasmid, a cos gene for packing phage DNA into protein coats, an ampicillin resistance gene (ampR), a region containing restriction sites for cloning (cosmid pJB8 – BamHI, EcoRI, ClaI, and HindIII).
- Cos sequence, ~200 base pairs long and essential for invitro packaging phage DNA into head protein coat.
- Cosmids can contain 37 to 52 (normally 45) kb of DNA, limits based on the normal bacteriophage packaging size.
- they are used cloning vehicles for preparing genomic recombinant DNA libraries and gene isolation, expression vectors for both transient and stable transformation in tissue culture cells, and shuttle vectors between bacteria and mammalian cells.
Properties of cosmid vectors
- Features of both plasmid and lambda phage cloning vectors.
- Contain an Ori sequence, selectable marker (ampR), and multiple restriction sites for cloning (BamHI, EcoRI, ClaI, and HindIII).
- Contain Phage (lambda) cos sites which permits packaging into phage heads and therefore introduction to E. coli cells.
- Packaging can occur with 37-52 kb fragment,
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Cosmid pJB8
pJB8 is 5.4 kb in size and carries the ampicillin-resistance gene (amp R), a segment of λ (lambda) DNA containing the cos site, and an Escherichia coli origin of replication (ori).
Contents
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Advantages of cosmid vectors
- relatively large size of insert DNA (up to 45 kb) can be cloned;
- DNA can be introduced into the host using bacteriophages derived by in vitro packaging.
Limitation of Cosmid vector
- Slower replication
- Higher frequency of recombination inside bacterial host.
- Unstable inside E.coli host and thus easy to lose vector.
- in vitro packaging is needed to maintain cosmids inside the viral heads