27-11-2012, 05:06 PM
Linear plasmid vector for cloning of repetitive or unstable sequences in Escherichia coli
Linear plasmid vector.pptx (Size: 993.19 KB / Downloads: 28)
Introduction
Superhelical stress in circular plasmid generates secondary structures that are substrates for deletion, example short nucleotide repeats and AT rich sequences and genomes( Eg plasmodium genome – 80-85 % AT).
Transcription from clones promoters can interfere with plasmid stability, replication or expression of selectable marker.
Many gaps in genomes are difficult to capture in circular plasmids.
High copy number- ability to clone large fragments is low.
Solutions
Construction of linear plasmid , eg. pG591,pJAZZ.
Contains transcriptional terminators on both sites of cloning sites to minimize transcriptional interference between vector and insert.
Stably maintains a variety of inserts.
Decreased size bias in cloning.
More uniform representation of larger fragments in libraries.
pG591 and pJAZZ
Based on coliphage N15 linear dsDNA.
Phage enzyme protelomerase(TelN) cuts at specific site(telRL) linearizing phage DNA.
TelN seals and generates covalently closed hairpin ends.
Why to construct a new strain?
Cellular nucleases of E. coli degrade hairpin telomeres of incoming linear molecules.
Due to absence of partitioning system low copy number vector is lost.
How the new strain helps?
Degradation of linear DNA is prevented by binding of TelN to hairpin telomeres.
sopAB ensures stable inheritance of plasmids.
Copy number of plasmids is elevated by expression of antirepressor AntA.
Conclusion
Ability of pJAZZ vectors to maintain fragments of 30-40 Kb, even if they are rich in repetitive DNA will be useful to close genome gaps.
Due to presence of transcriptional terminators, transcriptional interference is inhibited. This allows cloning of large cDNAs or operons.