2009
DOI: 10.1093/nar/gkp1181
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Linear plasmid vector for cloning of repetitive or unstable sequences in Escherichia coli

Abstract: Despite recent advances in sequencing, complete finishing of large genomes and analysis of novel proteins they encode typically require cloning of specific regions. However, many of these fragments are extremely difficult to clone in current vectors. Superhelical stress in circular plasmids can generate secondary structures that are substrates for deletion, particularly in regions that contain numerous tandem or inverted repeats. Common vectors also induce transcription and translation of inserted fragments, w… Show more

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Cited by 94 publications
(106 citation statements)
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“…As a result, we have encountered instances where, due to the foreign gene products produced, particular constructs were unstable in E. coli (unpublished results). Likewise, cloned heterologous DNA from extremely AT-rich organisms is also known to be exceptionally unstable in E. coli, due to frequent deletions and rearrangements (11). Thus, a cloning-independent methodology should be particularly useful for constructing markerless mutations in these species.…”
Section: Discussionmentioning
confidence: 99%
“…As a result, we have encountered instances where, due to the foreign gene products produced, particular constructs were unstable in E. coli (unpublished results). Likewise, cloned heterologous DNA from extremely AT-rich organisms is also known to be exceptionally unstable in E. coli, due to frequent deletions and rearrangements (11). Thus, a cloning-independent methodology should be particularly useful for constructing markerless mutations in these species.…”
Section: Discussionmentioning
confidence: 99%
“…Strategies such as insertion of introns into the viral genome (25,26) or maintaining the genome in multiple small fragments (27-31) have been used to overcome the inherent instability of the ZIKV and other flavivirus genomes. Here, using a novel linear vector (32) (Fig. 1A) with bacterial transcription terminators flanking the cloning site, we generated stable full-length cDNA clones of the ZIKV genome.…”
mentioning
confidence: 99%
“…Their high GC content causes amplification failure. A high concentration of housekeeping genes impedes the survival of such DNAs in BACs because of the transcription of the cloned DNA and selection against open reading frames [Godiska et al, 2010]. That is probably why chicken chromosome-specific BAC markers for the smallest MIs are missing in the available chicken genomic BAC-clone collections (CHORI-261 [https://bacpacresources.org], WAG [http://www.us.lifesciences.sourcebioscience.com]).…”
Section: Discussionmentioning
confidence: 99%