Advanced DNA assembly technologies in drug discovery

Tsvetanova, Billyana; Peng, Lansha; Liang, Xiquan; Li, Ké; Hammond, Linda E.; Peterson, Todd C.; Katzen, Federico

doi:10.1517/17460441.2012.672408

Cited by 2 publications

(2 citation statements)

References 33 publications

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“…In view of this, other more economical, non-sequence-directed, “seamless” recombinational cloning methods for joining two or more DNA fragments without the operation of extraneous sequences would represent an attractive alternative [34,35]. Nevertheless, in the most widely used seamless methods, DNA fragments to be assembled ought to be amplified by PCR with primers that provide some sequence overlap at their ends.…”

Section: Resultsmentioning

confidence: 99%

“…The distribution of these sequence overlaps among the fragments ensures they are stitched together in the required order. In other instances, “stitching” oligonucleotides can be used that act as a bridge encompassing the ends of the unrelated sequences to be joined [35]. In either case, the generation of a combinatorial cloning platform based on such methods would involve prior knowledge of the fusions that would be of interest in order to provide necessary tools in advance.…”

Section: Resultsmentioning

confidence: 99%

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A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector

et al. 2013

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BackgroundValuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous.ResultsWe present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit’s component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells.ConclusionsWe have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior modifications. Using this technology, any existing Gateway destination expression vector with its model-specific properties could be easily adapted for expressing fusion proteins.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%