A rapid cloning method employing orthogonal end protection.
We describe a novel in vitro cloning strategy that combines standard tools in molecular biology with a basic protecting group concept to create a versatile framework for the rapid and seamless assembly of modular DNA building blocks into functional open reading frames. Analogous to chemical synthesis strategies, our assembly design yields idempotent composite synthons amenable to iterative and recursive split-and-pool reaction cycles. As an example, we illustrate the simplicity, versatility and efficiency of the approach by constructing an open reading frame composed of tandem arrays of a human fibronectin type III (FNIII) domain and the von Willebrand Factor A2 domain (VWFA2), as well as chimeric (FNIII)(n)-VWFA2-(FNIII)(n) constructs. Although we primarily designed this strategy to accelerate assembly of repetitive constructs for single-molecule force spectroscopy, we anticipate that this approach is equally applicable to the reconstitution and modification of complex modular sequences including structural and functional analysis of multi-domain proteins, synthetic biology or the modular construction of episomal vectors.
Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
Arjen J
AJ
Jakobi
Eric G
EG
Huizinga
D000595:Amino Acid Sequence; D001483:Base Sequence; D014408:Biomarkers, Tumor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002135:Calcium-Binding Proteins; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D005353:Fibronectins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D044366:Transition Temperature
D014408:Biomarkers, Tumor; D002135:Calcium-Binding Proteins; D005353:Fibronectins; D011993:Recombinant Fusion Proteins; C496188:VWA2 protein, human; D004247:DNA