Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes.
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This paper reports a new strategy, recursive directional ligation by plasmid reconstruction (PRe-RDL), to rapidly clone highly repetitive polypeptides of any sequence and specified length over a large range of molecular weights. In a single cycle of PRe-RDL, two halves of a parent plasmid, each containing a copy of an oligomer, are ligated together, thereby dimerizing the oligomer and reconstituting a functional plasmid. This process is carried out recursively to assemble an oligomeric gene with the desired number of repeats. PRe-RDL has several unique features that stem from the use of type IIs restriction endonucleases: first, PRe-RDL is a seamless cloning method that leaves no extraneous nucleotides at the ligation junction. Because it uses type IIs endonucleases to ligate the two halves of the plasmid, PRe-RDL also addresses the major limitation of RDL in that it abolishes any restriction on the gene sequence that can be oligomerized. The reconstitution of a functional plasmid only upon successful ligation in PRe-RDL also addresses two other limitations of RDL: the significant background from self-ligation of the vector observed in RDL, and the decreased efficiency of ligation due to nonproductive circularization of the insert. PRe-RDL can also be used to assemble genes that encode different sequences in a predetermined order to encode block copolymers or append leader and trailer peptide sequences to the oligomerized gene.
Published Version (Please cite this version)
McDaniel, Jonathan R, J Andrew Mackay, Felipe García Quiroz and Ashutosh Chilkoti (2010). Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes. Biomacromolecules, 11(4). pp. 944–952. 10.1021/bm901387t Retrieved from https://hdl.handle.net/10161/4022.
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Ashutosh Chilkoti is the Alan L. Kaganov Professor of Biomedical Engineering and Chair of the Department of Biomedical Engineering at Duke University.
My research in biomolecular engineering and biointerface science focuses on the development of new molecular tools and technologies that borrow from molecular biology, protein engineering, polymer chemistry and surface science that we then exploit for the development of applications that span the range from bioseparations, plasmonic biosensors, low-cost clinical diagnostics, and drug delivery.
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