Show simple item record McDaniel, JR Mackay, JA Quiroz, FG Chilkoti, A
dc.coverage.spatial United States 2011-06-21T17:25:53Z 2010-04-12
dc.identifier.citation Biomacromolecules, 2010, 11 (4), pp. 944 - 952
dc.description.abstract 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.
dc.format.extent 944 - 952
dc.language eng
dc.language.iso en_US en_US
dc.relation.ispartof Biomacromolecules
dc.relation.isversionof 10.1021/bm901387t
dc.subject Cloning, Molecular
dc.subject DNA Restriction Enzymes
dc.subject Elastin
dc.subject Escherichia coli
dc.subject Genes
dc.subject Humans
dc.subject Peptides
dc.subject Phase Transition
dc.subject Plasmids
dc.subject Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
dc.title Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes.
dc.title.alternative en_US
dc.type Journal Article
dc.description.version Version of Record en_US 2010-4-0 en_US
duke.description.endpage 952 en_US
duke.description.issue 4 en_US
duke.description.startpage 944 en_US
duke.description.volume 11 en_US
dc.relation.journal Biomacromolecules en_US
pubs.issue 4
pubs.organisational-group /Duke
pubs.organisational-group /Duke/Pratt School of Engineering
pubs.organisational-group /Duke/Pratt School of Engineering/Biomedical Engineering
pubs.organisational-group /Duke/School of Medicine
pubs.organisational-group /Duke/School of Medicine/Institutes and Centers
pubs.organisational-group /Duke/School of Medicine/Institutes and Centers/Duke Cancer Institute
pubs.organisational-group /Duke/Trinity College of Arts & Sciences
pubs.organisational-group /Duke/Trinity College of Arts & Sciences/Chemistry
pubs.publication-status Published
pubs.volume 11
dc.identifier.eissn 1526-4602

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