Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes.
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.
Type
Journal articleSubject
Cloning, MolecularDNA Restriction Enzymes
Elastin
Escherichia coli
Genes
Humans
Peptides
Phase Transition
Plasmids
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Permalink
https://hdl.handle.net/10161/4022Published Version (Please cite this version)
10.1021/bm901387tPublication Info
McDaniel, Jonathan R; Mackay, J Andrew; Quiroz, Felipe García; & Chilkoti, Ashutosh (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.This is constructed from limited available data and may be imprecise. To cite this
article, please review & use the official citation provided by the journal.
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Show full item recordScholars@Duke
Ashutosh Chilkoti
Alan L. Kaganov Distinguished Professor of Biomedical Engineering
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 bio

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