Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes.

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • McDaniel, JR; Mackay, JA; Quiroz, FG; Chilkoti, A

Published Date

  • April 12, 2010

Published In

Volume / Issue

  • 11 / 4

Start / End Page

  • 944 - 952

PubMed ID

  • 20184309

Electronic International Standard Serial Number (EISSN)

  • 1526-4602

Digital Object Identifier (DOI)

  • 10.1021/bm901387t


  • eng

Conference Location

  • United States