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

Biomacromolecules. 2010 Apr 12;11(4):944-52. doi: 10.1021/bm901387t.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cloning, Molecular*
  • DNA Restriction Enzymes / metabolism
  • Elastin / chemistry
  • Elastin / genetics*
  • Elastin / metabolism
  • Escherichia coli / metabolism
  • Genes / genetics*
  • Humans
  • Peptides / metabolism
  • Phase Transition
  • Plasmids / genetics*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • Peptides
  • Elastin
  • DNA Restriction Enzymes