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

Publication ,  Journal Article
McDaniel, JR; Mackay, JA; Quiroz, FG; Chilkoti, A
Published in: Biomacromolecules
April 2010

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.

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Published In

Biomacromolecules

DOI

EISSN

1526-4602

ISSN

1525-7797

Publication Date

April 2010

Volume

11

Issue

4

Start / End Page

944 / 952

Related Subject Headings

  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Polymers
  • Plasmids
  • Phase Transition
  • Peptides
  • Humans
  • Genes
  • Escherichia coli
  • Elastin
  • DNA Restriction Enzymes
 

Citation

APA
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ICMJE
MLA
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McDaniel, J. R., Mackay, J. A., Quiroz, F. G., & Chilkoti, A. (2010). Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes. Biomacromolecules, 11(4), 944–952. https://doi.org/10.1021/bm901387t
McDaniel, Jonathan R., J Andrew Mackay, Felipe García Quiroz, and Ashutosh Chilkoti. “Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes.Biomacromolecules 11, no. 4 (April 2010): 944–52. https://doi.org/10.1021/bm901387t.
McDaniel JR, Mackay JA, Quiroz FG, Chilkoti A. Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes. Biomacromolecules. 2010 Apr;11(4):944–52.
McDaniel, Jonathan R., et al. “Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes.Biomacromolecules, vol. 11, no. 4, Apr. 2010, pp. 944–52. Epmc, doi:10.1021/bm901387t.
McDaniel JR, Mackay JA, Quiroz FG, Chilkoti A. Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes. Biomacromolecules. 2010 Apr;11(4):944–952.
Journal cover image

Published In

Biomacromolecules

DOI

EISSN

1526-4602

ISSN

1525-7797

Publication Date

April 2010

Volume

11

Issue

4

Start / End Page

944 / 952

Related Subject Headings

  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Polymers
  • Plasmids
  • Phase Transition
  • Peptides
  • Humans
  • Genes
  • Escherichia coli
  • Elastin
  • DNA Restriction Enzymes