Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes.
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.
McDaniel
Jonathan R
JR
Department of Biomedical Engineering, Duke University, Durham, North Carolina, 27708-0181, USA.
Mackay
J Andrew
JA
Quiroz
Felipe García
FG
Chilkoti
Ashutosh
A
D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004549:Elastin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005796:Genes / Q000235:genetics*; D006801:Humans; D010455:Peptides / Q000378:metabolism; D044367:Phase Transition; D010957:Plasmids / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010455:Peptides; D004549:Elastin; D004262:DNA Restriction Enzymes