title: Hierarchical ligation-independent assembly of PCR fragments.
abstract: The emerging field of synthetic biology requires novel cloning techniques that allow the rapid assembly of multiple expression units to build artificial genetic circuits. Here, we describe a rapid, flexible, and cost-efficient cloning method that requires only standard laboratory equipment and skills. Our technique relies on the 3'-5' exonuclease activity of T4 DNA polymerase to generate 20 nt single-stranded DNA overhangs that allow annealing and ligation-independent cloning (LIC) of four DNA fragments in one tube. The resulting intermediate-size constructs can be reused to hierarchically assemble constructs of more than 24 kb by the same method.
authors.affiliation: Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, Bonn, Germany.
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Schmid-Burgk
authors.forename: Zhen
authors.initials: Z
authors.lastname: Xie
authors.forename: Yaakov
authors.initials: Y
authors.lastname: Benenson
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011786:Quality Control; D013997:Time Factors
chemical_list: D017931:DNA Primers; D004277:DNA, Single-Stranded; D004259:DNA-Directed DNA Polymerase