This method enables virtually error-free joining of DNA fragments, even those with 5´ and 3´ restriction enzyme mismatches. Three enzymatic activities are employed: a 5’ exonuclease generates terminal cohesive ends (overhangs), a polymerase fills in the gaps of the annealed single-stranded regions, and a DNA ligase seals the nicks.Īnother method utilizing a proprietary high fidelity polymerase is NEBuilder HiFi DNA Assembly.
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His exonuclease-based method is performed under isothermal conditions after linear insert and vector are prepared by PCR and/or restriction digestion. One seamless cloning method is the Gibson Assembly method, originally described by Daniel G. Additionally, the ability to join 5-10 fragments in a predetermined order, with no sequence restrictions or scars, provides a powerful technique for synthetic biology endeavors, such as moving whole operons for metabolic engineering or whole genome reconstructions. The ability to quickly join a single insert to a plasmid at any sequence in the vector without a scar, makes these technologies very appealing cloning methods.
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Various commercial systems, such as NEBuilder HiFi DNA Assembly, In-Fusion ® and GeneArt ®, employ the Polymerase Chain Reaction (PCR) to amplify the gene of interest, an exonuclease to chew back one strand of the insert and vector ends, and either a ligase, recombination event, or in vivo repair to covalently join the insert to the vector through a true phosphodiester bond. The group of cloning methods we refer to as "seamless cloning" typically combine attributes of more established cloning methods to create a unique solution to allow sequence-independent and scarless insertion of one or more fragments of DNA into a plasmid vector.