Seamless Cloning

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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. 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 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. 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.

One seamless cloning method is the Gibson Assembly method, originally described by Daniel G. Gibson, of the J. Craig Venter Institute. His exonuclease-based method is performed under isothermal conditions after linear insert and vector are prepared by PCR and/or restriction digestion. 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.

Another method utilizing a proprietary high fidelity polymerase is NEBuilder HiFi DNA Assembly. This method enables virtually error-free joining of DNA fragments, even those with 5´ and 3´ restriction enzyme mismatches. This method has also been used to assemble either single-stranded oligonucleotides or different sizes of DNA fragments with varied overlaps (15–80 bp).

Golden Gate Assembly is another method of seamless cloning that exploits the ability of Type IIS restriction enzymes (such as BsaI-HF®v2) to cleave DNA outside of the recognition sequence. The inserts and cloning vectors are designed to place the Type IIS recognition site distal to the cleavage site, such that the Type IIS restriction enzyme can remove the recognition sequence from the assembly. The advantages of such an arrangement are three-fold:

  1. The overhang sequence created is not dictated by the restriction enzyme and, therefore, no scar sequence is introduced,
  2. The fragment-specific sequence of the overhangs allows for orderly assembly of multiple fragments simultaneously,
  3. The restriction site is eliminated from the ligated product, so digestion and ligation can be carried out simultaneously. The net result is the ordered and seamless assembly of DNA fragments in one reaction.

Seamless Cloning Workflow

Note that times are based on estimates for moving a gene from one plasmid to another. If the source for gene transfer is gDNA, add 2 hours to calculation for the traditional cloning method. Total time does not include transformation, isolation or analysis. **70 minutes for recombination occurs on second day.