Gibson Assembly
Gibson Assembly is a process that allows plasmids to be constructed from constituent parts (in a similar way to type IIs Golden Gate cloning). It's sort of a combination of a restriction digest and PCR, occurring at the same time.
In a single reaction, containing PCR-amplified DNA inserts, a 5' exonuclease, DNA polymerase, and DNA ligase, the correct overhanging (sticky) ends are given to all of the DNA components. The DNA polymerase then infills the gaps between the annealed inserts and plasmid backbone before ligase completes the backbone to give a complete plasmid construct.
This process can be used to assemble large synthetic genomes from large numbers of individual sections of DNA. 1
Gibson assembly is a scar-less technique, removing the restriction site from the final construct. Any DNA can be used in Gibson assembly, as restriction sites are not necessary.
Unlike Golden Gate cloning, Gibson assembly does not rely on specific restriction sites being present in the DNA inserts. This makes the reaction faster and easier to do, especially where large numbers of inserts are being used. Golden Gate cloning can have low efficiencies, and involve slow reactions. 2 GG cloning also requires specific restriction sites to be present in the DNA inserts and plasmid backbone (although these are present in all of the BioBricks present in the iGEM parts registry).
Questions:
[ ] How does Gibson assembly ensure the DNA inserts assemble in the correct order if the sticky ends are different?
Is this done through the PCR reaction to give complementary ends? How does this give scar-less cloning?
[ ] Another method is in-fusion cloning. See 2 for more detail.