Team:EPF-Lausanne/Tools/Gibson assembly

From 2011.igem.org

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(Principle)
(Principle)
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Combined together, these 3 enzymes allow overlapping DNA sequences (sequences A and B) to be ligated together in a single and isothermal reaction. First, the T5 exonuclease chews up bases at the 5' end on each strand, creating single-strands on the edges. Due to the sequence overlap, the single strand from A will anneal with the single strand from B. Then, the Phusion polymerase extend the DNA sequence from the 3' of the AB overlap. Finally, when there all the gaps have been filled by the polymerase, the Taq ligase ties up the A and B fragments.
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Combined together, these 3 enzymes allow overlapping DNA sequences (sequences A and B) to be ligated together in a single and isothermal reaction. First, the T5 exonuclease chews up bases at the 5' end on each strand, creating single-strands on the edges. Due to the sequence overlap, the single strand from A will anneal with the single strand from B. Then, the Phusion polymerase extends the DNA sequence from the 3' of the AB overlap. Finally, when there all the gaps have been filled by the polymerase, the Taq ligase ties up the A and B fragments.
[[File:EPFL_GibsonAssemblystrandscoloured.jpg|600px]]
[[File:EPFL_GibsonAssemblystrandscoloured.jpg|600px]]

Revision as of 07:59, 12 September 2011