Polymerase Cycling Assembly - Gibson Assembly

Gibson Assembly

A modification of this method, Gibson assembly, described by Gibson et al. allows for single-step isothermal assembly of DNA with up to several hundreds kb. By using T5 exonuclease to 'chew back' complementary ends, an overlap of about 40bp can be created. The reaction takes place at 50°C, a temperature where the T5 exonuclease is unstable. After a short timestep it is degraded, the overlapps can anneal and be ligated. Cambridge University IGEM team made a video describing the process.

Read more about this topic:  Polymerase Cycling Assembly

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