Gibson Assembly Several enzymes are present in the Gibson a

Gibson Assembly – Several enzymes are present in the Gibson assembly reaction. For each of the following enzymes, define the role of the enzyme at room temperature (when the reaction is set up) and at 50 °C (the temperature for the 30 minute incubation after set up). Phusion DNA polymerase , T5 exonuclease , Taq DNA ligase

Solution

Gibbson assembly is molecular cloning method found by Daniel Gibson in 2009. Gibson assembly is a molecular cloning method which occurs at a rapid rate. it is a reaction accomplished by mixing of components in a single tube and incubated at 50⁰C for one hour. Small DNA fragments of 20 to 40 base pairs, enzymes including DNA polymerase, exonuclease and DNA ligase and other buffer components are mixed in a tube and incubated. After incubation, overlapping fragments of DNA are joined together to form a DNA strand. When the reaction is set up mixing all components initially at room temperature, the enzymes are not active. When the mixture is incubated at 50⁰C, the T₅ exonuclease removes some terminal nucleotides at 3\' OH group, the phusion DNA polymerase adds complementary bases and Taq DNA ligase fills the gaps by sealing the nickes to form continuous DNA strand.