Question

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).

a. Phusion DNA polymerase ,
b. T5 exonuclease ,
c. Taq DNA ligase

Answer 1

Answer with explanation:

Gibson Assembly is a method of molecular cloning which join multiple DNA fragments in a single reaction. It was created by Daniel G. Gibson, Chief Technology Officer and co-founder of SGI-DNA. (See attached picture)

a. Role of Phusion DNA Polymerase

It bring the enzymes closer to DNA fragment and help enzymes to make PCR product with speed and more accurately. Moreover it has the ability of amplifying long templates. (See attached picture)

a. Role of T5 exonuclease

It is an exonuclease enzyme which means it remove the nucleotide from DNA stand in 5´ to 3´ direction. It create nicks in the double stranded DNA for the incorporation of other fragments. Furthermore, it also work fine in single stranded DNA. (See attached picture)

a. Role of Taq DNA ligase

It is a thermostable enzyme which catalyzes the phosphodiester bond formation between 5´-phosphate and the 3´-hydroxyl of two adjacent DNA strands. (See attached picture)