Question

Betimar is studying resistance genes to a fungus that attacks wheat. She managed to find a resistance candidate gene and would like to study it in more detail. Vivian, who is his collaborator in this research, gave him the idea of ​​cloning this gene in a vector. The vector they chose has 3 regions where restriction enzymes can cut, however, they noted that this resistance candidate gene did not have those restriction regions. What solution would you give to Betimar and Vivian so that they can use this vector and clone their resistance candidate gene?

Answer 1

In order to clone the resistance candidate gene,

1.Designing of primer -They should design the forward and reverse primer adding the unique restriction enzyme sites (only present in multiple cloning site(MCS) region). Here unique restriction sites means that it should be present in MCS region and  should not be present in any other region of vector upstream or downstream to MCS.

2. PCR amplification -Amplification of the resistance genes by using PCR (utilizing the forward and reverse primers and template of resistance gene).Amplified PCR product will have unique sites at 5'----3'.

4. Restriction digestion Now they need to perform the restriction digestion reactions for the vector and PCR amplified resistance genes with the unique restriction enzyme to generate the staggered end.

5. Ligation reaction - The digested resistance gene product and vector can be run through the gel electrophoresis and the both can be gel eluted. The eluted product can be ligated by the ligase enzyme reaction -( vector : resistance gene product =1:3 or 1:5, ligase buffer and ligase).

6. Bacterial transformation - The ligated product will be the clone of  resistance candidate gene in desired vector, which can be transformed in bacteria to get the DNA plasmid of the clone product.