Please answer the following question: You are studying a mouse gene MtoA. You kn
ID: 266335 • Letter: P
Question
Please answer the following question:
You are studying a mouse gene MtoA. You know the protein product of localizes to mitochondria in mice. Your goal is to know whether the mouse MtoA protein is capable of localizing to mitochondria if produced in yeast cells. You decide to clone a gene into a translational fusion vector that will add a jellyfish DNA sequence encoding the GFP protein (238 amino acids) in place of the nomal stop codon. Unfortunately, you can't find any restriction enzyme recognition sequences in the MtoA gene that would allow you to clone your DNA into the multiple cloning site.
What method can you use to amplify the gene?
Breifly describe a variation to this method that would help solve your problem.
Explanation / Answer
We want to clone the mouse gene MtoA into translational fusion vector. However, in the multple cloning site of the vector there is no resriction enzyme present which can clone the DNA fragment . To overcome the problem we can add sites for restriction enzyme at the both sides of the MtoA gene. Howerver, we have to be careful that there should not be any restriction enzyme present in the multiple clong site of the vector which can cut the mtoA gene. If we add the sequence stretch to the 5' site of the primer, MtoA gene can be amplified with the help of PCR and same time restriction site will be aded