Final answer:
To clone the mammalian expression vector pmv2 bmpr, appropriate restriction endonucleases must be selected to cut the vector without cleaving the gene of interest. Sticky ends created by these enzymes facilitate the annealing of the DNA insert to the vector, which is then sealed by DNA ligase. DpnI can be used post-PCR to remove the methylated template plasmid, leaving only desired mutants.
Step-by-step explanation:
The cloning of a mammalian expression vector like pmv2 bmpr involves the use of restriction endonucleases to cut the vector and insert a DNA sequence of interest, such as a gene. After PCR amplification, the DNA of interest needs to be inserted into a vector that has been cut with restriction enzymes that produce complementary ends, or 'sticky ends'. These sticky ends allow the foreign DNA and cloning vector to anneal. This is followed by the action of an enzyme called DNA ligase, which 'glues' the annealed fragments together, ensuring the continuous DNA sequence necessary for expression.
It is crucial to select restriction enzymes that do not cleave the DNA sequence of interest. For instance, if the ykkCD toxin sensor needs to be inserted into pUC19, one would have to choose restriction enzymes from the polylinker MCS that do not cut within the ykkCD sequence. Common restriction enzymes found in polylinker MCS include EcoRI, SacI, KpnI, SmaI, and BamHI.
To clone pmv2 bmpr, the same considerations apply. One must first identify restriction sites within the pmv2 bmpr vector that are suitable for the insertion of the gene of interest without disrupting essential elements within the vector or the gene itself. Then, enzymes that recognize these sites will be used for the cloning process. Additionally, DpnI may be employed post-PCR amplification to digest the methylated template plasmid, leaving only vectors containing the desired mutations.