Question

In pBR322, foreign DNA has to be introduced in $tet^R$ region. From the restriction enzymes given, which one should be used and why: Pvul, EcoRI, BamHI.

Answer 1

Hint: Restriction endonuclease is a bacterial enzyme that cuts dsDNA into fragments after recognizing specific nucleotide sequences known as recognition or restriction sites. These enzymes restrict the entry of foreign DNA inside the bacteria. It cuts only double-helical segments that contain a particular nucleotide sequence of four or eight pairs in length known as a restriction site.

Complete answer:
Vectors are the DNA molecules that act as transporting vehicles that carry foreign DNA into a host cell for the purpose of cloning and expression. The cloning vectors are used to clone foreign DNA.

There are vectors of E.Coli based on a plasmid(naturally occurring extrachromosomal replicating DNA, present in prokaryotes)DNA. One such example of a cloning vector is- pBR322.

• It replicated E.coli. In this, p stands for plasmid and br stands for its developer Bolivar and Rodriguez. 322 is the distinguishing character from others as it was formed in the same laboratory.
• It is small, containing 4361 base pairs.
• It has genes for ampicillin resistance and tetracycline resistance.
• Restriction enzyme BamHI should be used as the restriction site for this enzyme is present within the gene coding for tetracycline resistance.
• There are antibiotic resistance genes such as ampR and $tet^R$ are derived from plasmid R1 and R6-5.

Hence, the correct answer is BamHI.

Additional information:
Pvul will not be used because the restriction site for this region is not present in $tet^R$ but instead it is present in the ampR region.
EcoRI- It will not be used as the restriction site for this enzyme is not present in the $tet^R$ region.
The origin of replication is derived from pMB1.

Note: The job of a vector is to transport foreign DNA into a host cell and hence cloning takes place. There are different vectors that are specific to different hosts and hence the use of any other enzyme such as Pvul or EcoRI, would not lead to insertional inactivation of tetracycline resistance coding gene, which will lead to recombinant identification.