13. (a) Development of recombinant DNA technology has opened gates to many breakthroughs in the fields of medicine and agriculture. This has enabled scientists to isolate, sequence and manipulate individual genes obtained from diverse living or dead cells. Given below is a diagram showing the basic steps involved in genetically modifying an organism. Study the given diagram and answer the questions that follow:
(i) Are two different types of restriction endonucleases used, one to cut the vector DNA and another to cut the desired DNA to be cloned ? Support your answer, giving reason.
(ii) Which enzyme is used at step (X) to integrate the foreign DNA with the vector DNA?
(iii) What is the term used for step (Y) showing multiple copies of the foreign DNA being formed in transformed E. coli ?
(iv) Draw a diagram of E. coli cloning vector pBR322 to show the following
(1) Any one restriction endonuclease site in tetracycline resistance gene
(2) Any one restriction endonuclease site in ampicillin resistance gene
(3) 'ori' site
(v) What does "rop" code for in plasmid pBR322?
Solution:
i) No, different types of restriction endonucleases are not used.
The vector and source DNA should be treated with the same restriction enzyme so as to produce DNA fragments carrying same (sticky or blunt) ends. These ends are then sealed by the enzyme ligase.
ii) The enzyme used in X is ligase.
Ligases are the enzymes that catalyse the linking together of two compounds; e.g., enzymes catalysing the joining of C − O, C − S, C − N bonds.
iv)
(1) Any one restriction endonuclease site in tetracycline resistance gene: It is shown in pink colour.
i) No, different types of restriction endonucleases are not used.
The vector and source DNA should be treated with the same restriction enzyme so as to produce DNA fragments carrying same (sticky or blunt) ends. These ends are then sealed by the enzyme ligase.
ii) The enzyme used in X is ligase.
Ligases are the enzymes that catalyse the linking together of two compounds; e.g., enzymes catalysing the joining of C − O, C − S, C − N bonds.
DNA ligase join the DNA fragments by forming phosphodiester bond.
iii) Cloning is represented in step (Y) showing multiple copies of the foreign DNA being formed in transformed E. coli.
iii) Cloning is represented in step (Y) showing multiple copies of the foreign DNA being formed in transformed E. coli.
iv)
(1) Any one restriction endonuclease site in tetracycline resistance gene: It is shown in pink colour.
(2) Any one restriction endonuclease site in ampicillin resistance gene: It is depicted in red colour.
(3) 'ori' site: It is shown in green colour.
v) The plasmid pBR322 contains a Rop gene coding for the Rop protein, which promotes conversion of the unstable RNA I – RNA II complex to a stable complex.
v) The plasmid pBR322 contains a Rop gene coding for the Rop protein, which promotes conversion of the unstable RNA I – RNA II complex to a stable complex.