1. Why sticky ended ligations are carried out at temperatures lower than room temperature?
a) It is so because the vibrational and kinetic energy of the molecules at room temperature is lower than that of the energy required to break the bonds holding the ends
b) The energy required to break the bonds holding the ends is very less than that of the kinetic and vibrational energy at room temperature
c) The enzyme carrying out ligation is unstable at low temperature
d) The sticky ends created, don’t just relegate at low temperature
Explanation: The sticky ends are held together by the hydrogen bonds. These hydrogen bonds are weak in nature and the energy required to break them is very less than the kinetic and vibrational energy of the molecules at room temperature. Hence the reaction is carried at 4 degrees
2. Ligation reaction can be both intramolecular and intermolecular in nature.
a) True
b) False
Explanation: Ligation reaction can be both intramolecular and intermolecular in nature. The intermolecular reaction is that which ends ligated are of two different molecules. The intramolecular ligation is that which ends ligated belong to the same molecule.
3. If a ligation reaction is being carried out and recircularization is observed, which type of reaction is being carried out?
a) Intramolecular
b) Intermolecular
c) Both observe recircularization equally
d) Recirculation is not possible in any of the cases
Explanation: Recircularization is the phenomenon of joining the ends of the same molecule. It happens in the case of intramolecular ligation reaction
4. What is the kinetics of the intramolecular and intermolecular ligation reactions?
a) Second order kinetics for intramolecular and first order for intermolecular
b) First order kinetics for intramolecular and second order for intermolecular
c) Both are first order
d) Both are second order
Explanation: As the intramolecular reaction requires only the type of reaction species, the reaction is first order.Whereas, on the other hand intermolecular reaction requires collision between two different species and hence it is second order reaction.
5. What are the effects of increasing concentration of reaction components?
a) It increases chances of ligation in both intramolecular and intermolecular reactions
b) It increases chances of ligation only in intermolecular and no effect on intramolecular
c) It decreases chances of ligation in intramolecular and increases in that of intermolecular
d) It decreases chances of ligation in both types of reaction
Explanation: As the concentration of reaction components is increased, there are increased chances of ligation in intermolecular reaction because of frequency of collision of two different molecules increases. The intramolecular reaction is unaffected because the probability of meeting the ends of a molecule remains the same.
6. Enzyme commonly used for carrying out ligation reaction is ____________
a) Transferase
b) Reverse transciptase
c) Ligase
d) DNase
Explanation: Ligase is the enzyme which is commonly used for carrying out the ligation reaction. They can either be obtained from E. coli or from cells that have been infected by the virus.
7. Which of the following statements is correct with respect to T4 DNA ligase?
a) It can carry out only blunt ended ligations
b) It doesn’t requires ATP
c) It requires a phosphate group at 3’ end and a hydroxyl group at 5’ end for the molecule to be joined
d) It is obtained from T4 bacteriophage upon infection by E. coli
Explanation: T4 DNA ligase is obtained from T4 bacteriophage upon infection by E. coli. It carries out ligation both in blunt ended and sticky ended molecules and requires ATP. It requires a phosphate group at 5’ end and a hydroxyl group at 3’ end.
8. If blunt ended ligations are to be carried out. Which of the following enzymes can be used?
a) E. coli DNA ligase
b) T4 DNA ligase
c) Both of these enzymes act equally in carrying out blunt ended ligations
d) None of them is able to carry out blunt ended ligations
Explanation: If blunt ended ligations are to be carried out, T4 DNA ligase should be used. E. coli DNA ligase is unable to carry out blunt ended ligation
9. E. coli DNA ligase doesn’t require a cofactor.
a) True
b) False
Explanation: The E. coli DNA ligase requires a cofactor nicotamine adenine dinucleotide (NAD), along with 5’ phosphate group and 3’ hydroxyl group.
10. Mechanism of ligation for both T4 DNA ligase and E. coli DNA ligase makes use of Adenosine Monophosphate (AMP). Which of the steps is involved in the ligation mechanism?
a) AMP is added to the 5’ phosphate of one of the DNA molecule
b) It leads to the liberation of pyrophosphate from NAD and nicotinamide mononucleotide from ATP
c) The AMP is further displaced by an electrophilic attack
d) The AMP is further displaced by nucleophilic attack by 3’ hydroxyl of the same DNA molecule
Explanation: AMP is added to the 5’ phosphate of one of the DNA molecule. It leads to the liberation of pyrophosphate from ATP and nicotanamide mononucleotide from NAD. It is further displaced by nucleophilic attack by 3’ hydroxyl of the other DNA molecule