Correct option is A
The correct answer is: (1) Treatment of vector with Mung Bean Nuclease followed by treatment with Shrimp Alkaline Phosphatase
Explanation:
To facilitate the ligation of a blunt-ended insert fragment with a vector digested with EcoRI (which creates sticky ends), the best intermediate enzymatic reaction is to first treat the vector with Mung Bean Nuclease. This enzyme removes any overhanging ends on the vector, resulting in blunt ends that are compatible with the blunt-ended insert. After that, Shrimp Alkaline Phosphatase is used to remove the 5' phosphate groups from the vector. This step ensures that the vector will not self-ligate and will only ligate with the insert, allowing for a successful ligation.
Analysis of Other Options:
Option 2 (Treatment of insert with Klenow DNA Polymerase and vector with Polynucleotide Kinase): This option is ineffective because Klenow DNA polymerase does not provide a significant advantage for preparing the insert, and while Polynucleotide Kinase can phosphorylate the vector, it does not activate the vector in a way that allows for proper ligation with a blunt-ended insert.
Option 3 (Treatment of vector with Polynucleotide Kinase and insert with Mung Bean Nuclease): Polynucleotide Kinase does not efficiently prepare the vector for ligation in this scenario, and Mung Bean Nuclease is not effective on the insert, making this approach less suitable.
Option 4 (Treatment of insert with Klenow DNA polymerase followed by treatment with Shrimp Alkaline Phosphatase): Klenow DNA polymerase is not effective for modifying the insert in this case, and Shrimp Alkaline Phosphatase should be used on the vector, not the insert, to prevent self-ligation of the vector.
