Question

Joining two DNA molecules using an adapter involves the following steps
A. Artificial synthesis of an adapter
B. Treating with polynucleotide kinase
C. Blunt end ligation to add an adapter to a blunt-ended DNA molecule
D. Joining two DNA molecules with sticky ends
What is the correct sequence in which these steps are performed? [Question ID = 658][Question Description = 158_34_PLB_AUG22_Q58]

• A. B, A, D, C [Option ID = 2629]
• B. A, C, B, D [Option ID = 2630]
• C. D, B, C, A [Option ID = 2631]
• D. C, A, D, B [Option ID = 2632]

Hint:

Adapters have "pre-existing" sticky ends. Linkers, by contrast, are blunt-ended and must be cut with a restriction enzyme after ligation to create sticky ends.

Answer 1

The Correct Option is B

Step 1: Understanding the Concept:
An adapter is a short, chemically synthesized, double-stranded DNA molecule that has one blunt end and one sticky end. It is used to convert blunt-ended DNA fragments (which are hard to ligate) into fragments with sticky ends (which are easy to ligate).

Step 2: Detailed Explanation:
The logical sequence of this cloning strategy is as follows:
1. A: Artificial synthesis of an adapter: The specific oligonucleotide sequence must be synthesized first.
2. C: Blunt end ligation: The blunt end of the adapter is ligated to the blunt-ended DNA molecule of interest. At this stage, the adapter is typically synthesized without a \(5^{\prime}\)-phosphate to prevent it from ligating to itself (forming dimers).
3. B: Treating with polynucleotide kinase: After the adapter is attached to the target DNA, Polynucleotide Kinase (PNK) is used to add phosphate groups to the \(5^{\prime}\) ends of the sticky portion of the adapter. This makes the molecule ready for the next ligation step.
4. D: Joining two DNA molecules with sticky ends: Now that the target DNA has "phosphorylated sticky ends," it can be easily ligated into a vector that has complementary sticky ends.
Thus, the correct order is A \(\rightarrow\) C \(\rightarrow\) B \(\rightarrow\) D.

Step 3: Final Answer:
The methodological sequence is Synthesis \(\rightarrow\) Blunt Ligation \(\rightarrow\) Phosphorylation \(\rightarrow\) Sticky Ligation. Option 2 is correct.