Question

A researcher wants to insert a linear DNA fragment into a plasmid DNA. The linear DNA fragment has two MfeI restriction sites at its ends. The plasmid DNA has only one EcoRI restriction site. The plasmid was digested with EcoRI and the DNA fragment was digested with MfeI. The recognition sequences of the restriction enzymes on one strand are given.

(arrow indicates enzyme cut sites)
Based on this information, the DNA fragments generated by EcoRI and MfeI

• A. can ligate to each other, but the resultant product can neither be digested by EcoRI nor MfeI.
• B. cannot ligate to each other because the overhangs generated are from two different enzymes.
• C. can ligate to each other, and the resultant product can be digested by EcoRI but not MfeI.
• D. can ligate to each other, and the resultant product can be digested by MfeI but not EcoRI.

Hint:

Restriction enzymes that produce identical overhangs (isocaudomers) can produce compatible sticky ends that ligate together. However, the resulting hybrid site is almost always asymmetrical and cannot be cut by either of the original enzymes.

Answer 1

The Correct Option is A

Step 1: Understanding the Question:
The question asks about the feasibility of ligating DNA fragments generated by digesting a linear DNA fragment with MfeI and a plasmid DNA with EcoRI, and the properties of the resulting recombinant product.

Step 2: Key Formula or Approach:
Restriction enzymes EcoRI and MfeI recognize palindromic sequences and cut them as shown:

• EcoRI site: $5'-G \downarrow AATTC-3'$

• MfeI site: $5'-C \downarrow AATTG-3'$

We need to determine the single-stranded overhangs (sticky ends) produced by these enzymes and see if they can base-pair and ligate.

Step 3: Detailed Explanation:

• Let us analyze the cleavage of the EcoRI site: \[ 5'-G \ \ \ \ \ AATTC-3' \ \rightarrow \ 5'-G-3' \ \ \text{and} \ \ 5'-AATTC-3' \] This leaves a $5'$ single-stranded overhang of $5'-AATT-3'$.

• Let us analyze the cleavage of the MfeI site: \[ 5'-C \ \ \ \ \ AATTG-3' \ \rightarrow \ 5'-C-3' \ \ \text{and} \ \ 5'-AATTG-3' \] This also leaves a $5'$ single-stranded overhang of $5'-AATT-3'$.

• Because both EcoRI and MfeI generate identical single-stranded overhangs ($5'-AATT-3'$), these cohesive ends are fully compatible and can easily hybridize and ligate to each other in the presence of DNA ligase.

• Let us look at the sequence at the junction after ligation: itemize

• On one strand, the EcoRI end $5'-G-3'$ is joined to the MfeI end $5'-AATTG-3'$, forming the sequence: \[ 5'-GAATTG-3' \]

• The complementary strand will have the sequence: \[ 3'-CTTAAC-5' \quad \text{which is} \quad 5'-CAATTC-3' \]

The resulting hybrid junction sequence is $5'-GAATTG-3'$.
This sequence is neither recognized by EcoRI (which requires $5'-GAATTC-3'$) nor by MfeI (which requires $5'-CAATTG-3'$).
Therefore, once ligated, the junction is destroyed and can neither be cleaved by EcoRI nor by MfeI.
itemize

Step 4: Final Answer:
Therefore, they can ligate to each other, but the resultant product can neither be digested by EcoRI nor MfeI.