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NEST2026BiologyBiotechnology - Principles and Processes

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. EcoRI site: 5' -G AATTC- 3' MfeI site: 5' -C AATTG- 3' (arrow indicat

Options

  1. Acannot ligate to each other because the overhangs generated are from two different enzymes.
  2. Bcan ligate to each other, but the resultant product can neither be digested by EcoRI nor MfeI.
  3. Ccan ligate to each other, and the resultant product can be digested by EcoRI but not MfeI.
  4. Dcan ligate to each other, and the resultant product can be digested by MfeI but not EcoRI.

Correct answer

B. can ligate to each other, but the resultant product can neither be digested by EcoRI nor MfeI.

Step-by-step solution

The restriction enzyme EcoRI recognizes the sequence 5' -GAATTC- 3' and cuts between G and A, generating a 5' -AATT- 3' sticky end. The restriction enzyme MfeI recognizes the sequence 5' -CAATTG- 3' and cuts between C and A, also generating a 5' -AATT- 3' sticky end. Since both enzymes produce identical 5' -AATT- 3' overhangs, the DNA fragments have compatible sticky ends and can successfully ligate to each other. When the fragments ligate, the junctions formed will be a combination of the half-sites from each enzy

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