Two DNA fragments are cut with two different restriction enzymes that happen to produce the same 4-nucleotide 5' overhang. They are mixed and ligated together. Which statement best describes the resulting junction?
ABoth original restriction sites are fully regenerated at the junction, so either enzyme can cut there again
BThe junction contains a hybrid sequence from both original sites; it may not be recognized by either enzyme
CThe ligation fails because compatible sticky ends from different enzymes cannot pair
DThe junction is blunt-ended because ligation removes the single-stranded overhangs
Compatible sticky ends base-pair and are sealed by ligase regardless of which enzyme produced them. However, the full recognition sequence of a restriction enzyme extends beyond the overhang into the flanking DNA. When two fragments from different enzyme sites are joined, the resulting junction sequence is a hybrid of both sites' flanking sequences — and this hybrid may not match the complete recognition sequence of either original enzyme. This is actually useful in cloning: you can join fragments without worrying about re-cutting by the original enzyme.
Question 2 Multiple Choice
A restriction enzyme produces blunt-ended cuts. Compared to sticky-end-producing enzymes, blunt-end ligation in recombinant DNA work is...
AMore efficient, because blunt ends can join in any orientation without overhang constraints
BEqually efficient, since DNA ligase seals phosphodiester bonds identically in both cases
CLess efficient, because no single-stranded overhangs provide temporary base-pairing to bring the ends together
DImpossible without a different class of ligase specialized for blunt-end joins
Sticky ends have complementary single-stranded overhangs that base-pair with each other, transiently holding the two DNA molecules in close proximity before ligase seals the backbone. This dramatically increases ligation efficiency. Blunt ends have no such stabilization — the two fragments must collide and be held together long enough for ligase to act. Blunt-end ligation is possible but far less efficient, often requiring higher DNA concentrations and longer reaction times. Directionality is also lost with blunt ends since either end can join to either other end.
Question 3 True / False
A bacterium's own DNA is immune to cleavage by its restriction enzymes because its genome lacks the palindromic recognition sequences that restriction enzymes target.
TTrue
FFalse
Answer: False
This is a common misconception. The bacterium's genome DOES contain restriction sites — the same palindromic sequences the enzyme recognizes. Protection comes from a companion methyltransferase that adds methyl groups to adenine or cytosine bases within those recognition sequences. Methylated DNA is not cut by the restriction enzyme. Invading phage DNA, which lacks the host's methylation pattern, is recognized as foreign and cleaved. This restriction-modification system is a molecular 'self vs. non-self' detection mechanism, not an absence of target sequences.
Question 4 True / False
Restriction enzymes recognize palindromic DNA sequences, meaning the sequence reads identically on both strands in the 5' to 3' direction.
TTrue
FFalse
Answer: True
A DNA palindrome is defined by reading both strands 5' to 3'. For EcoRI's site: the top strand 5'-GAATTC-3' and the bottom strand (complementary, antiparallel) also reads 5'-GAATTC-3'. This palindromic symmetry is what allows the enzyme to bind with identical contacts on both sides of the double helix — it sees the same sequence on each strand. The two-fold symmetry of the recognition site matches the two-fold symmetry of the homodimeric enzyme, which is why most restriction enzymes are dimers that cut both strands.
Question 5 Short Answer
Explain why sticky ends — rather than blunt ends — are preferred for constructing recombinant DNA molecules, even though both can be ligated.
Think about your answer, then reveal below.
Model answer: Sticky ends have short single-stranded overhangs that base-pair specifically with complementary overhangs, providing temporary stabilization that dramatically increases ligation efficiency. They also confer directionality: a specific sticky end from one enzyme will only join with a compatible partner, not with any random blunt end. Blunt ends can join in any orientation and with any other blunt end, sacrificing specificity. The base-pairing of sticky ends also increases the local concentration of the two fragments relative to each other, making productive ligase encounters far more frequent.
In molecular cloning, efficiency and specificity both matter. Sticky-end ligation can be 10–100× more efficient than blunt-end ligation. The directional specificity ensures that an insert goes into a vector in the correct orientation — something blunt ends cannot guarantee. This control over orientation is critical when the insert must be expressed in a particular reading frame.