Please consider the restriction enzymes ApaI and PspOMI (OMG, who THINKS of these names???). Anyway, these enzymes have the following recognition/cut sequences, with the * indicating the cut site: ApaI: 5’GGCCC3’ 3’CCGGGG5’ PspOMI: 5’GGGCCC3’ 3’CCCGGGG5’ For each of these, please state whether the overhangs are 5’ ends or 3’ ends. Another enzyme, EagI, cuts at the sequence: 5’CGGCCG3’ 3’GCCGGC5’ Can a molecule that has been cut with EagI be annealed for one that is cut with ApaI, PspOMI, neither, or both? Please demonstrate or explain verbally.
The Correct Answer and Explanation is:
1. ApaI:
Recognition site:
- 5’ GGCC*C 3’
- 3’ C *CCGGG 5’
Cutting pattern:
ApaI cuts after the C on the top strand and before the C on the bottom strand. This leaves a 3’ overhang of the sequence C.
Resulting ends:
- 5’ GGCC
- 3’ CCGGG (with a 3′ overhang of “GGG”)
Therefore, ApaI produces a 3’ overhang.
2. PspOMI:
Recognition site:
- 5’ G*GGCCC 3’
- 3’ CCCGGG* 5’
Cutting pattern:
PspOMI cuts before the G on the top strand and after the G on the bottom strand, leaving a 5’ overhang.
Resulting ends:
- 5’ G (overhang)
- 3’ CCCGGG
Therefore, PspOMI produces a 5’ overhang.
3. EagI:
Recognition site:
- 5’ C*GGCCG 3’
- 3’ GCCGG*C 5’
Cutting pattern:
EagI cuts between C and G on both strands symmetrically, producing the overhang GGCC on the 5’ end.
Resulting ends:
- 5’ CGGCC
- 3’ GCCGG
(Leaving a 5’ overhang of “GGCC”)
Therefore, EagI produces a 5’ overhang.
Can a molecule cut with EagI anneal with ApaI or PspOMI?
Let’s compare the sticky ends:
- EagI sticky end: 5’ overhang of GGCC
- ApaI sticky end: 3’ overhang, not complementary to GGCC
- PspOMI sticky end: 5’ overhang, but G (single base), not complementary to GGCC
Conclusion:
The sticky ends from EagI are not compatible with either ApaI or PspOMI, because the sequences and overhang types (length and base sequence) do not match. For sticky ends to anneal, the overhangs must be complementary and of the same type (both 5’ or both 3’).
Final Answers:
- ApaI: 3’ overhang
- PspOMI: 5’ overhang
- EagI’s product can anneal with: neither ApaI nor PspOMI
