A student added a 5' exonuclease enzyme instead of a restriction enzyme to digest his purified plasmid DNA sample. What is he likely to observe when…
A student added a 5' exonuclease enzyme instead of a restriction enzyme to digest his purified plasmid DNA sample. What is he likely to observe when he runs his plasmid digest on an agarose gel?
Select an option to attempt
Correct Answer: Option D
Explanation
5' exonucleases require a FREE 5' terminus to initiate degradation — they bind the 5' end and processively cleave nucleotides from the 5' end toward the 3' end. CIRCULAR plasmid DNA (covalently closed circle, CCC) has NO FREE ENDS (both 5' and 3' ends of each strand are covalently joined). Therefore, a 5' exonuclease CANNOT bind or degrade circular DNA. The result: the plasmid will appear unchanged on the agarose gel — no degradation. Note: If the plasmid is linearised (by restriction enzyme) first, 5' exonuclease could then degrade from the free 5' ends. BAL-31, T7 exonuclease, and RecBCD are examples of enzymes that prefer or require linear DNA with free ends.