When choosing restriction enzymes for insert verification in molecular cloning, why is it preferable to select an enzyme that makes multiple cuts in the gene sequence?

Question

BlackTom AI · Accepted Answer

When planning restriction enzyme strategies for insert verification in molecular cloning, it matters what pattern of cuts is produced and how those fragments will appear on a gel or in analysis.
Option 1: 'To guarantee complete digestion of the plasmid.' A single enzyme that cuts multiple times in the gene does not inherently guarantee that the entire plasmid will be completely digested; plasmid digestion efficiency depends on many factors (enzyme activity, reaction conditions, methylation, etc.). Relying on multiple cuts in the gene to ensure complete plasmid digestion is not a sound rationale.
Option 2: 'To increase the efficiency of the digestion process.' In practice, having an enzyme that cuts multiple times in the insert can complicate the digestion pattern and may not improve overall digestion efficiency; efficiency is more about enzyme activity and reaction conditions, not simply the number of cut sites within the insert.
Option 3: 'To ensure the insert is present in the correct orientation.' Orientation verification typically relies on using specific restriction sites that flank the insert or sequencing, not on the insert having multiple internal cut sites. Multiple cuts in the gene do not by themselves guarantee that the insert is oriented correctly within the plasmid.
Option 4: 'To create a distinct banding pattern for easy identification.' This is the most practical reason: multiple cuts in the gene yield a characteristic fragmentation pattern when analyzed by gel electrophoresis, making it easier to confirm the presence and integrity of the insert through size-based identification of expected fragments. Distinct banding helps distinguish correct clones from partial digestions or incorrect constructs.
In summary, recognizing the goal of verification through fragment pattern leads to the conclusion that a multi-cut enzyme in the insert is advantageous for producing a identifiable, diagnostic banding pattern on a gel, which is why option 4 is the most appropriate choice.

AGRI30048_2025_SM2 Sample Final Exam Style Questions - unmarked

When choosing restriction enzymes for insert verification in molecular cloning, why is it preferable to select an enzyme that makes multiple cuts in the gene sequence?

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