Below are various steps in the cloning process. Which is the correct order?

1. Transform vector into bacteria.

2. Cut the vector and chromosomal DNA with the same restriction enzyme.

3. Add DNA ligase.

4. Plate on growth media containing an antibiotic such as ampicillin.

Question

Below are various steps in the cloning process. Which is the correct order?

1. Transform vector into bacteria.

2. Cut the vector and chromosomal DNA with the same restriction enzyme.

3. Add DNA ligase.

4. Plate on growth media containing an antibiotic such as ampicillin.

BlackTom AI · Accepted Answer

Restriction enzymes must first generate compatible ends: cut the vector and chromosomal DNA with the same restriction enzyme (step 2). This creates matching sticky or blunt ends that allow the insert to be joined into the plasmid backbone.

1. Relevant knowledge points and concepts
- Restriction digestion: endonucleases cut DNA at specific sequences, producing compatible ends on vector and insert.  
- Ligation: DNA ligase covalently joins the sugar-phosphate backbones to form a stable recombinant plasmid.  
- Transformation: introduction of the recombinant plasmid into competent bacterial cells.  
- Selection: plating on antibiotic-containing medium selects only bacteria that received a plasmid carrying the antibiotic resistance marker.

2. Step-by-step reasoning
- Step 2 first: both vector and target DNA must be cut with the same restriction enzyme(s) so ends are compatible. Without compatible ends, ligation is inefficient or impossible.  
- Step 3 next: add DNA ligase to join the insert into the opened vector, producing recombinant plasmid molecules. Ligation must occur before getting DNA into cells.  
- Step 1 then: transform the ligated plasmids into bacteria (e.g., chemically competent E. coli or by electroporation). Bacteria uptake plasmid DNA during transformation.  
- Step 4 last: plate transformed bacteria on growth medium containing an antibiotic (e.g., ampicillin). Only cells that have taken up plasmids carrying the resistance gene will grow, enabling selection.

3. Intermediate steps/calculations
- No numeric calculations are required. Intermediate experimental steps typically include verifying digestion (gel electrophoresis), optional vector dephosphorylation to reduce self-ligation, and recovering ligation products prior to transformation.

4. Why the correct answer is correct
- The molecular logic requires cutting before ligation and ligation before transformation; selection (plating on antibiotic) can only happen after bacteria have been transformed. Thus the chronological sequence 2 → 3 → 1 → 4 is required.

5. Why other options are incorrect
- "1, 2, 3, 4": transforming before cutting/ligation is impossible.  
- "2, 3, 4, 1": plating before transformation makes no sense—there are no transformed cells to plate.  
- "4, 1, 2, 3": plating first and cutting/ligating last is out of logical/experimental order.

6. Conclusion
The correct sequence is 2, 3, 1, 4, which matches the provided answer.

Below are various steps in the cloning process. Which is the correct order? 1. Transform vector into bacteria. 2. Cut the vector and chromosomal DNA with the same restriction enzyme. 3. Add DNA ligase. 4. Plate on growth media containing an antibiotic such as ampicillin. 单项选择题

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