QUESTION 143 (7 marks)
A vector is 3000 bp. The intended gene insert is 600 bp. One complete digest cuts each circular plasmid once, showing total length. A second complete digest cuts on both sides of the insert. A gene-specific PCR should produce a 200 bp band if the tested region of the intended gene is present. All positive-control reactions work; empty-vector controls give no gene-specific product.
| Clone | Single-cut band / bp | Insert-release bands / bp | Gene-specific PCR / bp |
|---|---|---|---|
| A | 3000 | 3000 | None |
| B | 3600 | 3000, 600 | 200 |
| C | 3600 | 3000, 600 | None |
a) Choose the clone best supported as carrying the intended gene and justify with two independent observations. [3 marks]
b) Explain why the digest data alone cannot distinguish B from C as carrying the intended gene. [1 mark]
c) Describe the roles of restriction enzymes and DNA ligase in making the recombinant plasmid. [2 marks]
d) State one property of a successful protein-producing clone that these tests do not establish. [1 mark]
Practice marking scheme
Answer
Clone B is best supported. Its 3600 bp total length and 600 bp released insert match the intended construction, and the PCR detects the tested gene region. Clone C has an insert of the expected size but lacks the diagnostic PCR product.
Working
B and C have the expected size increase over the vector, so size alone does not distinguish the insert’s identity. B also yields the expected gene-specific band. Restriction enzymes cut the chosen recognition sites; DNA ligase seals the insert/vector backbone. Neither a short PCR product nor insert size confirms the full insert sequence or successful protein expression.
Marking criteria
- Selects B. [1 mark]
- Uses expected total/insert lengths 3600/600 bp. [1 mark]
- Uses its expected 200 bp gene-specific PCR product. [1 mark]
- Explains that equal insert sizes do not establish equal sequences/identity. [1 mark]
- Describes sequence-specific DNA cutting by restriction enzymes. [1 mark]
- Describes backbone joining/sealing by ligase. [1 mark]
- Names full sequence accuracy, intact regulatory context, suitable expression, or functional protein production. [1 mark]
Practice question aligned to the current QCAA syllabus; review the worked solution and marking criteria.
View the QCAA syllabusCompare your working with the guide above.