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E.coli cloning vector pBR322 contains antibiotic resistance genes for Ampicillin (ampR) and Tetracycline (tetR) as selectable markers. Selectable markers help in selecting those host cells which contain the transformed vectors and eliminate the non-transformants. Plasmid pBR322 has a variety of unique recognition sites for restriction endonucleases. Two unique sites—PstI and PvuI—are located within the ampR gene and BamHI and SalI are within the tetR gene.

1. Insertion of the DNA fragment into the plasmid using enzyme PstI or PvuI places the DNA insert within the gene ampR, which makes ampR non-functional.
2. Bacterial cells containing such a recombinant pBR322 will be unable to grow in the presence of ampicillin but will grow on tetracycline.
3. Insertion of the DNA fragment into the plasmid using enzyme BamHI or SalI places the DNA insert within the gene tetR, which makes tetR non-functional.
4. Bacterial cells containing such a recombinant pBR322 will be unable to grow in the presence of tetracycline but will grow on ampicillin.

Correct answer: Insertion of the DNA fragment into the plasmid using enzyme PstI or PvuI places the DNA insert within the gene ampR, which makes ampR non-functional.

Solution

PstI and PvuI cut within the ampR gene, so inserting DNA there disrupts ampicillin resistance. The recombinant plasmid therefore cannot protect bacteria from ampicillin, while tetracycline resistance remains intact.

Related NEET Biology questions

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• Which one is a true statement regarding DNA polymerase used in PCR
• The separated DNA fragments can be visualised only after staining the DNA with Ethidium bromide followed by exposure to UV radiation.

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