Recombinant DNA technologyAQA A-Level Biology: Subtopic test

1. 1
   Beta cells of the human pancreas make large amounts of mRNA for insulin. Researchers want bacteria to make human insulin. Bacterial cells cannot remove introns from RNA. The researchers use mRNA from the beta cells as the starting material to obtain a copy of the insulin gene.

   (a)

   Which enzyme is used to make cDNA from mRNA?

   [1 mark]

   • ADNA ligase
   • BReverse transcriptase
   • CRestriction endonuclease
   • DRNA polymerase

   (b)

   Why is cDNA made from mRNA preferred to cutting the gene from human DNA for this work?

   [1 mark]

   • AmRNA is easier to cut with restriction enzymes
   • BThe mRNA has a stop codon that the gene lacks
   • CThe mRNA is inserted directly into the plasmid
   • DThe introns have been removed from mRNA, so the cDNA has no introns

   (c)

   Explain why the human insulin gene can be transcribed and translated to make insulin in the bacterial cell.

   [2 marks]

   Total for question 1: 4 marks

2. 2
   A plasmid vector has a single recognition site for the restriction endonuclease EcoRI, which cuts DNA leaving short single-stranded ends. A fragment of human DNA containing a gene is cut out of a chromosome using the same enzyme and then mixed with the cut plasmids.

   (a)

   Why are the gene fragment and the plasmid cut with the same restriction endonuclease?

   [1 mark]

   • AIt leaves complementary single-stranded ends that can base pair
   • BIt cuts the DNA at random places
   • CIt makes the DNA completely double stranded
   • DIt joins the fragment to the plasmid

   (b)

   What is the role of DNA ligase in making the recombinant plasmid?

   [1 mark]

   • AIt separates the two strands of DNA
   • BIt adds nucleotides to complementary bases
   • CIt joins the sugar-phosphate backbones of the fragment and plasmid
   • DIt cuts DNA at the recognition sites

   (c)

   Explain why EcoRI cuts DNA only at one specific base sequence.

   [2 marks]

   Total for question 2: 4 marks

3. 3
   A forensic laboratory has only a few molecules of DNA from a crime scene. It uses the polymerase chain reaction (PCR) to make many copies of a short target sequence before analysis. The reaction mixture contains the DNA, primers, free nucleotides and a heat-stable DNA polymerase.

   (a)

   Describe what happens in one cycle of PCR.

   [3 marks]

   (b)

   Calculate the number of copies of the target sequence after 25 cycles of PCR from one molecule, and explain why a heat-stable DNA polymerase is used.

   [4 marks]

   Total for question 3: 7 marks

4. 4
   A biotechnology company wants to make human growth hormone in Escherichia coli. The human gene is cut out and inserted into a plasmid that carries a gene for ampicillin resistance and a gene for green fluorescent protein (GFP). The restriction site used lies in the middle of the GFP gene. The plasmids are mixed with E. coli treated with calcium ions and a heat shock, and the bacteria are spread on agar containing ampicillin. Some colonies glow green and some do not.

   (a)

   Explain how the human gene is inserted into the plasmid, and how the bacteria containing the recombinant plasmid are identified.

   [6 marks]

   (b)

   Compare the use of PCR with the culture of transformed bacteria as methods of obtaining many copies of the human gene.

   [6 marks]

   Total for question 4: 12 marks

End of questions