Genetic Technology: Biology 9700 (Cambridge International AS & A Level)

Which statement correctly describes the roles of restriction endonuclease and DNA ligase in genetic engineering?

A biotechnology company wants to produce human insulin using genetically engineered bacteria. Scientists extract messenger RNA (mRNA) for the insulin gene from human pancreatic cells, then use this mRNA to prepare a gene that can be inserted into a bacterial plasmid.

(a) Describe how reverse transcriptase and DNA polymerase are used together to produce a double-stranded copy of the insulin gene, starting from insulin mRNA. [3]

(b) The double-stranded insulin gene and a bacterial plasmid are both cut with the same restriction endonuclease before being joined using DNA ligase. Explain how this produces a molecule of recombinant DNA. [3]

(c) Explain why a promoter sequence must also be inserted into the plasmid alongside the insulin gene. [2]

(d) Only some of the bacteria mixed with the recombinant plasmids actually take up a plasmid. Explain how a marker gene could be used to identify which bacterial colonies contain the recombinant plasmid. [2]

A researcher has only a very small sample of DNA extracted from a plant leaf. Before this DNA can be studied further, the researcher first uses the polymerase chain reaction (PCR) to make many copies of a particular gene, then separates the resulting DNA fragments using gel electrophoresis.

(a) The PCR reaction mixture contains the DNA sample, primers, free nucleotides and a heat-stable DNA polymerase. Describe what happens during each of the three temperature stages of one PCR cycle, stating an appropriate temperature for each stage. [6]

(b) Explain why a heat-stable polymerase, such as Taq polymerase, is used in PCR rather than the DNA polymerase found in human cells. [2]

(c) After 30 cycles of PCR, the amplified DNA is loaded onto an agarose gel and an electric current is applied. Explain how gel electrophoresis separates DNA fragments of different lengths, and explain why the DNA fragments move towards the positive electrode. [4]

Which statement correctly distinguishes genetic screening from gene therapy?

A gene from the soil bacterium Bacillus thuringiensis, which codes for a protein toxic to certain insect larvae, has been inserted into cotton plants to produce insect-resistant cotton. A government is deciding whether to approve widespread planting of this genetically modified cotton to help meet rising demand for cotton fibre.

(a) Explain how inserting this bacterial gene into cotton plants can help to increase the yield of cotton fibre. [2]

(b) Insect-resistant cotton is only one example of how genetic engineering is used to improve crop or animal productivity. Describe one other named example, and explain how the genetic modification improves productivity. [2]

(c) Discuss the ethical and social implications of growing genetically modified insect-resistant cotton. [4]

Which statement correctly distinguishes gene editing from the gene transfer techniques of genetic engineering, such as inserting a gene into a bacterial plasmid?

A biotechnology company wants to produce human growth hormone (hGH) using genetically engineered bacteria. The plasmid vector used carries two different antibiotic-resistance genes: a gene for ampicillin resistance, and a gene for tetracycline resistance. Before the plasmid is used to transform a culture of bacteria, the gene for hGH is inserted into the middle of the tetracycline-resistance gene, disrupting its base sequence.

(a) Explain why inserting the hGH gene into the middle of the tetracycline-resistance gene means that bacteria containing the recombinant plasmid become sensitive to tetracycline but remain resistant to ampicillin. [3]

(b) Describe how replica plating can be used to identify which bacterial colonies contain the recombinant plasmid (with the hGH gene inserted). [3]

(c) Explain why bacteria that failed to take up any plasmid at all would not be expected to survive on agar containing ampicillin. [2]

A DNA microarray (gene chip) is a slide onto which thousands of microscopic spots have been fixed, each spot containing many copies of a single-stranded DNA probe of known, different base sequence, at a known position.

(a) A hospital laboratory uses a microarray to test a patient's DNA sample for the presence of several different disease-associated alleles at the same time. Describe how the patient's DNA sample is prepared and applied to the microarray, and explain how the results reveal whether any of the disease-associated alleles are present. [4]

(b) In a separate investigation, mRNA extracted from a tumour sample is converted to cDNA and labelled with a red fluorescent marker, and mRNA from a healthy tissue sample is converted to cDNA and labelled with a green fluorescent marker. Equal amounts of both labelled samples are applied together to the same microarray. Explain how the colour produced at a particular spot would show that the corresponding gene is expressed more highly in the tumour sample than in the healthy sample. [3]

A child has severe combined immunodeficiency (SCID) caused by a faulty allele of a gene needed for their lymphocytes to develop and function normally, leaving the child unable to fight infection effectively. Doctors consider treating the child with gene therapy, delivering a functional copy of the gene into the child's bone marrow stem cells.

(a) Describe how a functional copy of the gene could be delivered into, and expressed by, the child's bone marrow stem cells during this gene therapy. [3]

(b) Explain two problems that can limit the long-term success of this type of gene therapy. [4]

Which of the following is a valid social or ethical concern that has been raised about genetic screening for inherited disease?