19.1. Principles of genetic technology
A subsection of Biology, 9700, through 19. Genetic technology
Listing 10 of 43 questions
Genetic technology uses many different enzymes and techniques. Restriction endonucleases are used in genetic modification. These enzymes occur naturally in prokaryotic cells. More than 3500 different restriction endonucleases have been identified and it is thought there are many more to discover. Name two domains that are a source of restriction endonucleases. Originally, the method used to obtain a restriction endonuclease was to: • grow large numbers of the specific prokaryotic cells that are the source of the enzyme • break open the cells and extract and purify the restriction endonuclease. This original method produced only a small quantity of restriction endonuclease and was not economical. The newer method for large-scale production is to: • obtain the gene coding for a specific restriction endonuclease • introduce the gene into Escherichia coli, with a promoter that allows the gene to be expressed continuously. The newer method increases the quantity of specific restriction endonuclease produced. Suggest and explain the steps needed to carry out the newer method for large-scale production of a specific restriction endonuclease. Describe the advantages of databases for the study and use of restriction endonucleases. Electrophoresis is a technique used in genetic technology. Paper chromatography is a technique used to investigate the photosynthetic pigments found in chloroplasts. Compare the similarities and differences between electrophoresis and chromatography.
9700_s23_qp_42
THEORY
2023
Paper 4, Variant 2
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The artificial plasmid, pBR322, was constructed to act as a vector. It has often been used to insert human genes, such as the human insulin gene, into the bacterium, Escherichia coli. The plasmid was constructed to include two genes, each giving resistance to a different antibiotic: an ampicillin resistance gene and a tetracycline resistance gene. The plasmid also has a target site for the restriction enzyme, BamHI, in the middle of the tetracycline resistance gene. A pBR322 plasmid was cut using BamHI and the cDNA gene for human insulin inserted into it. shows pBR322 and the recombinant plasmid. pBR322 recombinant plasmid human insulin gene tetracycline resistance gene G C G C A T T A C G C G ampicillin resistance gene target site for BamHI With reference to , describe how a cDNA human insulin gene can be inserted into pBR322 that has been cut by BamHI. Bacteria were then mixed with the recombinant plasmids. Those bacteria which had successfully taken up recombinant plasmids were identified using the following steps: step 1 – the bacteria were spread onto culture plates containing nutrient agar and ampicillin and incubated to allow colonies to form step 2 – some bacteria from each of the colonies growing on these plates were transferred to plates containing nutrient agar and tetracycline, as shown in . sterile sponge which transfers some of each colony, in the same relative positions, from plate A to plate T plate A plate T after incubation nutrient agar with tetracycline bacterial colony nutrient agar with ampicillin Explain why the bacteria were first spread onto plates containing ampicillin. Explain why it is important, for identifying bacteria that have successfully taken up the recombinant plasmid, that on pBR322 the target site for BamHI is in the middle of the tetracycline resistance gene. Use a label line and the letter C to identify, on , a colony of bacteria that contain the recombinant plasmid. Put your answer onto on page 5. Plasmid vectors carrying antibiotic resistance genes are now rarely used in gene technology. Explain why antibiotic resistance genes are now rarely used. State one type of gene that has replaced antibiotic resistance genes in plasmid vectors and indicate how its presence can be detected. type of gene detection
9700_w12_qp_43
THEORY
2012
Paper 4, Variant 3
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A marker gene can be used when a gene of interest is introduced into a plant by genetic engineering. Describe and explain how the use of a marker gene coding for a fluorescent product can show that the introduced gene of interest is being expressed in plants. Give two advantages of genetically engineering herbicide resistance in crop plants such as soybean. DNA microarray analysis is a technique used in genetic technology that involves fluorescence. A DNA microarray has single-stranded probes attached to its surface. These probes hybridise to the fluorescently tagged single-stranded DNA that is added. Explain why DNA hybridisation occurs between the probe DNA and the added DNA. A DNA microarray analysis can be used to identify the level of expression of some genes. Describe and explain how the level of expression of some genes can be identified using the DNA microarray analysis technique.
9700_w23_qp_42
THEORY
2023
Paper 4, Variant 2
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A number of diseases, such as dengue fever, are spread by mosquitoes. The incidence of this disease has increased dramatically in recent years and this has been linked with the spread of the mosquito, Aedes aegypti. In an attempt to reduce the numbers of A. aegypti, genetically modified (GM) male mosquitoes were produced. One of the genes added to these mosquitoes, when switched on, results in the production of a protein which is toxic to mosquitoes. In 2010, in the Cayman Islands and in Malaysia, GM male mosquitoes were released into the wild to mate with females. All the resulting offspring died in the larval stage. About 3 million GM male mosquitoes were released in the Cayman Islands. Suggest why releasing such large numbers of male mosquitoes did not immediately increase the risk of transmission of dengue fever. In Malaysia, both GM male and non-GM male mosquitoes were released in order to compare their dispersal and life span in the wild. The GM mosquitoes could be identified because they also carried a gene for green fluorescent protein (GFP). Explain why, in many examples of gene technology, fluorescent markers are used in preference to antibiotic resistance genes. In addition to the gene for GFP, the DNA that has been added to the GM mosquitoes consists of • a promoter • a gene coding for a toxic protein, tTA • a binding site for tTA. When a GM mosquito larva hatches from an egg, the promoter induces the production of only a small amount of tTA, so that the larva does not die immediately. In a process of positive feedback, the tTA produced binds to the DNA as shown in . This increases the expression of the gene until the increased concentration of tTA kills the larva. binding site for tTA gene coding for tTA tTA produced promoter Suggest why this process is called positive feedback. Explain why, in gene technology, a promoter needs to be transferred along with the desired gene. Switching on the gene coding for tTA in the mosquito larvae, rather than in the eggs, increases the effectiveness of this method of controlling mosquito numbers. Suggest why this is so. GM mosquitoes carrying the tTA gene can live and reproduce normally when fed on a diet containing an added chemical, A. With reference to : suggest how A could prevent death of the GM mosquitoes suggest how large numbers of adult GM male mosquitoes can be produced for release into the wild, from an original stock of GM males suggest why there is little danger of the gene carried by these GM mosquitoes being passed to other organisms from GM mosquitoes which escape or are released into the wild.
9700_s13_qp_41
THEORY
2013
Paper 4, Variant 1
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The pink bollworm moth, Pectinophora gossypiella, is a pest of cotton crops. The size of its population can be reduced by releasing large numbers of sterile male moths into cotton fields. The sterile male moths mate with wild females from the cotton fields, but no offspring are produced. Over a period of three years, 20 million genetically modified (GM) sterile male moths were released in the USA. Each insect contained a gene coding for a red fluorescent protein (DsRed) taken from a species of reef coral. The added DNA also included a promoter. Explain why, in gene technology: genes for fluorescent proteins such as DsRed are now more commonly used as markers than are genes for antibiotic resistance a promoter needs to be included when transferring a gene from a coral into an insect. DsRed is visible at all stages of the life cycle of the moth, but the presence of the gene in a particular individual can be confirmed by genetic fingerprinting, using gel electrophoresis. Outline the principles of gel electrophoresis. Explain how the presence of the gene for DsRed in a moth can be confirmed once electrophoresis is complete. DsRed allows sterile male moths to be distinguished from wild moths when caught in an insect trap in a field of cotton plants. Suggest why it is important to be sure whether a moth caught in such a trap is a released sterile male or a wild insect. The United States Department of Agriculture has ruled that the release of sterile males to control insect pest numbers is environmentally preferable to all other alternatives. Suggest what information would be needed to determine whether the release of the sterile male moths, carrying the gene for DsRed, has a damaging effect on the environment.
9700_s13_qp_42
THEORY
2013
Paper 4, Variant 2
View
A number of diseases, such as dengue fever, are spread by mosquitoes. The incidence of this disease has increased dramatically in recent years and this has been linked with the spread of the mosquito, Aedes aegypti. In an attempt to reduce the numbers of A. aegypti, genetically modified (GM) male mosquitoes were produced. One of the genes added to these mosquitoes, when switched on, results in the production of a protein which is toxic to mosquitoes. In 2010, in the Cayman Islands and in Malaysia, GM male mosquitoes were released into the wild to mate with females. All the resulting offspring died in the larval stage. About 3 million GM male mosquitoes were released in the Cayman Islands. Suggest why releasing such large numbers of male mosquitoes did not immediately increase the risk of transmission of dengue fever. In Malaysia, both GM male and non-GM male mosquitoes were released in order to compare their dispersal and life span in the wild. The GM mosquitoes could be identified because they also carried a gene for green fluorescent protein (GFP). Explain why, in many examples of gene technology, fluorescent markers are used in preference to antibiotic resistance genes. In addition to the gene for GFP, the DNA that has been added to the GM mosquitoes consists of • a promoter • a gene coding for a toxic protein, tTA • a binding site for tTA. When a GM mosquito larva hatches from an egg, the promoter induces the production of only a small amount of tTA, so that the larva does not die immediately. In a process of positive feedback, the tTA produced binds to the DNA as shown in . This increases the expression of the gene until the increased concentration of tTA kills the larva. binding site for tTA gene coding for tTA tTA produced promoter Suggest why this process is called positive feedback. Explain why, in gene technology, a promoter needs to be transferred along with the desired gene. Switching on the gene coding for tTA in the mosquito larvae, rather than in the eggs, increases the effectiveness of this method of controlling mosquito numbers. Suggest why this is so. GM mosquitoes carrying the tTA gene can live and reproduce normally when fed on a diet containing an added chemical, A. With reference to : suggest how A could prevent death of the GM mosquitoes suggest how large numbers of adult GM male mosquitoes can be produced for release into the wild, from an original stock of GM males suggest why there is little danger of the gene carried by these GM mosquitoes being passed to other organisms from GM mosquitoes which escape or are released into the wild.
9700_s13_qp_43
THEORY
2013
Paper 4, Variant 3
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Questions Discovered
43