19.1. Principles of genetic technology
A subsection of Biology, 9700, through 19. Genetic technology
Listing 10 of 43 questions
Green fluorescent protein (GFP) is a small protein that emits bright green fluorescence in blue light. It was first isolated from the jellyfish, Aequorea victoria. The gene coding for GFP can be expressed in bacteria, such as Escherichia coli, and so it is often used as a marker to show successful uptake of a gene by the bacterium. Outline how a gene from another species can be inserted into E. coli. Explain how a marker gene, such as the gene for GFP, is used to show successful uptake of a gene for a wanted protein. Genes for enzymes that produce fluorescent substances are often used as markers in gene technology. GFP is not an enzyme. Suggest one disadvantage of using the gene for GFP to produce easily detectable fluorescence, rather than using a gene for an enzyme that produces a fluorescent substance. Explain your answer. disadvantage explanation
9700_w12_qp_42
THEORY
2012
Paper 4, Variant 2
View
Traditional techniques for genetically modifying organisms use three enzymes: • restriction endonuclease • reverse transcriptase • DNA ligase. For example, these enzymes have been used to produce genetically modified pigs containing the GFP gene coding for green fluorescent protein, originally sourced from jellyfish. Outline how these three enzymes could be used in genetically engineering a transgenic pig containing the GFP gene. A new technique that aims to cause a deletion in a gene uses an enzyme called Cas9 nuclease. It is injected into zygotes along with an RNA sequence (the guide RNA) that is complementary to a target gene. The Cas9 nuclease causes a deletion in the target gene in the zygotes, preventing the expression of that gene. The toxicity and efficiency of the new technique was tested on four groups of pig zygotes. These pig zygotes were produced by IVF using: • ova from a female non-transgenic pig. • sperm from a male transgenic pig whose somatic cells contained one copy of the GFP gene per cell. The pig zygotes in three groups were injected with different concentrations of Cas9 nuclease and guide RNA targeted at the GFP gene. The fourth group of pig zygotes (control group) was not injected with Cas9 nuclease and guide RNA. Explain why the GFP gene was chosen for testing the new technique. Some of the zygotes in each group survived and after six days each had developed into a group of cells called a blastocyst. The blastocysts were counted using a light microscope. A filter was then added to the microscope, so that only blastocysts expressing the green fluorescent protein showed up. These were counted and the results are summarised in Table 5.1. Table 5.1 concentration of Cas9 nuclease and guide RNA / ng mm–3 number of blastocysts seen under white light number of blastocysts seen under filter 0 68 Calculate the percentage of zygotes in the control group that were transgenic. Show your working. % Explain whether the percentage you calculated for is higher or lower than expected. Name a statistical test that would allow you to test the significance of the difference between the percentage you calculated in and the expected percentage. State the best concentration of Cas9 nuclease and guide RNA to use to cause a deletion in the GFP gene and give reasons for your choice. shows the results from a second trial of the new technique, analysed by electrophoresis. • Lanes 1–4 show DNA from four pigs born after Cas9 nuclease was used to cause a deletion in a target gene coding for a cell surface protein. • Lane 5 shows DNA from their surrogate mother. • Lane 6 shows DNA from another normal pig for comparison. The size of the DNA fragments is given in kilobase pairs as shown in . 1 kbp is 1000 base pairs of DNA. The target gene measures 6 kbp and codes for a cell surface protein that is essential for the disease virus PRRSV to infect cells in the pig’s body. 6 kbp 4 kbp Explain what indicates about the success of the new technique in causing a deletion in a gene in pigs so that they show resistance to PRRSV.
9700_w18_qp_42
THEORY
2018
Paper 4, Variant 2
View
Scientists use many different techniques in genetic engineering. Sometimes the gene for genetic engineering cannot be extracted from the donor organism. Instead, the gene is synthesised using one of two different methods. Outline the two methods for synthesising a gene for use in genetic engineering. DNA ligase and DNA polymerase are two enzymes that are used in genetic engineering. Complete Table 8.1 to show the roles of DNA ligase and DNA polymerase in genetic engineering. Use a tick (✓) if the enzyme has the role or a cross (✗) if the enzyme does not have the role. Table 8.1 role in genetic engineering DNA ligase DNA polymerase joins two sections of sugar phosphate backbone in DNA adds a gene to a plasmid adds free activated DNA nucleotides to a polynucleotide The polymerase chain reaction (PCR) is used to make many copies of a gene. Three temperatures are used in a PCR cycle. State the three temperatures that are used, and outline what happens at each temperature during a PCR cycle.
9700_w24_qp_41
THEORY
2024
Paper 4, Variant 1
View
Scientists use many different techniques in genetic engineering. Sometimes the gene for genetic engineering cannot be extracted from the donor organism. Instead, the gene is synthesised using one of two different methods. Outline the two methods for synthesising a gene for use in genetic engineering. DNA ligase and DNA polymerase are two enzymes that are used in genetic engineering. Complete Table 8.1 to show the roles of DNA ligase and DNA polymerase in genetic engineering. Use a tick (✓) if the enzyme has the role or a cross (✗) if the enzyme does not have the role. Table 8.1 role in genetic engineering DNA ligase DNA polymerase joins two sections of sugar phosphate backbone in DNA adds a gene to a plasmid adds free activated DNA nucleotides to a polynucleotide The polymerase chain reaction (PCR) is used to make many copies of a gene. Three temperatures are used in a PCR cycle. State the three temperatures that are used, and outline what happens at each temperature during a PCR cycle.
9700_w24_qp_43
THEORY
2024
Paper 4, Variant 3
View
To identify the function of a gene, scientists can insert a copy of the gene into a plasmid to create recombinant DNA. The plasmid is then transferred into a host bacterium to express the gene. Define recombinant DNA. One plasmid used by scientists for this purpose is pIRES2-EGFP. shows the main features of pIRES2-EGFP. multiple cloning site (MCS) promoter region gene for antibiotic resistance gene coding for green fluorescent protein (GFP) This plasmid includes a gene that codes for a fluorescent protein, GFP. Explain the purpose of including a gene for a fluorescent protein in the plasmid. A gene with unknown function was inserted into the multiple cloning site (MCS) of pIRES2-EGFP. The MCS contains the target nucleotide sequence for a number of different restriction endonucleases. The nucleotide sequence of the MCS in pIRES2-EGFP is shown in . 5' 3' 3' 5' C G T A C G A T G C A T T A C G C G T A T A T A T A T A T A T A T A T A C G C G C G T A C G G C G C G C C G G C G C C G C G C G C G C G C G C G C G G C G C G C C G C G C G G C G C G C G C G C G C G C A T A T A T G C A T A T A T A T A T A T The nucleotide sequences targeted by six restriction endonucleases and the way in which these enzymes cut DNA are shown in . 5' 3' 3' 5' G C G C A T T A C G C G BamHI 5' 3' 3' 5' A T G C A T T A C G T A Bg/II 5' 3' 3' 5' G C A T A T T A T A C G EcoRI 5' 3' 3' 5' G C T A C G G C A T C G Sa/I 5' 3' 3' 5' C G C G C G G C G C G C SmaI 5' 3' 3' 5' C G T A C G G C A T G C XhoI Describe the type of end produced when DNA is cut using the restriction endonuclease SmaI. Scientists used two of the restriction endonucleases shown in to obtain the gene with unknown function for inserting into the MCS of pIRES2-EGFP. shows the gene obtained after cutting with these two restriction endonucleases, including the nucleotide sequences of the two ends. The DNA START codon, ATG, and DNA STOP codon, TAA, are shaded. 5' 3' T C G A G C A T T A G C 3' 5' T A A T A T G C A G C T DNA sequence of gene Name the two restriction endonucleases in that were used to cut the MCS of pIRES2-EGFP so that the gene shown in could be inserted. The nucleotide sequence of the MCS in pIRES2-EGFP is shown in . On , draw around the group of nucleotides in the MCS that were removed to insert the gene shown in . To identify the function of the gene, it is important that the gene can be easily inserted into the plasmid and, once inserted, that it is expressed. Suggest one reason why scientists used different restriction sites at the 5' end and 3' end of the gene for inserting the gene into the plasmid. Name the enzyme used to join the cut ends of the gene to the cut ends of the plasmid.
9700_m20_qp_42
THEORY
2020
Paper 4, Variant 2
View
Use Describe the role of insulin in the regulation of blood glucose concentration. Question 4 continues on page 8 Examiner’s Use shows some of the steps involved in the production of bacteria capable of synthesising human insulin. mRNA for human insulin isolated DNA coding for human insulin produced DNA coding for human insulin cloned DNA coding for human insulin inserted into a plasmid vector plasmid vector inserted into bacterium State the role of each of the following enzymes in the production of bacteria capable of synthesising human insulin, reverse transcriptase DNA polymerase restriction enzymes (restriction endonucleases) DNA ligase.
9700_s07_qp_4
THEORY
2007
Paper 4, Variant 0
View
Describe the role of insulin in the regulation of blood glucose concentration. State two advantages of treating diabetes with insulin produced by gene technology. One of the steps in the production of bacteria capable of producing human insulin is the insertion of the gene coding for human insulin into a plasmid vector. shows one of the artificial plasmids constructed to act as a vector. ampicillin resistance gene DNA of plasmid tetracycline resistance gene target site for the restriction enzyme BamHI G G A T C C C C T A G G With reference to , explain the importance of the plasmid having a single target site for a particular restriction enzyme, such as BamHI. The genes for ampicillin resistance and tetracycline resistance on the plasmid allow the genetic engineer to distinguish between bacteria that have taken up different circles of DNA. Complete the table to show whether bacteria which have taken up each different circle of DNA are, or are not resistant to ampicillin, to tetracycline or to both. Show presence of resistance with a tick (✓) and absence of resistance with a cross (✗). circle of DNA taken up by bacteria bacteria resistant to ampicillin bacteria resistant to tetracycline unaltered plasmids recombinant plasmids that have taken up the wanted gene circles of the wanted gene Explain why genes for antibiotic resistance are now rarely used as markers in gene technology. Describe the use of one alternative marker gene that can be used instead of an antibiotic gene.
9700_s09_qp_4
THEORY
2009
Paper 4, Variant 0
View
Questions Discovered
43