19. Genetic technology
A section of Biology, 9700
Listing 10 of 107 questions
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
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The treatment of many diseases has been improved by the use of genetic technology in medicine. Explain what is meant by genetic engineering. Leber Congenital Amaurosis (LCA) is an inherited eye disease. In LCA, the photoreceptor cells in the retina die at an early age. This causes impaired vision (reduced eyesight) in children, which can progress to blindness. Mutations in different genes cause different forms of LCA. One form of this disease, LCA2, is caused by a mutation in the RPE65 gene. Gene therapy has been used to treat LCA2. Outline how an inherited eye disease, such as LCA2, is treated with gene therapy. Suggest why the eye is a suitable organ for gene therapy. LCA10 is a different form of LCA caused by a recessive mutation in the CEP290 gene. This gene codes for the protein CEP290, which is involved in the correct functioning of photoreceptor cells in the retina. The mutation in CEP290 causes an error to be made when the primary transcript is spliced to form messenger RNA (mRNA). The abnormal mRNA that is formed has an extra sequence of RNA nucleotides, known as exon X, between exon 26 and exon 27. Exon X contains a STOP codon. compares the effect of the mutation in CEP290 with the normal gene expression. normal LCA10 exon intron exon section of DNA of CEP290 exon mutation exon exon intron exon primary transcript exon mutation exon mRNA exon exon X exon CEP290 polypeptide product shortened CEP290 polypeptide In 2022, research was carried out into possible treatment of LCA10 using genetic technology. A human clinical trial investigated a treatment of LCA10 using a short RNA nucleotide sequence known as Sepofarsen. Sepofarsen binds to the section of the primary transcript containing the CEP290 mutation so that normal splicing occurs and functional CEP290 protein is synthesised. • People in the clinical trial received regular treatment with Sepofarsen to the eye with the greatest loss of vision (treated eye) for a period of 12 months. • Changes in the light perception (visual acuity) of both eyes were measured using a vision chart. • A negative change in the visual acuity score shows an improvement in visual acuity. shows the results of the clinical trial over 12 months. exon exon Another method being investigated to treat LCA10 is to use a gene editing tool known as the CRISPR/Cas9 system. The CRISPR/Cas9 system uses a short length of RNA called guide RNA. Guide RNA is complementary to the target DNA and is linked to a nuclease enzyme called Cas9. Cas9 breaks phosphodiester bonds in DNA. The cell repair mechanisms repair the cut in DNA after the modification has taken place. • A vector delivers Cas9 and two specific guide RNAs to the photoreceptor cells. • They act on the section of DNA which contains the mutation. • Exon X is no longer added to the mRNA. Explain how this method used to treat LCA10 is an example of gene editing.
9700_w24_qp_42
THEORY
2024
Paper 4, Variant 2
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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
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The steps involved in a method of production of human insulin by gene technology are listed in Table 5.1. The steps are not listed in the correct order. Table 5.1 step description A DNA coding for human insulin inserted into cut plasmid vector B genetically modified bacteria identified C mRNA for human insulin isolated in β cells D plasmid vector inserted into bacterium E genetically modified bacteria cloned F DNA for human insulin cloned G human insulin harvested H cDNA coding for human insulin synthesised Complete Table 5.2 to show the steps in the correct order. Two of the steps have been done for you. Table 5.2 correct order letter of step C D Name the enzymes responsible for the following steps: step A step H Explain two advantages of treating diabetes with human insulin produced by gene technology rather than using insulin from animals.
9700_w11_qp_43
THEORY
2011
Paper 4, Variant 3
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The diagram outlines how a gene coding for human insulin is produced by genetic engineering techniques. mRNA for human insulin gene isolated step 1 step 2 X Y step 3 step 4 single strand of complementary DNA (cDNA) produced single-stranded DNA made into double-stranded DNA additional non-coding DNA added Name the enzymes X and Y. X Y Explain why the starting point in this procedure is mRNA. Suggest why some methods of manufacturing genetically engineered insulin use eukaryotic yeast cells rather than prokaryotic bacterial cells. State three advantages of using human insulin produced by genetic engineering.
9700_w14_qp_43
THEORY
2014
Paper 4, Variant 3
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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
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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
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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
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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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Questions Discovered
107