Principles of genetic technology

Describe the core principles of the polymerase chain reaction (PCR).

Feb/March 2017

The $b2$-globin gene specifies the $b2$-globin polypeptide found in haemoglobin. It exists in two allelic forms, $Hb^{A}$ (normal) and $Hb^{S}$ (sickle cell). The sickle cell allele differs from the normal one because of a base substitution mutation, and that mutation causes one amino acid in the $b2$-globin polypeptide to change. A man and woman who both carry sickle cell trait may decide to have children through IVF. This makes it possible to determine the genotype of embryos by gene testing before implantation. Embryos with the normal genotype can then be chosen and placed into the mother. One method that can be used to gene test an embryo for the $Hb^{S}$ allele is restriction fragment length polymorphism (RFLP) analysis.

Feb/March 2018

Explain what is meant by bioinformatics and outline the role of bioinformatics after the sequencing of the genomes of humans and parasites.

Feb/March 2021

Factor VIII may be produced as a recombinant human protein.

Feb/March 2022

Array comparative genome hybridisation (aCGH) is a method that uses a microarray to examine an entire genome or selected regions of a genome.

Feb/March 2023

Gentamicin is an antibiotic that is used to treat serious bacterial infections in children.

Feb/March 2025

CD28 and CD40 glycoproteins are present on the surface of T-lymphocytes (T-cells). They act as binding sites for cell-signalling molecules and are needed to trigger T-cell cloning during an immune response. A monoclonal antibody (mAb), which could inhibit the CD40 signalling pathway, was made from hamsters by the hybridoma method. Outline the procedure, beginning with a hamster, for producing mAbs that are suitable for use in another mammal, for example a mouse.

May/June 2010

The disc diffusion method can be used to determine how sensitive bacteria are to antibiotics. First, a bacterial inoculum is spread over agar culture plates, then filter paper discs soaked with antibiotic are placed on the agar surface. The plates are incubated. The bacteria form a ‘lawn’ across the agar, but a circular clear area (the zone of inhibition) is seen around any disc where bacterial growth has been prevented. Two bacterial species, A and B, were cultured on separate plates with three kinds of filter paper disc: 1 - no antibiotic (control); 2 - penicillin V, a natural penicillin; 3 - carboxypenicillin, a synthetic penicillin. The appearance of the incubated plates is shown in Fig. 3.1.

May/June 2010

When gold occurs alongside mineral ores such as iron sulfide, the sulfides need to be oxidised in order to free the gold particles. Since the mid 1990s, gold has been obtained from these ores by bioleaching. Appropriate bacteria oxidise iron sulfide to soluble iron sulfate, releasing $\text{Fe}^{3+}$ and $\text{SO}_4^{2-}$ ions. This reaction gives out heat energy, and the temperature inside a heap of ore undergoing bioleaching (a bioheap) may reach $70\,^{\circ}\text{C}$ or above. Table 2.1 gives examples of bacteria used in this bioleaching.

May/June 2011

Variable number tandem repeats (VNTRs) are repeated, non-coding stretches of DNA. The same VNTR is found at the same locus in different people, but the repeat number within that VNTR is not the same in every individual.

May/June 2012

To sequence a gene’s DNA, the DNA is first denatured so that the two strands separate. Next, in the presence of a large supply of each of the four nucleotides, DNA polymerase copies the single-stranded DNA.

May/June 2012

Variable number tandem repeats (VNTRs) are repeating, non-coding stretches of DNA. The same VNTR is found at the same locus in different individuals, but the repeat count in that VNTR differs from person to person.

May/June 2012

Several diseases, including dengue fever, are carried by mosquitoes. In recent years, the number of cases has risen sharply, and this has been associated with the spread of the mosquito, Aedes aegypti. To try to cut down the population of A. aegypti, genetically modified (GM) male mosquitoes were created. One of the genes inserted into these mosquitoes, when activated, makes a protein that is poisonous to mosquitoes. In 2010, GM male mosquitoes were released into the wild in the Cayman Islands and in Malaysia so that they could mate with females. Every offspring produced died while still in the larval stage.

May/June 2013

Fusarium venenatum, a filamentous fungus, may be cultivated in a fermenter and collected as mycoprotein. This food is marketed in several countries. The fungus is maintained in continuous culture in $150000\,\text{dm}^3$ airlift fermenters, where bubbles of compressed air supply oxygen and mix the contents. As it grows as thin, branched filaments, the harvested mycoprotein has a naturally chewy, fibrous texture. About $300\,\text{kg}$ of fungus may be harvested each hour.

May/June 2013

The pink bollworm moth, $\textit{Pectinophora gossypiella}$, is a pest of cotton crops. Its population can be lowered by releasing very 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 three-year period, 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 inserted DNA also included a promoter.

May/June 2013

Describe how penicillin acts on bacteria.

May/June 2014

Describe how penicillin is produced by the batch culture method.

May/June 2014

Describe how penicillin affects bacteria.

May/June 2014

Russian scientists found the fruits of the flowering plant, Silene stenophylla, in the food cache of a ground squirrel burrow within frozen deposits in Siberia. Dating methods indicate that the ground squirrel stored the fruits about $32\,000$ years ago, just before the ground became permanently frozen. Samples of tissue were removed from the fruits and cultured in a nutrient medium. After applying plant hormones to encourage root and shoot formation, $36$ whole plants were obtained. These ‘regenerated’ plants, which appeared identical to each other, flowered and, after cross-pollination, produced seeds that could germinate.

May/June 2014

In order to prepare infertile women for in-vitro fertilisation (IVF), the development and maturation of a number of ovarian follicles must be encouraged. This is achieved by administering daily injections of the glycoprotein hormone follicle stimulating hormone (FSH). Each FSH molecule has quaternary structure and is formed from two different polypeptide chains, $\alpha$ and $\beta$.

May/June 2015

Section B. Choose and answer one question.

May/June 2015

Describe how an enzyme may be immobilised in alginate and discuss the advantages of using an immobilised enzyme.

May/June 2015

When infertile women are being prepared for in-vitro fertilisation (IVF), several ovarian follicles need to be encouraged to grow and mature. This is achieved by giving daily injections of the glycoprotein hormone follicle stimulating hormone (FSH). Each FSH molecule has quaternary structure and is built from two different polypeptide chains, $\alpha$ and $\beta$.

May/June 2015

Answer any one question.

May/June 2015

Malaria is a severe, often deadly infectious disease caused by Plasmodium. Medicines such as chloroquine are commonly used to lower the chance of malaria and to treat patients who have been infected. However, in many regions of the world, Plasmodium populations have developed resistance to chloroquine. Sequencing the Plasmodium genome, together with bioinformatics, has identified several new targets for the development of anti-malarial drugs.

May/June 2016

Malaria is a dangerous disease that is often fatal and is spread by the mosquito Anopheles gambiae. One way to lower the incidence of malaria is to reduce the population size of these mosquitoes. In mosquitoes, as in humans, males carry one X chromosome and one Y chromosome, whereas females possess two X chromosomes. Researchers explored whether they could produce genetically modified (GM), fertile male mosquitoes in which most sperm carried a Y chromosome rather than an X chromosome. They predicted that releasing these males into an A. gambiae population could sharply cut the number of females in each generation and so lower the number of eggs laid.

May/June 2016

Malaria is a severe infectious disease that is often fatal and is caused by Plasmodium. Medicines such as chloroquine are commonly used both to lower the chance of malaria infection and to treat people who have already been infected. However, in many regions of the world, Plasmodium populations have developed resistance to chloroquine. Determining the genome sequence of Plasmodium and using bioinformatics has revealed a number of new targets for the development of anti-malarial drugs.

May/June 2016

Oil seed rape (canola), Brassica napus, has been genetically altered so that it is resistant to herbicides containing glufosinate ammonium. The genetically modified (GM) oil seed rape carries the bar gene, which came from a soil bacterium. This gene codes for an enzyme that changes glufosinate ammonium into a non-toxic compound.

May/June 2017

Oil seed rape (canola), $\textit{Brassica napus}$, has been genetically altered so that it is resistant to herbicides containing glufosinate ammonium. The genetically modified (GM) oil seed rape carries the $bar$ gene, which was obtained from a soil bacterium. This gene codes for an enzyme that changes glufosinate ammonium into a non-toxic compound.

May/June 2017

Soybean, Glycine max, is a major crop used for human food and for animal feed. Two soybean varieties are Vinton 81 and GTS 40-3-2. Vinton 81 was produced by the conventional method of selective breeding (artificial selection), whereas GTS 40-3-2 is an example of a genetically modified (GM) organism.

May/June 2019

Describe how the polymerase chain reaction is used to clone and amplify DNA.

May/June 2020

Section B. Answer one question.

May/June 2020

Genetic engineering uses naturally occurring enzymes and biological processes to alter genetic material. List the enzymes used in genetic engineering (genetic modification) and outline what each does in natural processes.

May/June 2021

In 1973, a genetic engineering method was employed for the first time. Recombinant DNA was produced with a plasmid, and this was then successfully introduced into an organism. By 2012, a different genetic engineering method, known as gene editing, had been developed.

May/June 2022

The first use of a technique for genetic engineering took place in 1973. Recombinant DNA was produced with the aid of a plasmid, and it was then transferred successfully into an organism. A later technique for genetic engineering, known as gene editing, was created in 2012.

May/June 2022

Genetic technology makes use of a wide range of enzymes and techniques.

May/June 2023

White-clawed crayfish, Austropotamobius pallipes, are found in rivers and lakes across Europe. In the 1850s, the North American signal crayfish, Pacifastacus leniusculus, was brought into Europe. The introduced species carried a pathogen that leads to a disease called crayfish plague. This disease kills A. pallipes. Since 1850, the population size of A. pallipes has fallen in many parts of Europe because crayfish plague has spread.

May/June 2024

Genetic engineering is a method for altering the genetic material of a chosen organism in order to change a characteristic.

May/June 2024

Some human diseases can be managed with recombinant human proteins. These proteins are made using recombinant DNA technology.

May/June 2025

Recombinant DNA technology is employed to produce recombinant human proteins. Two methods that can be used to obtain the gene of interest are: • excising the gene from genomic DNA with restriction enzymes • collecting messenger RNA (mRNA) from cells expressing the gene, then using reverse transcriptase to synthesise complementary DNA (cDNA). Plasmids may act as vectors to move the gene of interest into a host organism.

May/June 2025

Genetic engineering often involves transferring a gene into an organism.

May/June 2025

Insulin is secreted by the islets of Langerhans in the pancreas, and this causes the liver to lower the blood glucose concentration.

Oct/Nov 2010

The islets of Langerhans in the pancreas secrete insulin, and this causes the liver to lower the blood glucose concentration.

Oct/Nov 2010

Fig. 3.1 illustrates a way of producing monoclonal antibodies.

Oct/Nov 2010

Green fluorescent protein (GFP) is a compact protein that produces bright green fluorescence under 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 commonly used as a marker to indicate successful uptake of a gene by the bacterium.

Oct/Nov 2012

Green fluorescent protein (GFP) is a compact protein that gives off intense green fluorescence when it is illuminated with blue light. It was first isolated from the jellyfish, $Aequorea$ $victoria.$ The gene coding for GFP can be expressed in bacteria, such as $Escherichia$ $coli,$ so it is commonly used as a marker to indicate that a gene has been successfully taken up by the bacterium.

Oct/Nov 2012

The artificial plasmid pBR322 was designed to function as a vector. It has frequently been used to transfer human genes, for example the human insulin gene, into the bacterium Escherichia coli. This plasmid was made with two genes, each conferring resistance to a different antibiotic: an ampicillin resistance gene and a tetracycline resistance gene. It also contains a recognition site for the restriction enzyme, BamHI, positioned in the centre of the tetracycline resistance gene. A pBR322 plasmid was cut with BamHI and the cDNA gene for human insulin was inserted into it. Fig. 2.1 shows pBR322 and the recombinant plasmid.

Oct/Nov 2012

The waste water from the textile industry may contain azo-dyes, which are used to provide colour to textiles. Azo-dyes are organic pollutants and may be carcinogenic. White-rot fungi are valuable in environmental pollution treatment because they release extracellular enzymes that can decompose a range of organic pollutants, including azo-dyes. The extracellular enzymes made by white-rot fungi are primary metabolites that are produced in large quantities in continuous culture.

Oct/Nov 2014

Gold ions ($\text{Au}^{3+}$) are poisonous to most microorganisms. Even so, the bacterium Delftia acidovorans is often found in layers known as biofilms that develop on the surface of gold deposits. D. acidovorans makes a peptide synthase that catalyses the formation of a small peptide named delftibactin. Once isolated, delftibactin can cause $\text{Au}^{3+}$ ions to precipitate as tiny particles of metallic gold.

Oct/Nov 2015

Gold ions ($\text{Au}^{3+}$) are poisonous to most microorganisms. Even so, the bacterium \emph{Delftia acidovorans} is often present in slimy coatings, known as biofilms, that develop on the surfaces of gold deposits. \emph{D. acidovorans} makes a peptide synthase that catalyses the synthesis of a small peptide called delftibactin. Once isolated, delftibactin can cause $\text{Au}^{3+}$ ions to precipitate as tiny particles of metallic gold. Delftibactin is a secondary metabolite.

Oct/Nov 2015

The blackcap, Sylvia atricapilla, is a small song bird. It visits parts of northern Europe in summer, where it reproduces. A large number of blackcaps spend the winter (overwinter) in southern Europe, especially in Spain. Because many people leave food out for garden birds, some birds are able to get through the winter in the UK. Scientists compared the genetic variation among blackcaps from two woodland sites in Germany that were 800 km apart. At both sites there were birds that had overwintered in Spain and birds that had overwintered in the UK. The measurements were taken soon after the birds came back from their winter feeding grounds.

Oct/Nov 2015

PCR, or the polymerase chain reaction, is a technique for making very large quantities of DNA from a tiny starting sample. Fig. 3.1 shows the main stages of PCR.

Oct/Nov 2016

The polymerase chain reaction (PCR) is used to make large quantities of DNA from a very small starting sample. The main stages of a PCR are shown in Fig. 3.1.

Oct/Nov 2016

Vitamin A deficiency is a serious health issue in many parts of the world where children eat a restricted diet. Rice enriched with pro-vitamin A has been created by genetic engineering. This rice, named Golden Rice, contains high levels of $\beta$-carotene, which the human body uses to make vitamin A. From this Golden Rice, newer Golden Rice varieties have been developed by selective breeding. Fig. 3.1 illustrates how Golden Rice was first produced by genetic engineering.

Oct/Nov 2016

Leber’s congenital amaurosis (LCA) is an autosomal recessive eye disease. It causes eye disorders present from birth, including severe loss of vision. LCA has been treated successfully by gene therapy, using a virus rather than a plasmid as the vector. Adeno-associated virus (AAV) vectors carrying the therapeutic allele were injected straight into the retina, the layer at the back of the eye that contains the photoreceptor cells. People who had been blind from a young age were able to see again. There is a risk linked to the injection technique used to deliver the vectors, because it could make the retina detach and so damage vision. This delivery method was first applied to LCA before being tested on other retinal diseases that gradually reduce people’s vision as they grow older.

Oct/Nov 2018

Conventional methods for genetically modifying organisms rely on three enzymes: restriction endonuclease, reverse transcriptase and DNA ligase. These enzymes have been used to make genetically modified (transgenic) pigs that carry the GFP gene coding for green fluorescent protein, which was originally obtained from jellyfish.

Oct/Nov 2018

Leber’s congenital amaurosis (LCA) is an autosomal recessive disorder of the eye. LCA leads to eye disorders, including a severe reduction in vision at birth. LCA has been treated successfully with gene therapy, with a virus used rather than a plasmid as the vector. Adeno-associated virus (AAV) vectors carrying the therapeutic allele were injected straight into the retina, the layer at the back of the eye that contains the photoreceptor cells. The injection technique used to deliver the vectors carries a risk, because it could make the retina detach and so damage vision.

Oct/Nov 2018

Part B

Oct/Nov 2020

Sickle cell anaemia is a chronic, non-infectious disease. If it is not treated, sickle cell anaemia can cause severe pain and may be life-threatening. Sickle cell anaemia results from a base substitution mutation in the gene that codes for the β-globin polypeptide of haemoglobin. This changes the primary structure of the polypeptide, because valine is found instead of glutamine. As a result, red blood cells become abnormally sickle-shaped and stick to one another inside blood vessels. Symptoms of sickle cell anaemia include painful episodes when red blood cells obstruct capillaries in tissues and organs.

Oct/Nov 2020

Describe and explain how plasmid properties make them suitable for gene cloning.

Oct/Nov 2020

In 1984, geneticist Alec Jeffreys developed a DNA testing method called DNA profiling, which gives a DNA banding pattern on a gel. This banding pattern, or profile, is unique to every individual. DNA profiling is useful in police forensic investigations to catch criminals. Since 1987, police in many countries have taken and kept DNA from crime scenes to build DNA profiles, which they then compare with the DNA profiles of criminal suspects.

Oct/Nov 2021

Genome-wide association studies detect connections between single nucleotide polymorphisms (SNPs) and phenotypic traits such as human diseases. SNPs are positions in DNA that differ within a population because of DNA base substitutions. A genome-wide association study examines how genetic variation affects a disease. A very large number of affected people and a very large number of healthy control individuals supply DNA. Microarray chips are then used to determine each individual's genotype at many SNPs. The Wellcome Trust Case Control Consortium (WTCCC) study was a major genome-wide association study. It used a microarray chip that determined each person's genotype at $500\,000$ different SNPs. The study searched for associations between SNPs and $7$ different diseases. For each disease, $2000$ people with the disease were tested. Their findings were compared with those from $3000$ healthy control individuals.

Oct/Nov 2021

In 1984, the geneticist Alec Jeffreys developed a DNA testing method called DNA profiling, which creates a DNA banding pattern on a gel. This banding pattern, or profile, is different for every individual. DNA profiling is used in police forensic work to identify criminals. Since 1987, police in many countries have gathered and stored DNA from crime scenes to build DNA profiles, then compared these with the DNA profiles of criminal suspects.

Oct/Nov 2021

Myosotis is a genus of small flowering plants. Many Myosotis species occur on the islands of New Zealand, which are an important centre for Myosotis evolution. Lowland Myosotis species live at low altitude, whereas alpine Myosotis species live at high altitude on mountain summits.

Oct/Nov 2022

A marker gene may be used when a gene of interest is inserted into a plant by genetic engineering. Describe and explain how a marker gene that codes for a fluorescent product can show that the introduced gene of interest is being expressed in plants.

Oct/Nov 2023

Scientists employ a variety of different techniques in genetic engineering.

Oct/Nov 2024

Scientists employ a variety of methods in genetic engineering.

Oct/Nov 2024

Metachromatic leukodystrophy (MLD) is a genetic disorder that affects the nervous system. MLD results from mutations in the ARSA gene on chromosome 22. The ARSA gene, which has a length of 3150 base pairs (bp), contains 8 exons and is shown in Fig. 5.1. A genetic test using DNA sequencing is available to detect mutations linked with MLD in the ARSA gene. The sequencing method can operate only when a DNA fragment is shorter than 1000 bp. The test has several stages: genomic DNA is extracted from a blood sample; polymerase chain reaction (PCR) with 5 pairs of primers isolates 5 separate DNA fragments of the ARSA gene; gel electrophoresis is performed on the DNA fragments; the DNA fragments are sequenced and examined for mutations. Table 5.1 gives the lengths of the DNA fragments and the exons contained in each DNA fragment.

Oct/Nov 2025