2010 Prelim 3 Essay Compilation

2010 CJC
4 (a) Describe how gel electrophoresis can be used to analysis nucleic acids. [8]
1. A loading dye is added to the DNA samples, which allows the process of electrophoresis to be tracked / helps DNA to sink into the wells.
2. DNA fragments are placed in wells at one end of an agarose gel with a micropipette.
3. Gel is submerged in a buffer solution that will conduct electricity.
4. An electric current is applied though electrodes at opposite ends of the gel.
5. DNA is negatively charged and moves towards the anode/positive electrode.
6. Rate of movement / speed / distance traveled in a given time depends on size of DNA fragment.
7. Gel electrophoresis separates DNA fragments by size / molecular weights, with the smallest fragments moving the fastest and furthest as the smaller fragments have lower resistance in moving through the pores of the gel, whereas the larger fragments move slower due to higher resistance.
8. A DNA ladder using known lengths of DNA can be used to calibrate the gel.
9. Ethidium bromide or fluorescent dyes can be used to bind to the invisible DNA fragments and UV light is used to visualize the DNA bands.
10. Complementary radioactive or fluorescent probes can be used to identify specific bands or sequences.
11. This enables identification of different alleles or mutations of genes and allows sequencing of DNA.


(b) With reference to a named example, explain how RFLP can be used in disease detection. [7]
1. Human DNA is cut with a restriction enzyme (DdeI) was subjected to agarose gel electrophoresis followed by Southern blotting to transfer the DNA to a nylon membrane.
2. This is followed by hybridisation with a radioactive probe specific for the β-globin gene.
3. The membrane is laid under photographic film, allowing any radioactive areas to expose the film (autoradiography).
4. Black bands on the film correspond to the locations on the membrane where DNA has hybridized to the probe.
5. Differences in DNA fragment sizes (polymorphisms) are detected by autoradiography depending on where the probe binds to, i.e. it binds to complementary sequences in the β- globin gene.
6. A healthy person with two copies of the normal β-globin allele (βAβA) shows two bands at 175 and 201 bp.
7. A person with sickle cell anaemia (βSβS) would have two copies of the mutant β-globin allele and show one band at 376 bp.
8. A person considered a “carrier” would have one normal and one defective β-globin allele (AS) but would not have sickle cell anaemia because there is one functioning copy of the β-globin allele. For the carrier, bands show at 376, 201 and 175 bp.


(c) Discuss the ethical concerns that have arisen about the human genome project. [5]

Statements made must be accompanied with some discussion.
1) Privacy and confidentiality of genetic information
• difficult to determine who owns and controls genetic information;

2) Fairness in the use of genetic information
• by insurance companies, employers, courts, schools, adoption agencies, and the military;
• difficult to determine who should have access to personal genetic information and how it will be used;
3) Commercialization of products
• including property rights (patents, copyrights, and trade secrets) and accessibility of data and materials;
• difficult to determine who owns genes and other pieces of DNA;
• difficult to ascertain if patenting DNA sequences will limit their accessibility and development into useful products;

4) Fairness in access to advanced genomic technologies.
• difficult to ascertain who will benefit or if it will result in major worldwide inequities;

5) Clinical issues
• including the education of doctors and other health-service providers, people identified with genetic conditions, and the general public about capabilities, limitations, and social risks; and implementation of standards and quality control measures;
• problems associated with the evaluation and regulation for accuracy, reliability and utility of genetic tests;
• the need to prepare healthcare professionals for the new genetics as well as the public to make informed choices;

6) Uncertainties associated with gene tests for susceptibilities and complex conditions
• e.g., heart disease, diabetes, and Alzheimer’s disease – questions related to whether:
 tests should be performed when no treatment is available;
 parents have the rights to have their minor children tested for adult-onset diseases;
 genetic tests are reliable and interpretable by the medical community;

7) Reproductive issues
• including adequate and informed consent and use of genetic information in reproductive decision making;
• possibility of improper counselling of parents by healthcare personnel about the risks and limitations of genetic technology;
• difficult to determine the reliability and usefulness of fetal genetic tests;

8) Psychological impact, stigmatization, and discrimination
• due to an individual’s genetic differences;
• possibility that personal genetic information as perceived by society, may affect an individual in various ways;

9) Conceptual and philosophical implications
• regarding human responsibility, free will vs genetic determinism, and concepts of health and disease;
• some of the related perceptions and uncertainties include:
 whether behaviour is determined by genes;
 what is the acceptable level of diversity;
 whether there is a clear distinction between medical treatment and enhancement;
[Total: 20]

2010 YJC
5 (a) Discuss the uses of plant tissue culture. [4]
The term plant tissue culture refers to the aseptic growing of excised plant parts in vitro.

1. Rapid production; of large numbers; of transgenic plants (transgenic/normal) from just one or a few stock plants can be achieved; through asexual means;

2. Plant diseases can be avoided. Production of virus-free plants; can be obtained by selecting only meristematic tissue of the transgenic plant for propagation;

3. If a new gene is introduced into a plant cell by genetic engineerin;, the modified cell can be grown into a whole plant; and then cloned to produce many new plants/mass production;, all containing that gene (transgenic plants) and are genetically identical;.

Recall that for transgenic plants to be made:
4. These transgenic plants can be produced at any time of the year;
a. can be put in cold storage, taking up relatively little space;
b. combined with rapid production, this gives great flexibility in supplying consumer demand;
c. plants can be produced out of season and people can buy plants at lower prices than usual;

5. Land are needed to grow these transgenic plants through tissue culture is much less; save space;.

6. Reliability; and quality control can be achieved; since growing conditions are standardized; and batch after batch of standard plants are produced.


As compared to normal reproduction: _____________________
7. Prevents “loss” of transgene due to sexual reproduction / through gamete formation; maintenance of transgene within entire crop;

8. Prevents transgenic plants from being completely wiped out by changing environmental factors; as the plants are always available as stored in the cold storage;

The transgenic plantlets produced are light and small in size; so they can be air-freighted and transported easily and cheap;, thus increasinginternational trade

(b) Discuss briefly, with specific examples, desirable genes that have been genetically engineered into crop plants and animals used as food. [7]

Genetic engineering serves to bring in desirable characteristics from other species (unrelated) to add characteristics to enhance the quality of the organism. It can be to

Choose any three
development of resistance to viral, bacterial, and fungal diseases; E.g. Development of plants resistant to pest / BTcorn resistant to corn borers (or Bt tomato). Plants will express the Bt toxin gene from the bacterium B. thuringiensis

Transgenic plants with important traits such as herbicide resistance can be introduced. 1. E.g., glyphosate-resistant soybean (Round-Up Ready ™)

The plants have been engineered to readily degrade the herbicide. Spraying of herbicides will kill weeds but not affect crop thus reducing competition for nutrients/light. Herbicide resistant plant  can spray herbicides to kill weeds without affecting crops


Enhancement of nutritional values. E.g., Golden rice with genes for enzymes converting a natural compound in rice to beta-carotene (e.g., daffodil)  rich in beta-carotene. Golden rice has been genetically engineered to produce beta-carotene a pre-cursor of vitamin A is a such a product. Vitamin A deficiency leads to blindness and susceptibility to disease


(c) Discuss the social and ethical concerns pertaining to the use of genetically modified animals. [4]

1. Environmental protection from GMOs; safeguards of products with GM components may not be adequate; to prevent transfer of genes/pollen from GM organisms to wild-type organisms;

2. Unknown effects of GM products on human health and disease; effects may only be known after long period of exposure; allergic reactions may occur if people unknowingly consume products containing introduced genes;

3. Tampering with nature / “playing God”; by mixing genes among species; genetic modifications of animals especially resulting in enhancement; draws parallels that human genetic modification for enhancement one day may occur;

4. Improper human use of animals - violations of animal rights; cruelty to animals; results in animal deformity / disease; (OWTTE) GM animals may suffer unnecessarily.E.g., oncomouse which may increased susceptibility to cancer.

5. Introduction of animal genes to plants; / porcine genes to other organisms; may result in objections from vegetarian; / specific religious groups; respectively especially when GM foods are unlabelled

6. Accountability of biotechnological and agricultural firms wrt to GMOs; labeling issues; Ownerships issues – unfair for large multinational companies to patent GM animals.

7. Release (accidental or otherwise) of GM animals into the wild may result in GM animals outcompeting wild types such that ecological balance is disrupted.E.g., larger transgenic salmon may be preferably selected as mates over smaller wild types.

8. Introduction of foreign gene(s) may result in production of secondary metabolites that may be toxic to animals themselves and/or livestock/humans that consume them.New proteins in GM animals may be potentially allergenic to humans that consume them

All other logical points accepted if appropriate elaboration given
2010 DHS
4 (a) Discuss the advantages and evolutionary consequences of using plant tissue culture to propagate transgenic plants. [10]
(b) Explain, with reference to Bt corn, the significance of genetic engineering in improving the world food situation. [6]
(c) Discuss the ethical issues involved with the production of genetically modified organisms. [4]

Advantages
1. Tthe modified cell can be grown into a whole plant and then cloned to produce many new plants/mass production, all containing that gene (transgenic plants) and are genetically identical.
2. Rapid production of large numbers of transgenic plants from just one or a few stock plants can be achieved through asexual means.
3. Plant diseases can be avoided / production of virus-free plants.
4. These transgenic plants can be produced at any time of the year and put in cold storage, taking up relatively little space. Combined with rapid production, this gives great flexibility in supplying consumer demand as plants can be produced out of season and people can buy plants at lower prices than usual.
5. Reliability and quality control can be achieved since growing conditions are standardised and batch after batch of standard plants are produced.
6. Land area needed to grow these transgenic plants through tissue culture is much less /saves space.
7. Prevents “loss” of transgene due to sexual reproduction / through gamete formation /maintenance of transgene within entire crop.
8. Prevents transgenic plants from being completely wiped out by changing environmental factors as the plants are always available as stored in the cold storage.
9. The transgenic plantlets produced are light and small in size so they can be air-freighted and transported easily and cheaply.
10. Produces rooted plantlets that are ready for growth.
11. Plantlets of a particular desired sex can be grown selectively.
Max 6

Evolutionary consequences
12. Desirable / beneficial genes are quickly passed on to subsequent generations / ensures integrity and maintenance of genetic composition as the transgenic plants can propagate rapidly under favourable environment.
13. Lack of genetic variation posed a problem to the survival of a species / unable to respond to unfavourable changes in selection pressure as a result of changing environment. Low evolutionary potential / No variation unless mutation occurs, thus no speciation / natural selection cannot occur without variation.
14. Thus there is a high risk of extinction of that species as all individuals are genetically identical / equally susceptible to pathogens / succumb to outbreak of epidemics / disease.
15. Decrease in gene pool due to loss of beneficial alleles.
16. Such transgenic plants produced, when reintroduced into the wild, might cross-pollinate and these might cause production of pesticide/herbicide resistance / generating “superweeds” that will disrupt the ecological balance / ecosystem.
Max 5
Overall max 10

(b)
1. Genetic engineering allows these organisms to acquire by genes from the same or different species that improves crop yield or nutritional value.
2. Bt delta endotoxin genes from Bacillus thuringiensis is inserted into corn crops via use of Ti plasmid of Agrobacterium tumefaciens.
3. Protein produced kills the larvae of the Lepidoptera species, including the European corn borer due to damage caused to the gut wall; hence Bt endotoxin specifically acts on species that ingest corn.
4. Hence they are unable to cause further damage to crops, this prevents yield loss due to corn borers.
5. Use of Bt corn reduces need for pesticide applications, hence reducing cost, labour, environmental damage and non-specific harming of other insect species.
6. This allows farmers to concentrate resources on aspects like fertilizers, improved farming practices etc. that result in further improved crop yield.
7. Implementation in developing countries, however, may not alleviate problems of world food situation due to unequal food distribution / prohibitive cost of GM seeds / public distrust of GMOs.
Max 6

(c)
1. Environmental protection / safeguards of products with GM components may not be adequate to prevent transfer of genes/pollen from GM organisms to wild-type organisms.
2. Unknown effects of GM products on human health and disease – effects may only be known after long period of exposure / allergic reactions may occur if people unknowingly consume products containing introduced genes;
3. Tampering with nature by mixing genes among species / genetic modifications of animals especially resulting in enhancement draws parallels that human genetic modification for enhancement one day may occur.
4. Improper human use of animals - violations of animal rights / cruelty to animals / results in animal deformity / disease.
5. Introduction of animal genes to plants / porcine genes to other organisms may result in objections from vegetarian / specific religious groups respectively.
6. Accountability of biotechnological and agricultural firms with relation to GMOs.
7. Labeling issues.
Max 4

2010 IJC
5 (a) Explain how gel electrophoresis is used to analyse DNA. [6]
1. agarose gel acting as molecular sieve to separate fragments based on molecular size;;
2. distance travelled by band is inversely proportional to molecular size;;
3. presence of TAE/TBE buffer provide the ions to support conductivity;;
4. application of direct current enables the neg charged DNA to travel to the positive electrode;;
5. gel stained with methylene blue to reveal positions of DNA bands;;
6. relative thickness of band is an indication of the amount of DNA with that molecular size;;
(b) Outline the process of nucleic acid hybridisation and explain how it can be used to detect and analyse restriction fragment length polymorphism (RFLP). [5]
1. Lay nitrocellulose paper on the electrophoresed gel submerged in NaOH with a stack of paper towels;;
2. dsDNA denatured into ssDNA, adhere to nitrocellulose paper;;
3. placed into bag with radioactive ssDNA probe with complementary sequences to target sequences;;
4. Excess probe washed off and placed on X-ray film, hybridized areas will show up as dark bands;;
5. banding patterns is an indication of number of fragments and fragment length that can be compared to other DNA samples;;

(c) Explain how RFLP analysis facilitated the process of linkage mapping, diseases detection and DNA fingerprinting. [9]
1. RFLP is based on variation in no. & length of restriction fragments btw individuals visible through gel electrophoresis / nucleic acid hybridization / Southern blotting;;
Linkage Map
2. shows the relationship between 2 or more genes located on the same chromosome
3. tightly linked RFLP with genes are used as DNA markers for respective genes where linkage are calculated based on the recombinant phenotype freq;;
Disease detection
4. disease causing allele differs from normal allele due to mutation, may result in the lost/creation of a restriction site in the mutated allele;;
5. RFLP marker is closely linked to or within disease causing allele, resultant fragments from RE digestion can indicate presence / inheritance of specific allele;;
DNA fingerprinting
6. DNA profiling process identifying individuals based on diff in repetitive DNA sequence e.g. VNTR
7. Different number of repeats within a restriction site results variable length of fragments formed after RE digestion;;
8. Analysis is based on matching DNA fingerprint between samples;;
9. perfect match in parentage identification/ criminal analysis or degree of similarity in evolutionary studies;;

[Total: 20]

2010 JJC
(a) With reference to the analysis of DNA molecules, describe the process and explain the use of Gel Electrophoresis and Nucleic Acid Hybridisation. [10]

Gel Electrophoresis
1. DNA fragments are loaded in a well;
2. at one end of the agarose gel;
3. Electric field or voltage applied across the gel;
4. DNA being negatively charged;
5. will move towards the positive electrode/anode;
6. DNA ladder is added to calibrate the size of DNA;
7. Distance traveled in a given time depends on the molecular weight of the DNA with larger DNA fragments travel slower and smaller fragments traveling faster;
8. More resistance for the larger fragment to move through the pores of the gel;
9. Ethidium Bromide and UV light/ fluorescent dyes are use to see the DNA bands;
10. This allows for separation of DNA fragments and identification of different alleles/ two different DNA molecules;;
[max 4m for description and 1 m for analysis of DNA]

Nuclei Acid Hybridisation
11. DNA fragments in the gel is denatured by alkaline solution;
12. resulting in single stranded DNA;
13. then transferred to the nitrocellulose membrane via capillary action;
14. a single stranded probe;
15. which consists of a radioisotope and short nucleotide sequence/oligonucleotide radioactively labeled probe;
16. which is complementary to the DNA sequence of the target DNA fragment is added;
17. the probe hybridise to the target fragment;
18. excess probe is wash off;
19. the membrane is then exposed to a X-ray film;
20. the region where probe bind to will give a dark band;
21. This allows for identification of different alleles/ two different DNA molecules/ detect and analyse Restriction Fragment Length Polymorphisms;;
[Any 4 points -4m for description and 1 m for analysis of DNA]

(b) Discuss the uses of Restriction Fragment Length Polymorphisms (RFLP) analysis.[10]

1. RFLP – refers to the genetic variation that is observed in the size of the DNA fragments that is obtained after the DNA (of different individuals) is digested by a specific restriction enzyme;
2. Different sized fragments can be separated by gel electrophoresis;
3. Followed by nucleic acid hybridization to make the bands visible;
Max 1

Disease detection;
4. Indirect screening for disease-causing alleles;
5. Due to loss or gain of restriction sites/ changes in DNA sequences that affects a restriction site;
6. Generating different size restriction fragments;
7. Use of RFLP as proxy for genetic diseases because the marker is closely linked to disease-causing allele;
8. Inherited together as crossing over is unlikely;
9. inheritance of the a particular RFLP can indicate the inheritance of the disease causing allele;
Max 3

Genomic mapping;
10. Use of RFLP as genetic markers;
11. The frequency with which two RFLP markers or an RFLP marker and a certain allele for a gene is inherited together;
12. is a measure of the closeness of the 2 gene loci on a chromosome /relative distance between the 2 gene loci;
13. Based on recombination frequencies/ frequency of crossing over between 2 RFLP markers/ cross-over values;
14. Can be used to construct linkage map;
Max 3

DNA fingerprinting/ genetic fingerprinting/ DNA profiling;
15. Ref to VNTR (variable number of tandem repeats or STR (short tandem repeats);
16. Different VNTR have different lengths;
17. VNTR highly polymorphic;
18. Each individual’s combination is unique, having inherited one VNTR locus from each parent;
19. Use to identify criminals/ determine paternity/ identify endangered animals etc;
Reject answers that are not clear eg. genetic fingerprinting use for criminal cases
Max 3
[Total: 20]

2010 PJC


(a) Question 4

Explain how RFLP analysis is used to support the process of linkage mapping.

1. A linkage map shows the relative location;
2. or the order of genes along a chromosome;
3. constructed on the assumption that the probability of a crossover between two genetic loci is proportional to the distance separating the loci;
4. Linkage maps are usually constructed with several thousand known genetic markers spaced evenly throughout the genome.
5. The RFLP markers can be any genes or any other identifiable DNA sequences, such as variation number tandem repeats (VNTR).
6. Linkage map must be done based on experimental crosses;
7. RFLP from parental and offspring organisms are analysed;
8. using Southern blot;
9. RFLP pattern obtained are used to calculate the total percentage of recombinants;
10. Give an indication of the distance between the RFLP sequences based on the recombination frequencies obtained;
11. the farther apart the two RFLP sequences are, the higher the probability that a crossover will occur between them and therefore
12. the higher the recombination frequency;
13. For example, if 70% of the progeny produced are parental and 30% were recombinant, the RFLP loci are 30 centi-Morgans (cM) apart from each other (1 mark for example)

(b) Describe how the micropropagation technique is used in cloning of plants.

1. In micropropagation, plant tissues or explants eg: meristematic cells are removed from plants ® explants only;
2. To produce thousands of plantlets which are clones of the original plant;
3. Surface of explants sterilised with dilute sodium hypochlorite (Clorox) to kill bacterial and fungal pathogens or organisms;
4. And grown in a containing sterile media containing nutrients and hormones needed for plant growth;
5. Eg: inorganic ions such as nitrogen,magnesium, iron, potassium), vitamins, carbohydrate source and
6. plant growth substances such as auxin and cytokinins
7. Containers holding the explants are then sealed and incubated for 1-9 weeks.
8. Conditions like temperature, light intensity and humidity are carefully controlled;
9. During this period, the cultured cells divide by mitosis
10. to form a mass of undifferentiated tissue called a callus.
11. As callus increases in size, pieces of callus is sliced off and grown on new medium composition (subculturing);
12. By adjusting concentration of plant hormones in growth medium,
13. cells in callus can be induced to differentiate into roots and shoots;
14. Further growth being encouraged by the use of plant growth substances until plantlets are large enough
15. To be weaned and planted in sterile soil in green house
16. After acclimatization in green house, the plant are transferred to soil for field planting

(c) Explain how gel electrophoresis can be used to provide evidence of molecular homology.

Must make the link to explain how you can compare samples from different organisms in order to establish molecular homology.

1. Define molecular homology: the similarities in DNA nucleotides and/or amino acid sequence of organisms.
2. the more closely related organisms are, the more similarities they share;
3. Gel electrophoresis is used as a tool to analyse the similarities in DNA sequences between organisms;
4. Isolate genomic DNA from different organisms;
5. Obtain cytochrome c or haemoglobin DNA as starting material using PCR using specific primers;
6. Cut amplified sample DNA using the same restriction enzyme
7. A loading dye is added to the DNA samples, which allows the process of electrophoresis to be tracked/ helps DNA to sink into the wells;
8. DNA standard / ladder is loaded to enable estimation of the sizes of the DNA bands obtained
9. DNA samples loaded into wells at negative end of an agarose gel;
10. Gel is submerged in a buffer solution that will conduct electricity;
11. An electric current is applied through electrodes at opposite ends of the gel;
12. Negatively-charged DNA fragments migrate to the positively charged electrode/anode;
13. Gel electrophoresis separates DNA fragments by size/ molecular weight;
14. with the smallest fragments moving the fastest and furthest;
15. as the smaller fragments have lower resistance in moving through the pores of the gel;
16. whereas the larger fragments move slower due to higher resistance;
17. invisible DNA bands can be viewed under UV light by staining the gel with a dye such as ethidium bromide;
18. Two samples of DNA with similar maps for the locations of restriction sites will produce similar banding patterns;
19. In contrast, two genomes that have diverged extensively since their last common ancestor will have a very different distribution of restriction sites, and the DNA will not match closely in the sites of restriction fragments.




2010 SAJC
(a) Discuss with specific examples, how desirable genes have been genetically engineered into crop plants and animals that are used as food. [7]

1 ref. gene transfer techniques for plants, eg. electroporation, liposomes on protoplast, Ti plasmid (not in syllabus), gene gun (not in syllabus)
2 ref. gene transfer techniques for animals, eg. microinjection, electroporation, liposomes
3 ref. plant tissue culture techniques
4 ref. insertion of gene construct into animal embryos

Animals with faster growth rate
5 Atlantic salmon with more active salmon growth hormone gene
6 grow to its full length in a shorter period of time
OR
7 Cattle with bigger muscles
8 more meat per animal

Pest-resistant plants
9 Eg. Corn / potato / broccoli / tomato plants which can produce the Bt toxin;
10 express the Bt toxin gene from the bacteria Bacillus thuringiensis;
11 specifically kills insect pests
12 crop losses can be reduced, leading increased profits in agriculture

Herbicide resistant plants
13 E.g. Glyphosate-resistant soybean / tomato
14 glyphosate works by inhibiting an enzyme EPSP synthetase, which plants require to make essential aromatic amino acids
15 can spray herbicides to kill weeds without affecting crops
16 crop losses can be reduced, leading increased profits in agriculture

Plants with improved nutritional qualities
17 E.g. Golden rice enriched with beta-carotene
18 produced by transplanting genes from daffodil and bacteria
19 help prevent Vitamin A deficiency which leads to blindness and susceptibility to disease

Plants with delayed ripening
20 Eg. Flavr-Savr tomato
21 polygalacturonase normally responsible for the ripening process
22 has antisense gene of enzyme polygalacturonase binds to the polygalacturonase mRNA, preventing it from being expressed
23 delay in fruit ripening / spoiling during transport / improved shelf life
24 larger and has greater flavour

OR
25 blocking the biosynthetic pathway for ethene
26 ethene is hormone responsible for fruit ripening
27 fruits can be allowed to grow on the vine for a longer period of time,
28 delay in fruit ripening / spoiling during transport / improved shelf life
29 larger and has greater flavour


(b) Discuss the social and ethical concerns pertaining to the use of genetically modified animals. [8]
1 release (accidental or otherwise) of GM animals into the wild;
2 may result in GM animals out competing wild types;
3 ecological balance is disrupted / severe impacts on the food-chain;
4 destabilises & hence threatens biodiversity;
5 E.g. larger transgenic salmon may be preferably selected as mates over smaller wild types;

6 introduction of foreign gene(s);
7 may result in production of secondary metabolites;
8 maybe toxic to animals themselves and/or livestock/humans that consume them;
9 new proteins in GM animals;
10 may be potentially allergenic to humans that consume them;

11 animal rights – GM animals may suffer unnecessarily;
12 E.g. oncomouse;
13 which may increased susceptibility to cancer;
14 rearing animals for transplant purposes;

15 religious implications in food choice;
16 especially when GM foods are unlabelled;
17 E.g. incorporation of pig genes into cows;
18 ownership issues – unfair for large multinational companies to patent GM animals / increased dependence of undeveloped countries on rich developed countries

2010 TPJC
(a) Explain the use of Restriction Fragment Length Polymorphism in disease detection and genomic mapping. [8]
Disease detection
1 RFLP is based on variation in the length of DNA sequence (variation in nucleotide sequences or VNTR) in an allele or non-coding region;
2 The different patterns of restriction fragments/genetic fingerprint of individuals is detected by the use of restriction enzymes, gel electrophoresis / Southern Blotting with some proper description ;
3 Can be used for comparison for similarity between affected and unknown individuals;
Indirect screening:
4 RFLP marker that is tightly-linked (located close to disease causing allele) can serve as a indicator for the disease-causing allele;
5 Crossing over between the marker and allele is unlikely to occur during meiosis, hence the marker and allele would always be inherited together;
Direct screening for disease allele:
6 A disease-causing allele differs from the normal allele due to mutation which alters the ability of the restriction enzyme to recognise and cut that site;
7 Mutation within in a restriction site within an allele may cause the restriction site to be destroyed;
8 resulting in the appearance of a larger fragment and the loss of the two smaller fragments;
OR
9 Mutation occurring in the gene may create a restriction site;
10 resulting in the loss of a larger fragment and the appearance of two smaller fragments;
[max 5]
Genomic mapping
11 RFLP markers are used to construct linkage / genetic maps of a several thousands markers evenly spaced throughout the chromosomes
12 RFLP markers serve as a genetic markers to order the sequence of genes;
13 RFLP markers can be used to determine the relative distances of genes or between genetic markers on chromosomes
14 based on the recombination frequency of two RFLP markers / an RFLP marker and a certain allele of a gene;
15 Recombination frequency is a measure of closeness of the two loci on the chromosome e.g. distance in cM; or give example: the higher the frequency of the two loci inherited together, the closer are the two loci / vice versa;
[max 3]


(b) Describe the genetic basis for X-linked Severe Combined Immunodeficiecy disease (SCID)
and explain how gene therapy can be used to treat the disease. [8]

Genetic basis for X-linked Severe Combined Immunodeficiecy disease
1. Due to mutations in the gene interleukin-2 receptor gamma chain (IL2RG) encoding the common gamma chain, a protein that is shared by the receptors for some interleukins.
2. These interleukins and their receptors are responsible for the differentiation/production of T and B cells.
3. Mutations in gene that result in non functional common gamma chain will result in defects in interleukin signaling  deficient immune system.
4. Gene that code for the common gamma chain is situated on the X chromosome.
5. X-linked SCID affects only males;
[ max 4 marks]

Gene therapy to treat the disease

6. Gene therapy is a technique for introducing normal / functional copy of the IL2RG allele / gene into cells of patients;
7. restores target cell to normal state / corrects function of cell / correct phenotype by the expression of functional IR2RG protein.
8. Preparation of genetically engineered retrovirus vector to contain a normal copy of the IL2RG gene
9. Retrovirus infects T lymphocytes/blood stem cells (can be ex vivo or in vitro) and transfers IL2RG gene into nucleus of cells.
10. May be integrated into the chromosomal DNA of the lymphocytes by integrase (depend on the type of virus used) .
11. Expression of functional interleukin gamma chain  functional interleukin receptor  functional T cells and B cells production  competent immune system.
[ max 4 marks]


(c) One of the treatments for SCID is bone marrow transplant.
Explain how the properties of hematopoietic stem cells allow them to be effectively used to fight a disease such as SCID. [4]

1. Haematopoietic stem cells from donor’s bone marrow contains a normal/functional copy of the allele ADA / Interleukin 2 receptor gamma chain which can be expressed to produce a functional protein ADA / IR2RG.
2. HSCs are multipotent which has the ability to differentiate into a variety of specialised blood cells which include functional T lymphocytes and B lymphocytes.
3. To replace diseased cells lacking ADA/IR2RG or give rise to competent immune system to fight SCID
4. HSCs are capable of self-renewal and can divide by mitosis;
5. to continue supplying functional cells for long-term treatment;
6. reference to asymmetric division; Any 4 marks