19. Genetic Technology Notes

Question 1

Recombinant DNA

Answer 1

DNA that contains genetic material from 2 different organisms

Question 2

Main stages used in gel electrophoresis to separate and locate DNA fragments cut with restriction endonuclease

(Paper 5)

Answer 2

1. Amplify DNA by PCR (should be done before cutting)
2. Make agarose gel & Add buffer solution
3. Add loading dye to each sample
4. Load samples into wells at negative end (cathode) of gel
5. Apply current between electrodes
6. Stain DNA using ETHIDIUM BROMIDE {fluoresces} & observe DNA under UV light
7. Hazard and safety precaution:
   - do not touch connectors with wet hands, to prevent electrocution
   - wear safety gloves b/c ethidium bromide is harmful
   * EtBr will bind to DNA, mutagen and carcinogenic

Question 3

How is gel electrophoresis used to distinguish between two alleles of a gene?

Answer 3

1. DNA is cut with restriction enzymes
2. Agarose gel is immersed in a buffer solution
3. DNA fragments are loaded into wells in the gel, at cathode
4. Current is applied, creating an electric field
5. Fragments are negatively charged, so they move towards the ANODE
6. Gel acts as a molecular sieve
7. Smaller fragments move faster and further than larger ones
8. Current is switched off
9. Carry out Southern blotting, by transferring fragments to a membrane {used to identify specific fragments}
10. Add radioactive probes for visualisation under X-ray / fluorescent dye under UV light
    * probes will hybridise to complementary DNA bases
11. Compare banding patterns; the alleles have different positions on gel

Question 4

Variations to gel electrophoresis for separating polypeptides

Answer 4

1. Gel is stained using Coomassie blue stain to be visible, as polypeptides are colourless
2. Western blotting
3. Identify specific polypeptides by using (monoclonal) antibodies tagged with fluorescent dye
4. We can differentiate 2 different polypeptides with a similar size by comparing their net charges

Question 5

How is microarray analysis done?

Answer 5

1. mRNA extracted from 2 samples (interest + control)
2. REVERSE TRANSCRIPTASE is used to produce cDNA from mRNA
3. cDNA amplified by PCR
4. DNA labelled with DIFFerent colour fluorescent dyes
5. Labelled DNA is denatured to produce ssDNA
6. Microarray chip has ssDNA PROBES - each probe is from a known gene
7. DNA hybridises to probes on microarray chip
   - Excess (unbound) cDNA is washed away after hybridisation
8. Microarray chip is scanned with UV light to record fluorescence pattern; fluorescence shows the expressed genes
   - Intensity of fluorescence shows level of gene expression
9. Compare fluorescence between the 2 samples

Question 6

Bioinformatics

Answer 6

Database collection for the analysis of biological information and data using computer software

Question 7

❖ Outline how sequencing the genome of Plasmodium and the use of bioinformatics can suggest new targets for anti-malarial drugs. [3m]

Answer 7

1. identify genes
2. predict primary structure of proteins
3. predict (& model) 3D/tertiary structure of proteins
4. identify function of protein from 3D structure
5. develop drugs that bind with protein to block activity of protein
6. develop drugs that prevent transcription of genes

Question 8

Explain how DNA sequencing could be used to compare the DNA of 2 species. [4m]

Answer 8

• DNA cut into fragments by restriction enzymes
• DNA denatured to produce ssDNA
• primers added for annealing, attach to start of DNA
• dideoxyribonucleotide chain termination is carried out
• using DNA polymerase & copies of different lengths are produced
• then, gel electrophoresis is carried out
• laser scanner is used to detect fluorescence
• sequence of bases read by computer

Question 9

5 advantages of producing human proteins by recombinant DNA techniques

eg.

• insulin
• factor VIII for the treatment of haemophilia
• ADENOSINE DEAMINASE (ADA) enzyme for treating severe combined immunodeficiency (SCID)

Answer 9

1. Unlimited supply
2. Less risk of transmitting disease
3. Cost of purification is lower
4. Lower risk of allergy/side effects
5. Potential to improve recombinant protein

Question 10

8 advantages of screening for genetic conditions

eg.

• genes for breast cancer, BRCA1 and BRCA2
• genes for haemophilia, sickle cell anaemia, Huntington’s disease and cystic fibrosis

Answer 10

1. Provide information about the increased risk of person having genetic conditions
   eg. breast cancer
2. Can prepare for late onset genetic conditions
   eg. Huntington’s disease
3. Identify whether foetuses are going to develop a genetic condition…
4. so can give early treatment when born
5. Allows parents to prepare for the birth of a child who will need treatment {eg. financially}
6. Identify carriers of genetic conditions
7. Help to provide early diagnosis
8. Allow couples who are both carriers of a genetic condition to make family planning decisions (can choose IVF)

Question 11

Discuss the social and ethical considerations of using gene testing (embryos for genetic diseases) and gene therapy in medicine

3 pros & 4 cons

Answer 11

Pros
+ can avoid having offspring with serious genetic disease
+ can avoid late abortions (if genetic disease discovered later in foetal development)
+ allow couples to have children who would otherwise choose not to due to risk of genetic disease

Cons

• viable embryos discarded
• may conflict with religious beliefs
• could lead to selection based on gender or specific traits (“designer babies”)
• ? may be waste of healthcare resources to test for genetic conditions that have no treatment available, eg. Huntington’s disease

Question 12

4 aims of gene therapy

What type of genetic disease is suitable for treatment with gene therapy and why?

Answer 12

1. insert a normal ALLELE to obtain normal protein
2. reduce symptoms of the disorder
3. restore cellular functions
   eg. enzyme reaction, blood clotting protein, membrane transport
4. improve quality of life / life expectancy

Genetic disease in which faulty allele is RECESSIVE.

• only 1 copy of normal, dominant allele needs to be inserted per cell
• to synthesise correct protein
• to cure disease
• no need to remove faulty allele, as would be the case if faulty allele was dominant

Question 13

7 symptoms of cystic fibrosis (CF)

Answer 13

1. thick, sticky mucus
2. build up in lungs
3. lung infections, which damage lungs
4. mucus blocks pancreatic duct and prevents secretion of digestive enzymes from pancreas,
5. causing malnutrition, due to inadequate digestion and absorption
6. reduced growth
7. mucus blocks sperm duct, sterile males

Question 14

How can cystic fibrosis be treated with gene therapy?

• an autosomal recessive disease

Answer 14

1. CF is caused by MUTATION of CFTR gene, making the CFTR gene defective & faulty CFTR protein is produced
2. so insert normal dominant CFTR allele into DNA…
3. …in cells of respiratory system
4. by using a VECTOR
5. use liposomes, which are sprayed and inhaled
6. use harmless virus - ADENOVIRUS
7. correct form of CFTR protein made
8. inserted into membrane
9. act as CHLORIDE CHANNEL to actively transport chloride ions out of cell
10. water potential decreases outside cell, so WATER leaves cell by osmosis
11. normal mucus formed
12. symptoms reversed
    eg. less chance of lung infections

Question 15

Outline how genetic diseases can be treated with gene therapy

Answer 15

1. obtain normal allele from healthy person {cut using restriction enzymes, sticky ends}
2. insert into vector
3. use liposomes as vector
   - liposomes are sprayed as an aerosol and inhaled {suitable for respiratory diseases}
   - liposomes fuse with host cell
4. use harmless virus as vector
   - may cause side effects
5. effects are short-lived & repeat treatments are needed

Question 16

Challenges in choosing appropriate vectors, such as viruses, liposomes and naked DNA

Answer 16

Viruses

1. only small amount of DNA can be carried
2. low probability of integration into host genome
3. can insert viral DNA randomly into host DNA, so can cause mutations in host DNA & may cause cancer/side effects
4. inserted allele may be inactivated
5. virus may not enter target cells or virus may enter non-target cells

Liposomes (tiny balls of lipids)
1. low ability to add genes into target cells genome

Naked DNA

1. has to be injected into target cell
2. low efficiency of cellular uptake
3. rapidly broken down

Question 17

Genetic engineering is significant in improving the quality and yield of crop plants and livestock in solving the demand for food in the world.
eg. Bt maize, vitamin A enhanced rice (Golden riceTM) and GM salmon

How can GM salmon be used to increase food production?

Answer 17

1. Allele for faster growth rate / growth hormone inserted
   + Promoter from another species also inserted
2. Growth occurs all year round, not just in spring & summer
3. GM salmon grow faster (reduced growth duration from 3 years to 18 months)

• Rear only sterile females to prevent mating between GM salmon and wild fish
• Reduced ability to compete with wild salmon in natural environment

Question 18

Herbicide-resistant oil seed rape & tobacco

4 possible environmental effects

Answer 18

Herbicide can kill weeds but not the GM crops, thus reduces competition from weeds & increases crop yield

1. GM crops may become weeds themselves / invasive
2. pollen transfer to wild relatives would TRANSFER resistance gene to non-GM crops, producing hybrid offspring that are invasive
3. intensive use of herbicide selects for herbicide-resistant weeds, leads to development of SUPERWEEDS
4. intensive use of herbicide reduces biodiversity

Question 19

Insect-resistant maize & cotton

4 effects

Answer 19

• Pesticides kill pests, but can also harm beneficial insects
  eg. pollinators, bees, predators of pests
• need to conserve biodiversity
• Genes coding for ‘Bt toxin’ protein are inserted into maize and cotton plants
• Protein is produced in plants and converted to toxin inside gut of insects that ate leaves, killing leaf-eating insects

+ less pesticide used, cost-effective (benefit to farmers)

• Insect pests may develop resistance
• may cause side effects in humans
• pest-resistant GM crops may harm other useful insects

Question 20

Vitamin A enhanced rice - Golden Rice TM

• vitamin A deficiency can cause blindness
• human body synthesises vitamin A from β-carotene (found in aleurone layer but not in endosperm)
• aleurone layer removed in production of white rice b/c it will go rancid

Answer 20

1. vitamin A found in aleurone layer of rice seeds
2. white rice does not contain vitamin A / β-carotene
3. genes coding for β-carotene extracted from both bacteria (Pantoea ananatis) & maize
4. genes inserted into plasmid vector
5. promoters added {needed for gene expression}
6. recombinant plasmids put into &laquo_space;Agrobacterium tumefaciens&raquo_space;
7. Agrobacterium tumefaciens mixed with rice EMBRYOS
8. some embryos take up bacteria and β-carotene gene
9. these embryos grow into adult plants and produce seeds with β-carotene in ENDOSPERM
   = Golden Rice TM

Question 21

❖ Suggest how children with ADA-deficient SCID can be treated with gene therapy using a virus. [3m]

• SCID is a group of life-threatening diseases
• caused by mutations that prevent the normal function of the immune system
• T-lymphocytes do not develop normally
• SCID infants at very high risk of infectious diseases

Answer 21

• obtain normal ADA allele
• insert allele into virus vector; retrovirus
• remove stem cells {from bone marrow}
• insert allele/virus into stem cells
• return stem cells to body {by injecting into blood}

*stem cells differentiate into T-lymphocytes with functioning ADA

Question 22

9 advantages of growing genetically modified crops + examples

Answer 22

1. Increase crop yield
2. Improve food quality
3. Crops may be more tolerant to climate change
4. Crops can be grown in poor quality land
5. Herbicide resistance reduces competition from weeds
6. Insect resistance increases crop growth
   eg. Bt maize/cotton kills leaf-eating insects
7. Less pesticide used
8. Benefit to famers; cost-effective
9. Add nutrients to crop to improve human health
   eg. Golden Rice TM for extra vitamin A

Question 23

6 disadvantages of using genetically modified crops

Answer 23

1. High cost of GM seeds, poor farmers cannot afford
2. Increase selling price, reduce efforts in relieving poverty
3. Possible allergic reactions in humans, eg. Bt toxin protein
4. Cross pollination with wild plants may give rise to herbicide-resistant weeds (superweeds)
5. Reduce biodiversity
6. Pest-resistant GM crops may harm other useful insects

Question 24

2 ethical and 3 social implications of using genetically modified organisms (GMOs) in food production

Answer 24

E1. GM crops relieve hunger/starvation
E2. GM crops reduce land area for crops, so conserve habitats / protect biodiversity

S1. GM crops increase food supply
S2. GM crops decrease food cost
S3. GM crops increase a country’s wealth