Genetic Engineering

Question 1

Define the term recombinant DNA.

Answer 1

DNA made by combining DNA from 2 or more different organisms

Question 2

explain what is meant by genetic engineering?

Answer 2

The transfer of genes from one organism into another (of the same/different species) to express the gene into its new host

Question 3

overview of gene transfer

Answer 3

1) identification of the desired gene
2) isolation of the desired gene by
- cutting from a chromosome using enzymes (restriction endonucleases)
- using reverse transcriptase to make a single strand of complementary DNA (cDNA) from mRNA
or synthesizing the gene artificially using nucleotides
3) multiplication of the gene (using polymerase chain reaction - PCR)
4) gene is inserted into a vector (e.g. plasmids, viruses, liposomes) which delivers the gene to cells of the organisms
5) identification of the cells with the new gene (by using a marker), which is then cloned

Question 4

Name the three ways in which genes can be generated for genetic engineering.

Answer 4

1) extracted directly from an organism’s DNA using restriction endonuclease
2) generated from a mRNA sequence using reverse transcriptase
3) synthesizing the gene artificially using nucleotides

Question 5

Explain why starting point in genetic engineering is mRNA
(pp)

Answer 5

• large copes of mRNA are readily available
• easier to obtain than extracting from cells DNA
• Introns already removed

Question 6

list the enzymes used in genetic engineering and outline their roles in natural processes
(pp)

Answer 6

1) restriction enzyme- cuts DNA
2) DNA ligase- forms phosphodiester bonds during DNA replication
3) reverse transcriptase- makes complementary DNA from mRNA
4) Taq polymerase- copies DNA

Question 7

Name two domains that are a source of restriction endonucleases
(pp)

Answer 7

bacteria and Archaea

Question 8

source of reverse transcriptase enzymes

Answer 8

retroviruses

Question 9

why are most recombinant human proteins produced using eukaryotic cells (eg. yeast, or mammalian cells in culture) rather than using prokaryotic cells

Answer 9

eukaryotic cells will carry out the post-translational modification (due to presence of Golgi Apparatus / enzymes) that is required to produce a suitable human protein

Question 10

advantage of using reverse transcriptase enzymes

Answer 10

easier for scientists to find mRNA with the specific characteristic because specialised cells make very specific types of mRNA (eg. β-cells of the pancreas produce many insulin mRNA) and mRNA does not contain introns

Question 11

transferring plasmids to host cells (bacteria)

Answer 11

1) the plasmids and bacteria are bathed in an ice-cold calcium chloride solution (high conc. of Ca ions) and then heat shocked, making the bacteria’s cell surface membrane more permeable

2) only a very small proportion of bacteria take up the plasmids with the gene (〜1%), those that do so are said to be transformed

Question 12

plasmid

Answer 12

small, circular pieces of double-stranded DNA

it is a vector used to carry DNA into host cell

Question 13

List and explain the properties of plasmids that allow them to be used as vectors
(pp)

Answer 13

1) small - can be inserted into cells
2) have restriction site- so new gene can be added
3) have marker genes- so recombinant cells which have been taken up can be recognised
4) self replicate- so can multiply and can be expressed
5) have promoter- so gene can be expressed
6) circular- so more stable

Question 14

properties of plasmids that allow them to be used in gene cloning

Answer 14

1) they occur naturally in bacteria so easier to extract from bacteria
2) can be cut using restriction endonuclease
3) can be produced artificially
4) may contain antibiotic resistance genes- used as marker genes which can help to identify transformed bacteria
5) replicate independently in bacteria

Question 15

role of promoter

Answer 15

1) ensures that RNA polymerase recognises the template strand
2) transcription start-point
3) the promoter is used to regulate gene expression because only if it is present will transcription and therefore the expression of the gene occur

Question 16

Explain why a promoter has to be introduced as well as the desired gene?/2
(pp)

Answer 16

• to start transcription. so it allows binding of RNA polymerase
• at all times

Question 17

examples of gene markers

Answer 17

1) antibiotic resistant genes (the gene for antibiotic resistance is replaced, therefore the ‘transformed’ bacteria would not be able to grow in a medium with an antibiotic present)

2) GFP (green fluorescent protein) which fluoresces under UV light

3) GUS (β-glucuronidase enzyme) which transforms colourless or non-fluorescent substrates into products that are coloured or fluorescent

Question 18

Explain why genes for antibiotic resistance are now rarely used in gene technology as marker?
(pp)

Answer 18

1) risk of antibiotic resistance genes spreading to other bacteria, producing pathogenic strains that can’t be killed by antibiotics
2) if the resistance spread to other bacteria this could make antibiotics less effective

Question 19

Explain the use of genes for fluorescence as markers in gene technology?
(pp)

Answer 19

• add marker gene to the plasmid
• gene of interest is inserted close to marker gene
• marker gene emits light
• visible colour change
• exposing to UV light
• easy to identify transformed bacteria
• for example GFP
• no known risk

Question 20

Explain why, in many examples of gene technology, fluorescent markers are used in preference to antibiotic resistance genes?
(pp)

Answer 20

1) they are easier to identify

2) more economical

3) no risk of antibiotic resistance being passed onto other bacteria

4) there are antibiotics that are no longer effective and therefore would not stop any bacteria from growing

Question 21

role of restriction endonucleases (restriction enzymes) in the transfer of a gene into an organism

Answer 21

1) isolate the desired gene
2) separate the DNA strands (at the same base sequence) in a vector so the desired gene can be inserted

Question 22

why are many different restriction endonucleases required

Answer 22

they bind to a specific restriction site (specific sequences of bases) on DNA, eg. HindIII will always bind to the base sequence AAGCTT

Question 23

how restriction endonuclease work

Answer 23

restriction enzymes either cut straight across the sugar-phosphate backbone to give blunt ends or they cut in a staggered fashion to give sticky ends

Question 24

what is the function of PCR?

Answer 24

method for the rapid production of a very large number of copies of a particular fragment of DNA

Question 25

Outline the substances required for PCR

Answer 25

1) DNA fragment to be amplified 2) primer (short nucleotide sequences) 3) DNA Taq polymerase 4) buffer solution - to provide the optimum pH for the reactions to occur in

Question 26

The three stages of PCR

Answer 26

1) denaturation: - the double-stranded DNA is heated to 95°C -breaks the hydrogen bonds that bond the two DNA strands together 2) annealing: -the temperature is 65°C -primers can attach to the ends of single- stranded DNA molecules 3) elongation: the temperature is 72°C as this is the optimum temperature for Taq polymerase to build the complementary strands of DNA to produce the new identical double-stranded DNA molecules

Question 27

Describe what happens to the DNA at each temperature in PCR (pp)

Answer 27

95C- DNA splits into single strands. Hydrogen bonds are broken 50C- primers bind to single stranded DNA 75C- complementary DNA strand is made

Question 28

features of Taq polymerase that enable it to be used in PCR

Answer 28

1) not destroyed in the denaturation step, so it does not have to be replaced each cycle 2) its high optimum temperature means the temperature for the elongation step does not have to be dropped below that of the annealing process so efficiency is maximised

Question 29

Describe the principles of PCR. (pp)

Answer 29

- PCR is the production of large number of copies of DNA - rapid process - only small samples of DNA are needed - DNA is denatured at 95C - primers are added to DNA at 65C - by complementary base pairing - Taq polymerase replicates the strand at 75C - heat again to separate strands - Taq polymerase is heat stable - Taq polymerase does not need replacing at each cycle

Question 30

What is the function of gel electrophoresis?

Answer 30

To separate DNA fragments, nucleic acids and proteins according to their size and charge

Question 31

Outline the principles of electrophoresis. (pp)

Answer 31

- cut DNA using reverse transcriptase - DNA is loaded into wells at the negative end of gel - direct current is applied - due to negatively charged phosphate groups, DNA gets attracted to anode - smaller the size, faster they travel - due to gel impedence - visualize DNA under UV light

Question 32

factors affecting the movement of charged molecules in gel electrophoresis

Answer 32

1) net (overall) charge - -vely charged molecules move to anode (+), +vely charged molecues move to cathode (-), highly charged molecules move faster than those with less overall charge 2) size - smaller molecules move faster than larger ones 3) composition of gel - size of pores within gel (e.g., agarose for DNA has different pore size than polyacrylamide for proteins) determines speed with which molecules move

Question 33

Explain why electrophoresis produced a DNA banding pattern on gel? (pp)

Answer 33

As the electric current is applied, DNA is attracted to positive electrode and as as DNA fragments are small, they move faster

Question 34

electrophoresis of proteins

Answer 34

1) the charge on proteins is dependent on the ionisation of the R groups of amino acids - the charge of the R groups depends on the pH and therefore buffer solutions are used during the separation of proteins to keep the pH constant. 2) gel electrophoresis is used to separate polypeptide chains produced by different alleles of the same gene e.g., the haemoglobin variants (α-globin, β-globin and the sickle cell anaemia variant of β-globin)

Question 35

Compare the similarities and differences between electrophoresis and chromatography. (pp)

Answer 35

similarities: - both are separating techniques - both are separated due to mass differences: - molecule separated: -electrophoresis: DNA - chromatography: pigments - medium of separation: -electrophoresis: gel - chromatography: paper - separated by: -electrophoresis: current - chromatography: solvent - visualisation due to: -electrophoresis: UV light - chromatography: different colours of pigment - identification by: -electrophoresis: use DNA probe - chromatography: Rf value

Question 36

use of PCR and DNA testing in forensic medicine and criminal investigations

Answer 36

1) electrophoresis of DNA is used in genetic profiling (fingerprinting) in forensic science 2) PCR is used in forensic science to solve crimes: used to amplify DNA from small tissue samples

Question 37

Outline how microarrays are used in the analysis of genomes? (pp)

Answer 37

1) restriction enzymes are used to cut the DNA into fragments l 3) the fragments are denatured to form single-stranded DNA molecules 4) the DNA is labelled with fluorescent tags 5) the labelled DNA samples are mixed together and allowed to hybridise with the probes on the microarray 6)probes are, single-stranded DNA 7) each probe is unique to a particular gene ; 8) any DNA that has not bound to the probes is washed off 9) the microarray is viewed under UV light, causing the tags to fluoresce 10) the presence of colour indicates that hybridisation has taken place (as the DNA fragments are complementary to the probes) ‣ red and green: DNA from one species has hybridised with probes ‣ yellow: DNA from both species hybridised (the two species have DNA with exactly the same base sequence) ‣ no colour/blue: no hybridisation, gene not present in either species 11) the microarray is scanned so data is read by a computer and stored

Question 38

Define the term bioinformatics. (pp)

Answer 38

- bioinformatics is the analysis of biological information - using computer software - from large databases of genes

Question 39

role of bioinformatics following the sequencing of genomes

Answer 39

1) comparisons can be made with other known genomes using the many databases available; sequences can be matched and degrees of similarity calculated 2) human genes such as those associated with development can be found in other organisms e.g, Drosophila 3) ways to control Plasmodium and gene sequencing is helping in the development of vaccines for malaria

Question 40

Explain what is meant by bioinformatics and outline the role of bioinformatics following the sequencing of genomes of humans and parasites?/6 (pp)

Answer 40

1) bioinformatics is the analysis of biological data using computer software 2) from large databases of, genes  3) it is fast and accurate 4) allows data to be, shared  5) can predict, amino acid sequences / protein structure (from DNA sequence data) 6) analytical tool ; e.g. BLAST 7) allows comparisons to determine the similarities and differences between genomes 8) used to find methods to control parasites 9) named example of control ; e.g. vaccine

Question 41

Give some examples of human proteins that have been produced by recombinant DNA technology

Answer 41

1) insulin to treat diabetes 2) factor VII to treat haemophilia 3) adenosine deaminase to treat severe combined immunodeficiency (SCID)

Question 42

Outline the benefits of producing human proteins by recombinant DNA technology

Answer 42

1) few practical and ethical problems as proteins do not have to be extracted from animal sources 2) the proteins are engineered to be identical to human proteins or have modifications that are beneficial 3) The rate at which proteins can be produced is much faster than other methods eg. blood transplants 4) The supply is reliable (production facilities do not require much space and the processes can be carried out anywhere in the world) 5) The gene product can be easily extracted and purified 6) large volume of products are produced

Question 43

Describe how the gene coding for human insulin can be obtained and inserted into plasmid vector? (pp)

Answer 43

1) obtain mRNA from B-cells of islets of Langerhans of Pancreas 2) use reverse transcriptase to make cDNA from mRNA 3) DNA polymerase use to make double stranded DNA 4) use DNA with restriction enzyme to make sticky ends 5) cut plasmid with same restriction enzyme to form complementary sticky ends 6) cDNA is mixed with plasmid 7) use DNA ligase to seal nicks in sugar phosphate backbone

Question 44

Explain the advantages of treating diabetes with human insulin produced by genetic engineering (pp)

Answer 44

1) identical to human insulin, 2) there is unlimited supply available to meet demand 3) fewer ethical, moral or religious concerns (proteins are not extracted from cows or pigs) 4) fewer rejection problems or side effects or allergic reactions 5) does not stimulate immune system 6) cheaper to produce in large volumes 7) more rapid response 8) can be produced without killing animals

Question 45

producing recombinant adenosine deaminase (ADA)

Answer 45

the enzyme adenosine deaminase is used to treat severe combined immunodeficiency (SCID) while patients are waiting for gene therapy or when gene therapy is not possible

Question 46

Give examples of genetic conditions that can be screen for

Answer 46

1) sickle cell anaemia 2) Breast cancer (BRCA1 and BRCA 2) 3) Haemophilia 4) cystic fibrosis 5) Huntington's disease

Question 47

Why is genetic screening carried out?

Answer 47

It is used to test for and diagnose genetic conditions, for example in an unborn fetus or an individual with a family history of a genetic condition

Question 48

Genetic screening for the faulty alleles of Brca-1 and Brca-2

Answer 48

Brca-1 and Brca-2 are genes that produce tumour suppressor proteins and thus they play an important role in regulating cell growth ‣ faulty alleles of these particular genes exist (can be inherited from either parent) which increase the risk of an individual developing breast cancer

Question 49

Advantages of screening for Brca-1 and Brca-2

Answer 49

1) preventative measures can be taken e.g., an elective mastectomy (breast removal) to reduce the risk of developing cancer 2) screening for breast cancer may begin from an earlier age or more frequently 3) enables the person to participate in research and clinical trials

Question 50

genetic screening for sickle cell anaemia

Answer 50

. sickle cell anaemia is an autosomal recessive disease that results in the haemoglobin molecule being less soluble if oxygen is not present, causing red blood cells to form a sickled shape ‣ this sickled shape reduces the ability of the red blood cell to carry oxygen and the cells are less flexible, and more prone to getting stuck in small capillaries

Question 51

Advantages of screening for anaemia

Answer 51

1) people with a family history of the disease can be tested if they are carriers 2) if the person is determined to be a carrier they can discuss with a genetic counsellor their options so they can make informed decision 3) if the person is undergoing IVF, they could use PGD to select an embryo that does not have the recessive alleles

Question 52

genetic screening for haemophilia

Answer 52

‣ haemophilia is a sex-linked recessive inherited disease where the body does not produce a blood protein (either factor VIII or factor IX) that is required for the blood to clot

Question 53

Advantages of screening for haemophilia

Answer 53

1) can determine whether they are carriers or not (as haemophilia is a recessive disease) 2) can help the women (and their partners) make decisions about future pregnancies (if they're carriers) 3) help the doctors take special precautions during the pregnancy (if a carrier) 4) women can use pre-implantation genetic diagnosis (PGD) during IVF to choose an embryo that is carrying the allele for the relevant blood clotting factor as opposed to embryos carrying the recessive alleles

Question 54

State possible advantages of using gene editing as a method of treating haemophilia (pp)

Answer 54

- precise - no risk of immune response - no need to introduce allele as person's own gene can be editied - no need to introduce promoter

Question 55

producing recombinant factor VIII

Answer 55

factor VIII is a blood-clotting protein that haemophiliacs cannot produce 1) kidney and ovary hamster cells have been genetically modified to produce factor VIII 2) once modified, the cells are cultured in fermenters 3) due to the optimal conditions in the fermenter, the hamster cells constantly express factor VIII which can then be extracted and purified, and used as an injectable treatment for haemophilia

Question 56

advantages of using recombinant factor VIII

Answer 56

1) fewer ethical, moral or religious concerns (proteins are not extracted from human blood) 2) less risk of transmitting infection (e.g. HIV) or disease 3) greater production rate

Question 57

genetic screening for Huntington's disease

Answer 57

‣ Huntington's disease is a late-onset neurodegenerative disease caused by an austosomal dominant allele

Question 58

advantages of screening for Huntington's disease

Answer 58

1) people to plan for the future (how they will live and be cared for) 2) couples to make informed reproductive decisions (as the risk that their children may inherit the disease is 50%) 3) people to participate in research and clinical trials

Question 59

Explain why gene editing is more suitable as compared to gene therapy as a potential cure for Huntingtin's disease (pp)

Answer 59

- HD is caused by dominant allele - adding a normal recessive allele would not work - gene editing can remove mutant allele

Question 60

explain what is meant by gene therapy? (pp)

Answer 60

- To treat disease caused by faulty allele - by delivering gene into target cells

Question 61

Outline the aims of gene therapy (pp)

Answer 61

- insert a normal allele - to obtain a functional protein - reduce symptoms of disorder - restore cellular functions

Question 62

most common vectors that are used to carry normal alleles to host cells (in gene therapy)

Answer 62

- viruses (retrovirus, lentivirus, HIV, adeno-associated virus) - liposomes -naked DNA

Question 63

retroviruses in gene therapy

Answer 63

‣ retroviruses insert their genes into the host's genome however do so randomly ‣ this means they may insert their genes within another gene or into the regulatory sequence of a gene (which may then activate a nearby gene causing cancer)

Question 64

lentivirus

Answer 64

‣ a virus characterized by a long incubation period (e.g., HIV) ‣ inserts genes randomly into host's genome, but this virus can be modified to not replicate

Question 65

adeno-associated virus (AAV)

Answer 65

‣ this virus does not insert its genes into the host genome and so they are not passed on to daughter cells when a cell divides ‣ this is a problem when cells are short-lived e.g. lymphocytes but can be used successfully with long-lived cells such as liver cells and neurones

Question 66

State ethical consideration of using retrovirus gene therapy (pp)

Answer 66

- reterovirus could be inserted in wrong place - could cause cancer - could cause immune response - could cause infection

Question 67

advantage of using naked DNA in gene therapy

Answer 67

removes problems associated with using vector

Question 68

social & ethical considerations of using gene therapy

Answer 68

1) the potential for side effects that could cause death (eg. the children who were treated for SCID developed leukaemia) 2) whether germ cell gene therapy should be allowed 3) genetic conditions where treatments already exist 4) the expense of treatments as multiple injections of the genes may be required if the somatic cells are short-live; this may make the cost of gene therapy accessible to a limited number of people 5) who has the right to determine which genes can be altered and which cannot

Question 69

germ cell gene therapy

Answer 69

attempts to alter alleles in cells involved in sexual reproduction

Question 70

gene therapy for Leber congenital amaurosis

Answer 70

‣ a form of hereditary blindness caused by retinal cells dying off gradually from a young age in males ‣ doctors injected adeno-associated viruses into the retina that contained the normal alleles of one of the genes that caused damage to the photoreceptors, improving their eyesight

Question 71

Outline the basic principles of gene therapy for the treatment of cystic fibrosis. (pp)

Answer 71

- CF caused by mutation of the CFTR gene - CFTR transporter protein becomes defective - Hence insert a normal dominant CFTR allele into the DNA in cells of respiratory system using a vector; e.g. liposomes taken as spray or a harmless virus - however, not all cells take up virus and may cause unpleasant side-effects - The effects only lasts for a short period of time, and the treatment needs repeating

Question 72

advantage of genetic screening for cystic fibrosis

Answer 72

1) t enables couples to make informed reproductive decisions (as both may be carriers and therefore not display any symptoms) 2) people can participate in research and clinical trials

Question 73

Outline the procedure used for gene therapy treatment of a person with SCID (pp)

Answer 73

1) drug given to increase number of stem cells 2) obtain stem cells 3) mix stem cells with vector containing a normal allele 4) drug to kill stem cells in bone marrow (to make space for new cells) 5) inject stem cells into blood 6) lymphocytes produce functioning ADA

Question 74

Suggest and explain why it may be appropriate to use enzyme replacement therapy to treat SCID instead of bone marrow transplant (pp)

Answer 74

- donor not needed - no risk of immune response - immediate effect - cheaper

Question 75

Describe the role of a genetic counsellor in dealing with genetic diseases in humans and discuss the circumstances in which a couple might be referred to a genetic counsellor. (pp)

Answer 75

counsellor: - pedigree analysis - genetic screening such as tissue samples from adults - explain results of tests - may discuss termination - may discuss treatments - may discuss financial implications - may discuss ethical issues couple referred if: - either has genetic disease in family or are carriers - older women - history of recurrent marriages

Question 76

crop plants have been genetically modified to be

Answer 76

1) resistant to herbicides - increases productivity / yield 2) resistant to pests - increases productivity / yield 3) enriched with vitamins - increases the nutritional value

Question 77

benefits of using genetic engineering rather than traditional selective breeding techniques to solve the global demand for food

Answer 77

1) organisms with the desired characteristics are produced more quickly 2) all organisms will contain the desired characteristic (there is no chance that recessive allele may arise in the population) 3) the desired characteristic may come from a different species / kingdom

Question 78

consequences of using genetically engineered organisms to solve the global demand for food

Answer 78

1) the development of resistance for the genes that have been introduced 2) the risk of the gene spreading to wild relatives 3) the modified organism may become a pest 4) the reduction in biodiversity 5) potential ecological effects (e.g. harm to non-targeted species)

Question 79

disadvantages of using genetically engineered organisms to increase the productivity of the crop

Answer 79

1) the development of resistance for the genes that have been introduced 2) the risk of the gene spreading to wild relatives 3) the modified organism may become a pest 4) the reduction in biodiversity 5) potential ecological effects (e.g. harm to non-targeted species) 6) possible risk to human health as an allergy (there are no long-term studies on the effect on human health)

Question 80

GM salmon

Answer 80

1) growth-hormone regulating genes from Pacific Chinook salmons and promoters from ocean pout are injected into the fertilised egg of an Atlantic salmon 2) this enables the salmon to produce growth hormone throughout the year and therefore grow all year instead of just the summer and spring 3) they reach market size in 18 months as opposed to 3 years of an unmodified fish 4) to prevent the GM salmon from reproducing in the wild, all the salmon are female and sterile

Question 81

detrimental effects on the environment of growing a herbicide-resistant crop

Answer 81

1) the genetically modified plant will become an agricultural weed 2) pollen will transfer the gene to wild relatives, producing hybrid offspring that are invasive weeds 3) herbicide-resistant weeds will evolve because so much of the same herbicide is used

Question 82

detrimental effects on the environment of growing an insect-resistant crop

Answer 82

1) the evolution of resistance by the insect pests 2) a damaging effect on other species of insects (however, less pesticide is used, less risk of spray carrying and affecting non-target species in other areas) 3) the transfer of added gene to other species of plant

Question 83

social implications of using genetically modified organisms in food production

Answer 83

) modified crop plants may become agricultural weeds or invade natural habitats 2) the introduced genes may be transferred by pollen to wild relatives whose hybrid offspring may become more invasive 3) the introduced genes may be transferred by pollen to unmodified plants growing on a farm with organic certification 4) the modified plants may be a direct hazard to other animals and humans by being toxic or producing allergies 5) the herbicide that can now be used leaves toxic resides on the crop 6) genetically modified seeds and herbicides are expensive and their cost removes any advantage of growing a resistant crop 7) growers need to buy new seeds every season, keeping costs high 8) danger of losing traditional varieties with their desirable background genes and possibly unknown traits which might be useful