Genetic Engineering Flashcards
Define the term recombinant DNA.
DNA made by combining DNA from 2 or more different organisms
explain what is meant by genetic engineering?
The transfer of genes from one organism into another (of the same/different species) to express the gene into its new host
overview of gene transfer
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
Name the three ways in which genes can be generated for genetic engineering.
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
Explain why starting point in genetic engineering is mRNA
(pp)
- large copes of mRNA are readily available
- easier to obtain than extracting from cells DNA
- Introns already removed
list the enzymes used in genetic engineering and outline their roles in natural processes
(pp)
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
Name two domains that are a source of restriction endonucleases
(pp)
bacteria and Archaea
source of reverse transcriptase enzymes
retroviruses
why are most recombinant human proteins produced using eukaryotic cells (eg. yeast, or mammalian cells in culture) rather than using prokaryotic cells
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
advantage of using reverse transcriptase enzymes
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
transferring plasmids to host cells (bacteria)
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
plasmid
small, circular pieces of double-stranded DNA
it is a vector used to carry DNA into host cell
List and explain the properties of plasmids that allow them to be used as vectors
(pp)
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
properties of plasmids that allow them to be used in gene cloning
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
role of promoter
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
Explain why a promoter has to be introduced as well as the desired gene?/2
(pp)
- to start transcription. so it allows binding of RNA polymerase
- at all times
examples of gene markers
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
Explain why genes for antibiotic resistance are now rarely used in gene technology as marker?
(pp)
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
Explain the use of genes for fluorescence as markers in gene technology?
(pp)
- 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
Explain why, in many examples of gene technology, fluorescent markers are used in preference to antibiotic resistance genes?
(pp)
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
role of restriction endonucleases (restriction enzymes) in the transfer of a gene into an organism
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
why are many different restriction endonucleases required
they bind to a specific restriction site (specific sequences of bases) on DNA, eg. HindIII will always bind to the base sequence AAGCTT
how restriction endonuclease work
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
what is the function of PCR?
method for the rapid production of a very large number of copies of a particular fragment of DNA
Outline the substances required for PCR
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
The three stages of PCR
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
Describe what happens to the DNA at each temperature in PCR
(pp)
95C- DNA splits into single strands. Hydrogen bonds are broken
50C- primers bind to single stranded DNA
75C- complementary DNA strand is made
features of Taq polymerase that enable it to be used in PCR
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
Describe the principles of PCR.
(pp)
- 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
What is the function of gel electrophoresis?
To separate DNA fragments, nucleic acids and proteins according to their size and charge
Outline the principles of electrophoresis.
(pp)
- 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
factors affecting the movement of charged molecules in gel electrophoresis
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
Explain why electrophoresis produced a DNA banding pattern on gel?
(pp)
As the electric current is applied, DNA is attracted to positive electrode
and as as DNA fragments are small, they move faster
electrophoresis of proteins
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)
Compare the similarities and differences between electrophoresis and chromatography.
(pp)
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
use of PCR and DNA testing in forensic medicine and criminal investigations
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
Outline how microarrays are used in the analysis of genomes?
(pp)
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
Define the term bioinformatics.
(pp)
- bioinformatics is the analysis of biological information
- using computer software
- from large databases of genes
role of bioinformatics following the sequencing of genomes
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
Explain what is meant by bioinformatics and outline the role of bioinformatics following the sequencing of genomes of humans and parasites?/6
(pp)
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
Give some examples of human proteins that have been produced by recombinant DNA technology
1) insulin to treat diabetes
2) factor VII to treat haemophilia
3) adenosine deaminase to treat severe combined immunodeficiency (SCID)
Outline the benefits of producing human proteins by recombinant DNA technology
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
Describe how the gene coding for human insulin can be obtained and inserted into plasmid vector?
(pp)
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
Explain the advantages of treating diabetes with human insulin produced by genetic engineering
(pp)
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
producing recombinant adenosine deaminase (ADA)
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
Give examples of genetic conditions that can be screen for
1) sickle cell anaemia
2) Breast cancer (BRCA1 and BRCA 2)
3) Haemophilia
4) cystic fibrosis
5) Huntington’s disease
Why is genetic screening carried out?
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
Genetic screening for the faulty alleles of Brca-1 and Brca-2
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
Advantages of screening for Brca-1 and Brca-2
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
genetic screening for sickle cell anaemia
. 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
Advantages of screening for anaemia
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
genetic screening for haemophilia
‣ 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
Advantages of screening for haemophilia
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
State possible advantages of using gene editing as a method of treating haemophilia
(pp)
- precise
- no risk of immune response
- no need to introduce allele as person’s own gene can be editied
- no need to introduce promoter
producing recombinant factor VIII
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
advantages of using recombinant factor VIII
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
genetic screening for Huntington’s disease
‣ Huntington’s disease is a late-onset neurodegenerative disease caused by an austosomal dominant allele
advantages of screening for Huntington’s disease
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
Explain why gene editing is more suitable as compared to gene therapy as a potential cure for Huntingtin’s disease
(pp)
- HD is caused by dominant allele
- adding a normal recessive allele would not work
- gene editing can remove mutant allele
explain what is meant by gene therapy?
(pp)
- To treat disease caused by faulty allele
- by delivering gene into target cells
Outline the aims of gene therapy
(pp)
- insert a normal allele
- to obtain a functional protein
- reduce symptoms of disorder
- restore cellular functions
most common vectors that are used to carry normal alleles to host cells (in gene therapy)
- viruses (retrovirus, lentivirus, HIV, adeno-associated virus)
- liposomes
-naked DNA
retroviruses in gene therapy
‣ 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)
lentivirus
‣ 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
adeno-associated virus (AAV)
‣ 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
State ethical consideration of using retrovirus gene therapy
(pp)
- reterovirus could be inserted in wrong place
- could cause cancer
- could cause immune response
- could cause infection
advantage of using naked DNA in gene therapy
removes problems associated with using vector
social & ethical considerations of using gene therapy
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
germ cell gene therapy
attempts to alter alleles in cells involved in sexual reproduction
gene therapy for Leber congenital amaurosis
‣ 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
Outline the basic principles of gene therapy for the treatment of cystic fibrosis.
(pp)
- 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
advantage of genetic screening for cystic fibrosis
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
Outline the procedure used for gene therapy treatment of a person with SCID
(pp)
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
Suggest and explain why it may be appropriate to use enzyme replacement therapy to treat SCID instead of bone marrow transplant
(pp)
- donor not needed
- no risk of immune response
- immediate effect
- cheaper
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)
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
crop plants have been genetically modified to be
1) resistant to herbicides - increases productivity / yield
2) resistant to pests - increases productivity / yield
3) enriched with vitamins - increases the nutritional value
benefits of using genetic engineering rather than traditional selective breeding techniques to solve the global demand for food
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
consequences of using genetically engineered organisms to solve the global demand for food
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)
disadvantages of using genetically engineered organisms to increase the productivity of the crop
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)
GM salmon
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
detrimental effects on the environment of growing a herbicide-resistant crop
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
detrimental effects on the environment of growing an insect-resistant crop
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
social implications of using genetically modified organisms in food production
) 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
