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Genetic technology, the plasmid is cut from the bacteria using a…
Genetic technology
Genetic screening
Describe what genetic screening is and when it can be done [ 6 marks ]
- genetic screening is the analysis of a person's DNA
- to check for the presence of a particular allele
- this can be done in adults,
- in a fetus,
- embryos in the uterus
- or in embryos during IVF
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Discuss the ethical and social considerations of gene testing embryos for genetic diseases [3 marks] marking points
PGD (Pre-implantation genetic diagnosis)
- mixing the father's sperm with the mother's eggs (oocytes) in a dish
- At the 8 cell stage, 1 of the cells from the tiny embryo is removed
- The DNA in the cell is analysed and used to predict whether or not the embryo will have a genetic disease for which both parents are carriers.
An embryo NOT carrying the allele that would cause the disease is chosen for implantation and the embryos that do have the allele is discarded to produce a child without a genetic disease.
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Gel electrophoresis
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Microarrays
How microarrays are used in the analysis of genomes and in detecting mRNA in studies of gene expression
Outline how a microarray enables the detection of particular alleles [4] marking points
- probes are short lengths of single-stranded DNA
- probes are complementary to the DNA
- many copies of 1 type of probe is placed in each cell of the microarray
- single stranded DNA made (from RNA)
- (target) alleles/DNA labelled with fluorescent tags
- (target) alleles/DNA hybridises with probes
- DNA that doesn't bind to the probes is washed off
- UV light is used to detect the presence of hybridised probes/particular alleles/DNA
DNA hybridises with probes (probes become hybridised)
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What are VNTR regions?
VNTR (Variable Number Tandem Repeats) sequence
= a region of DNA that varies between different people
Probes have base sequences complementary to the VNTR regions.
Cystic Fibrosis
Thick mucus is produced in the lungs and other parts of the body.
What 3 major issues does the sticky mucus cause?
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Examples of producing human proteins using recombinant DNA techniques:
- insulin to treat diabetes
- factor VIII to treat haemophilia
- adenosine deaminase (ADA) made by genetically modified insect larvae to treat SCID
Factor VIII
What is Haemophila?
Haemophilia - not being able to make the Factor VIII which causes blood clotting
How is Factor VIII obtained?
The human gene for making factor VIII is inserted into hamster kidney and ovary cells. The cells constantly produce Factor VIII which is extracted and used to treat people with haemophilia.
People with haemophilia need regular injections of Factor VIII, which before the availability of recombinant Factor VIII, came from donated blood.
What's the advantage of using recombinant Factor VIII instead of blood derived Factor VIII?
Using donated blood/blood derived Factor VIII carries risks of infection e.g. HIV.
Suggest why the host cell used to produce genetically engineered Factor VIII must be a mammalian cell and not a bacterial cell [1]
only mammalian cells have golgi
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Advantages of making human proteins using bacteria, yeast and cultures of mammalian cells
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Disadvantages of making human proteins using bacteria, yeast and cultures of mammalian cells
Outline how a gene mutation may occur [4]
- a random
- change in base/nucleotide sequence
- ref. to base substitution/deletion/addition
- ref. to frameshift mutations
- mutagens
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Promoters= the region of DNA to which RNA polymerase binds as it starts transcription
- promoters recognise which of the 2 DNA strands is the template strand
- the plasmid is cut from the bacteria using a restriction endonuclease
- the gene of interest is cut from a human pancreatic cell using the same restriction endonuclease
- gene of interest inserted into plasmid to produce recombinant plasmid
- joined by DNA ligase
- the cells are cultured to make large amounts of the gene, therefore the protein
- no ethical issues
- bacteria reproduce quickly
- bacteria are cheap to culture in large amounts
- large volumes of product are produced
- don't need much production space
- simple nutritional requirements (cells easy to feed/keep alive)
- using mammalian cells is expensive
- bacteria don't have a Golgi apparatus so they don't modify proteins
- a person with cystic fibrosis is very prone to infections because it's difficult for the mucus to be removed
- the pancreatic duct may become blocked
- around 90% of men with cystic fibrosis are sterile
Cystic fibrosis is caused by a recessive allele of the gene that codes for a transporter protein CFTR.
This protein sits in the cell surface membranes of cells in the alveoli (& elsewhere in the body) and allows chloride ions to pass out of the cells.
The recessive allele codes for a faulty version of this protein that doesn't act properly as a chloride ion transporter.
In a NORMAL PERSON, the cells lining the airways + in the lungs pump out Cl- ions through the channel in the cell surface membrane formed by the CFTR.
-> high conc. of Cl- ions outside the cells. This reduces the water potential so the water potential is lower outside of the cell.
-> water moves out of the cells by osmosis, down the water potential gradient
-> it mixes with the mucus, making it thin enough to be swept away by the cilia
In someone with CYSTIC FIBROSIS, less water moves out of the cells,
-> so mucus on the surfaces of cells stay thick and sticky.
-> the cilia or coughing can't remove it all.
- Cystic fibrosis is caused by the mutation of the CFTR
- CFTR transporter protein becomes defective
- insert a normal dominant CFTR allele into the DNA in cells in the respiratory system using a vector e.g. the normal allele is inserted into the vector - liposomes taken as a spray or a harmless virus (may experience unpleasant side effects due to infection by the virus)
- the effects only last for a short period of time, the treatment needs repeating
- allele is dominant, so it'll still be expressed when the normal recessive allele is present
- gene therapy is only used to treat recessive allele disorders, as it cannot remove the dominant allele
- dominant allele affects tissues in many parts of the body
Why did liposomes (tiny balls of lipid molecules) not work as a vector?
The normal allele was inserted into liposomes, which were then sprayed as an aerosol into the volunteers' noses so the allele was introduced into a few cells lining the nose, but the effect only lasted for 1 week, because these cells have a short life span.
Why did a viral vector not work as a vector?
Some of the volunteers experiences unpleasant side effects as a result of infection by the virus.
Gene therapy involves introducing a 'correct' allele into a person's cells as a treatment for a genetic disease
So far, all attempts to do this in humans have involved placing the allele in body cells, aka somatic cells
Another possibility would be to insert the allele into germ cells (cells that are involved in sexual reproduction like gametes or an early embryo)For example, in theory, a woman with cystic fibrosis could opt to conceive a baby using IVF. Eggs would be harvested, then the correct allele of the CFTR gene would be injected into the egg Germ cell gene therapy is illegal in humansThe problem:
- All of the cells of the child are produced from a genetically engineered zygote, and therefore will all carry the gene that's been inserted
- when the child grows up and produces eggs and sperm, these gametes will contain the allele and will be passed on for generations
- In some people with cystic fibrosis, the mutation in the gene has simply replaced one base with another.
- This has created a STOP codon in the middle of the gene
- mRNA is produced in the normal way
- The translation on the ribosomes stops when this codon is reached.
- This means only a short length of CFTR is made
- The PTC124 drug allows translation to just keep going across the STOP codon so the entire protein is made
3 reasons why germ cell gene therapy is illegal in humans
- Ethical issues
- How can 'good' and 'bad' uses of gene therapy be distinguished?
- Who decides which traits are normal and which constitute a disability or disorder?
- Will the high costs of gene therapy make it only available to the wealthy?
- Could the widespread use of gene therapy make society less accepting of those who are different?
- Should people use gene therapy to enhance basic human traits e.g. height, intelligence or athletic ability?
- Potential risk due to virus vector
- The viruses and other agents used to deliver the "good" genes, if a gene is added to DNA, it could be put in the wrong place which could affect other cells and cause cancer/damage
Natural rape seed oil contains substances (erucic acid + glucosinolates) that are undesirable in oil that is to be used in human or animal food.
A hybrid, bred in Canada to produce low concentrations of these undesirable substances was called Canola (Canadian oilseed low acid).
Gene technology has been used to produce herbicide-resistant strains of oil seed rape.
Oil see rape that is resistant to herbicide glyphosate is grown in many countries.
Glyphosate inhibits an enzyme involved in the synthesis of 3 amino acids: phenylalanine, tyrosine and tryptophan.
Glyphosate is absorbed by the plant's leaves and is transported to the growing tips.
The amino acids are needed for producing essential proteins, so the plant dies.
Various microorganisms have versions of the enzyme involved in the synthesis of phenylalamine, tyrosine and tryptophan that aren't affected by glyphosate.
The gene was transferred into crop plants from a strain of bacterium Agrobacterium.
Why is it economical to kill weeds with herbicides? What 4 things do crops compete for with weeds?
Killing weeds that would otherwise compete with the crop for space, light, water or ions will increase the yield of crops.
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What are the 3 detrimental affects of herbicide resistant crops?
- the genetically modified plant will become an agricultural weed
- pollen will transfer the gene to wild relatives, producing hybrid offspring that are invasive weeds
- herbicide-resistant weeds will evolve because so much of the same herbicide is used
The risk of pollen transfer (by wind/insects)
- Oil seed rape interbreeds easily with 2 related species - resistance can be passed on to their hybrid offspring
- Safe planting distances should be increased (over 4000m) to allow the organic farming industry to maintain its 'GM-free' certification.
What are the 3 detrimental affects of insect-resistant crops?
- the evolution of resistance by the insect pests
- a damaging effect on other species of insects
- the transfer of the added gene to other species of plant
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What are the positive effects of using less pesticide?
- Reduces the risk of spray carrying to and affecting non-target species of insects in other areas
- economical
- less bioaccumulation in food chains/ less risk to humans when they eat the crops
What is the Bt toxin?
- Is a toxin that's lethal to insects to eat it but harmless to other animals
- has been taken from a bacterium (Bacillus thuringiensis)
- crops plants that contain the Bt toxin gene produce their own insecticides
What are the advantages of Bt maize?
- Chemical pesticides don't have to be used
- less harm to humans from pesticides
- Once corn borers eat the maize they'll die
- increased yield of maize
Describe the steps in genetically engineering rice to produce golden rice
- genes for the production of carotene were extracted from daffodils and bacteria
- these genes together with promoters, were inserted into plasmids
- the plasmids were inserted into bacteria
- these bacteria naturally infect plants and so could introduce the genetically modified plasmid into rice cells
- the bacteria were mixed with rice embryos in petri dishes
- the rice embryos, now containing the carotene genes, were grown into adult plants. They produced seeds containing carotene in their endosperm
What are the benefits of golden rice?
- Golden rice is a good source of vitamin A
- Places where people are poor, many had a vitamin A deficiency so golden rice could provide people with proper nutrition
- Vitamin A deficiency causes blindness and can cause an immune deficiency syndrome
Why are some people against golden rice?
Non-governmental organisations have condemned golden rice as being the wrong way to solve the problem of people having diets that are short of vitamin A. Poverty should be eradicated.
Social implications of using GMOs
- the modified crop plants may become agricultural weeds or invade natural habitats
- the introduced genes may be transferred by pollen to wild relatives whose hybrid offspring may become more invasive (the hybrid offspring become invasive because they may be 'born' polyploidy so they're able to reproduce/are non-sterile)
- the introduced genes may be transferred by pollen to unmodified plants growing on a farm with organic certification
- the modified plants may be direct hazard to humans, domestic or other beneficial animals, by being toxic or producing allergies
- the herbicide that can now be used on the crop will leave toxic residues in the crop
- genetically modified seeds are expensive, as is herbicide so their cost may not be as advantageous as thought
- growers mostly need to buy seed each season, keeping costs high - unlike for non GMO plants where the grower keeps the seed from 1 crop to sow for the next
- there's a danger of losing traditional varieties with their desirable background genes and their possibly unknown traits that might be useful in a world where the climate is changing - seed banks and growing and harvesting traditional varieties are required
- if someone finds out they carry an allele for a disease and there's a possibility they could develop this disease later on => they could take preventative measures (e.g. having breasts removed to prevent breast cancer)
- can avoid having offspring with genetic disease
- preventative treatment may be cheaper than for the disease itself
- if alleles not present, removes worry
- may terminate the pregnancy
- may terminate the pregnancy of an embryo with a genetic disease even though the defect is minor so the child can still live a normal life
- some parents have decided to terminate the pregnancy simply because the child isn't the sex that they want or they have used PGD to select the sex of the embryo that they choose to implant
- net charge - negatively charged molecules move towards the anode (+) and positively charged molecules move towards the cathode; highly charged molecules move faster than those with a lower net charge
- size - smaller molecules move through the gel faster than larger molecules
- composition of the gel - the size of the 'pores' within the gel determines the speed with which proteins and fragments of DNA move
(polyacrylimide gel for proteins & agarose gel for DNA)
- easy to extract from bacteria
- can be cut using restriction endonucleases
- may contain genes for antibiotic resistance which are used as marker genes to identify bacteria with recombinant DNA
- acts as a vector and can have a promoter
- a gene can be inserted into the plasmid
- the quantity of DNA is increased by PCR
- the DNA is cut using restriction endonucleases
- DNA fragments are loaded into wells in agarose gel
- direct current applied
- the negative phosphate groups of DNA
- are attracted to the anode (+)
- the shorter DNA fragments will move faster through the gel
- the pieces are transferred onto absorbent paper placed on top of the gel
- the paper is heated to separate the DNA strands
- probes are added
- and the paper is placed on an X-ray film
- a pattern of stripes produced
- negatively charged DNA phosphate groups
- move to the anode
- due to electric field/when current applied
- longer DNA fragments move more slowly
- ref. to gel impedance/resistance
Pros:
- can avoid having offspring with genetic disease
- can avoid late abortions
- allows couples to have children who would otherwise choose not to due to the risk of genetic disease
Cons:
- viable embryos discarded
- may conflict religious beliefs
- could lead to selection based on gender or specific traits
- shows which genes are expressed by an organism
- a microarray is based on a small piece of glass
- short lengths of single-stranded DNA (probes) are attached at known positions to the slide/chip
- take DNA from a sample and cut using restriction endonucleases
- denature to make it single stranded
- the DNA is labelled with fluorescent tags
- the labelled DNA samples will hybridise with the complementary probes
- any DNA that doesn't bind to probes on the microarray is washed off
- the microarray is inspected using UV light causing the tags to fluoresce
- UV light is used to detect the presence of hybridised probes
- identifies which genes are expressed by an organism
- transcription of a gene produces mRNA (specific to question)
- probes are short lengths of single-stranded DNA
- single-stranded DNA is attached at known positions to a slide/chip
- cDNA binds to complementary probe
- hybridised probes are shown as fluorescent spots
- positions recorded by laser/scanner
- mRNA is taken from the B cells (in the islets of Langerhans in the pancreas)
- reverse transcriptase enzyme reverses transcription using mRNA as a template to
- make single stranded DNA
- DNA polymerase is used to make the single stranded DNA double stranded (by assembling nucleotides to make the complementary strand)
- restriction endonucleases cut the DNA to produce sticky ends
- plasmids are cut from bacteria using
- the same restriction endonuclease
- to produce complementary sticky ends
- the insulin gene is inserted into plasmid to produce recombinant plasmid
- joined by DNA ligase
Describe how the gene coding for human insulin can be obtained and inserted into a plasmid vector [8] marking points
Amniocentesis
Amniocentesis is used to obtain a sample of amniotic fluid at 15-16 weeks of pregnancy. Various tests can be carried out on this sample to check the health of the fetus.
Most amniocentesis samples, however, are to look for chromosomal mutations.
Chorionic villus sampling (CVS)
Can be carried out between 10-13 weeks of pregnancy, so it allows parents to get an earlier warning of any genetic abnormalities in the fetus than is possible with amniocentesis.
A small sample of part of the placenta (chorion) is removed by a needle.
Both procedures are monitored by ultrasound scanning, used to visualise the fetus and locate the position of the placenta, fetus + umbilical cord.
A suitable point for the insertion of the hypodermic syringe needle is chosen & marked on the abdominal skin surface.
- To get the plasmids the bacteria is treated with enzymes to breakdown their cell walls
- the 'naked' bacteria is spun at high speed in a centrifuge
- the plasmid is cut open using a restriction enzyme
- the same restriction enzyme is used to cut out the gene, to produce complementary sticky ends
- the opened plasmids & the lengths of DNA are mixed together causing pairing of sticky ends
- DNA ligase is used to link together the sugar-phosphate backbones of the DNA molecule & the plasmid, producing recombinant DNA
- the bacteria are put into a solution with a high conc. of calcium ions
- then cooled & given a heat shock to increase the chances of plasmids passing through the cell surface membrane
Herbicide-resistant crops - Oil seed rape
- gene technology has been used to produce herbicide resistant strains of oil seed rape
- oil seed rape has been made resistant to herbicide glyphosate
- the gene was transferred into crop plants from bacteria
How does herbicide glyphosate work?
- glyphosate inhibits an enzyme involved in the synthesis of 3 amino acids: phenylalanine, tyrosine + tryptophan
- glyphosate is absorbed by the plant's leaves & is transported to the growing tips
- the amino acids are needed for producing essential proteins, so the plant dies
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By using genetic markers:
- If the genetic marker is antibiotic resistance,...
- this is done by spreading bacteria on agar plates each containing a antibiotic
- you'll know if the DNA is recombinant DNA if the DNA isn't able to grow on the agar containing the specific antibiotic (because the gene is inserted into the plasmid replacing the antibiotic resistance gene) ?
- Use Jellyfish GFP gene - any transformed bacteria will glow green under UV
- Use E.Coli GUS gene - any transformed cell can react with a dye & it'll fluoresce