Flashcards in DNA technology Deck (113):
Why did old method of treatment of diseases that resulted from individuals being unable to produce for themselves various metabolic chemicals pose problems?
Previously involved extracting the chemical from a human or animal donor and introducing it into the patient. This presents problems such as rejection by the immune system and risk of infection. Cost is also considerable.
RECOMBINANT DNA TECHNOLOGY
General term that covers the processes by which genes are manipulated altered or transferred from organism to organism. Also known as genetic engineering.
The DNA of two different organisms that has been combined.
GENETICALLY MODIFIED ORGANISM
Organism that has had its DNA altered as a result of recombinant DNA technology.
Describe the 5 stages of the process of making a protein using the DNA technology of gene transfer and cloning.
1. ISOLATION of the DNA fragments that have the gene for the desired protein.
2. INSERTION of the DNA fragment into a vector.
3. TRANSFORMATION, that is, the transfer of DNA into suitable host cells.
4. IDENTIFICATION of the host cells that have successfully taken up the gene by use of gene markers.
5. GROWTH/CLONING of the population of host cells.
What are the two methods of identification and isolation of a gene?
-Using reverse transcriptase
-Using restriction endonucleases
What are retroviruses?
Retroviruses are a group of viruses of which the best known is human immunodeficiency virus (HIV). The genetic information of retroviruses is in the form of RNA, however they are able to synthesise DNA from their RNA using an enzyme called reverse transcriptase (called so because it catalyses the production of DNA to RNA which is the reverse of usually RNA to DNA)
What are restriction endonucleases?
Enzymes that cut up viral DNA?
How do bacteria use restriction endonucleases?
Bacteria are frequently invaded by viruses that inject their DNA into them in order to take over the cell. Some bacteria defend themselves by producing enzymes that cut up the viral DNA.
What do restriction endonucleases do and what are the two ways they do it?
There are many types of restriction endonucleases. Each one cuts a DNA double strand at a specific sequence of bases called a recognition sequence.
Sometimes, this cut occurs between two opposite base pairs.This leaves two straight edges known as blunt ends.
Other restriction endonucleases cut DNA in a staggered fashion. This leaves an uneven cut in which each strand of the DNA has exposed, unpaired bases.
What are the two ways of cloning fragments of DNA so that there is a sufficient quantity for medical or commercial use?
-in vivo, by transferring the fragments to a host cell using a vector.
-in vitro, using the polymerase chain reaction
the sequences of DNA that are cut by restriction endonucleases.
The nucleotides on the single strand at one side of the cut (sticky end are complementary..
to those at the other side because they were previously paired together.
What does it mean if the same restriction endonuclease is used to cut DNA?
Then all the fragments produced will have ends that are complementary to one another. This means that the single-stranded end of any one fragment van be joined (stuck) to the single stranded end of any other fragment.
What is DNA ligase used for?
Once the complementary bases of two "sticky ends" have paired up, an enzyme called DNA ligase is used to join the phosphate-sugar framework of the two sections of DNA and so unite them as one.
Why are sticky ends important?
Provided the same restriction endonuclease is used, we can combine the DNA of one organism with that of any other organism.
What is a vector used for?
To transport the DNA into the host cell.
What is the most common vector?
What are plasmids?
Circular lengths of DNA, found in bacteria, which are separate from the main bacterial DNA.
Describe the insertion of DNA fragment into a vector.
Once an appropriate fragment of DNA has been cut from the rest of the DNA, the next task is to join it into a carrying unit, known as a vector. The vector is used to transport DNA into a host cell.
Usually a plasmid.
Plasmids almost always contain genes for antibiotic resistance, and restriction endonucleases are used at one of these antibiotic-resistant genes to break up the plasmid loop.
The restriction endonucleases used is the same as the one that cut out the DNA fragment. This ensures that the "sticky ends" of the opened-up plasmids are complementary to the "sticky ends" of the DNA fragment.
When the DNA fragments are mixed with the opened-up plasmids, they may become incorporated into them. Where they are incorporated, the join is made permanent using the enzyme DNA ligase. These plasmids now have recombinant DNA.
What must happen after the incorporation of a DNA fragment into a vector? How does it work?
Once the DNA has been incorporated into at least some of the plasmids, they must then be reintroduced into bacterial cells.
This process is called transformation and involves the plasmids and bacterial cells being mixed together in a medium containing calcium ions.
The calcium ions, and changes in temperature, make the bacteria permeable, allowing the plasmids to pass through the cell membrane into the cytoplasm.
However, not all bacterial cells will possess the DNA fragments.
What are two reasons why not all bacterial cells will possess the DNA fragments?
-Only a few bacterial cells (as few as 1%) take up the plasmids when the two are mixed together.
-Some plasmids will have closed up again without incorporating the DNA fragment.
Describe how bacterial cells that have taken up the plasmid are identified. (R-plasmid with gene for resistance to ampicillin and tetracycline)
The task of finding out which bacterial cells have taken up the plasmids entails using the gene for antibiotic resistance, which is unaffected by the introduction of the new gene. This is the gene for resistance to ampicillin. The process works as follows:
-All the bacterial cells are grown on a medium that contains the antibiotic ampicillin.
-Bacterial cells that have taken up the plasmids will have acquired the gene for ampicillin resistance.
-These bacterial cells are unable to break down the ampicillin and therefore survive.
-The bacterial cells that have not taken up the plasmids will not be resistant to ampicillin and therefore die.
What is the issue with identification of the bacterial cells have taken up the plasmids?
Some cells will have taken up the plasmids and then closed up without incorporating the new gene, and these will also have survived. The next task is to identify these cells and eliminate them.Achieved using gene markers.
What do gene markers involve?
Using a second, separate gene on the plasmid which is easily identifiable.
Why is the second gene on a plasmid easily identifiable?
-It may be resistant to an antibiotic.
-It may make a fluorescent protein that is easily seen.
-It may produce and enzyme whose action can be identified.
How are the bacterial cells that have taken up the new gene identified?
This process uses the other antibiotic-resistant gene in the plasmid: the gene that was cut in order to incorporate the required gene. In the ampicillin/tetracycline example, this gene is tetracycline. As this gene has been cut, it will no longer produce the enzyme that breaks down tetracycline. In other words, the bacteria that have taken up the required gene will no longer be resistant to tetracycline. We can therefore identify these bacteria by growing them on a culture that contains tetracycline.
What is the problem with the treatment with tetracycline?
It will destroy the very cells that contain the required gene.
How does replica plating work?
Used to identify those cells with plasmids that have taken up the new gene.
-The bacterial cells that survived treatment with the first antibiotic (ampicillin) are known to have taken up the plasmid.
-These cells are cultured by spreading them very thinly on nutrient agar plates.
-Each separate cell on the plate will grow into genetically identical colony.
-A tiny sample of each colony is transferred onto a second (replica) plate in exactly the same position as the colonies on the original plate.
-This replica plate contains the second antibiotic (tetracycline), against which the antibiotic-resistance gene will have been made useless if the new gene has been taken up.
-The colonies killed by the antibiotic must be the ones that have taken up the required gene.
-The colonies in exactly the same position on the original plate are the ones that possess the required gene. These colonies are therefore made up of bacteria that have been genetically modified, that is, have been transformed.
Describe fluorescent markers.
Transference of gene from a jellyfish into the plasmid. The gene in question produces a green fluorescent protein (GFP). The gene to be cloned is transplanted into the centre of the GFP gene. Any bacterial cell that has taken up the plasmid with the gene that is to be cloned will not be able to produce GFP. Unlike the cells that have taken up the gene, these cells will not fluoresce. A the bacterial cells with the desired gene are not killed, there is no need for replica plating. Results can be obtained by simply viewing the cells under a microscope and retaining those that do not fluoresce.
Describe enzyme markers.
Another gene marker is the gene that produces the enzyme lactase. Lactase will turn a particular colourless substrate blue. The required gene is transplanted into the gene that makes lactase. If a plasmid with the required gene is present in a bacterial cell, the colonies grown from it will not produce lactase. Therefore, when these bacterial cells are grown on the colourless substrate they will be unable to change its colour. Where the gene has not transformed the bacteria, the colonies will turn the substrate blue.
POLYMERASE CHAIN REACTION
A method of copying fragments of DNA. The process is automated, making it both rapid and efficient.
What does PCR require? (5)
-THE DNA FRAGMENT to be copied
-DNA POLYMERASE- an enzyme capable of joining another tens of thousands of nucleotides in a matter of minutes. It is obtained from bacteria in hot springs and is therefore tolerant to heat (thermostable) and does not denature during the high temperatures of the process.
-PRIMERS- short sequences of nucleotides that have a set of bases complementary to those at one end of each of the two DNA fragments.
-NUCLEOTIDES- which contain each of the four bases found in DNA.
-THERMOCYCLER- a computer-controlled machine that varies temperature precisely over a period of time.
What are the three stages of PCR?
1. SEPARATION OF THE DNA STRAND. The DNA fragments, primers and DNA polymerase are placed in a vessel in the thermocycler. The temperature is increased to 95C, causing the two strands of the DNA fragments to separate.
2.ADDITION (annealing) OF THE PRIMERS. The mixture is cooled to 55C causing the primers to join (anneal) to their complementary bases at the end of the DNA fragment. The primers provide the starting sequences for DNA polymerase to begin DNA copying because DNA polymerase can only attach nucleotides to the end of an existing chain. Primers also prevent the two separate strands from simply rejoining.
3. SYNTHESIS OF DNA. The temperature is increased to 72C. This is the optimum temperature for the DNA polymerase to add complementary nucleotides along each of the separated DNA strands. It begins at the primer on both strands and adds the nucleotides in a sequence until it reaches the end of the chain.
Process is repeated
Why are two copies of the original fragment produced in PCR?
Both separated strands are copied.
Roughly how long doe the temperature cycle take?
What are the advantages of in vitro gene cloning?
-EXTREMELY RAPID. Within a few hours a 100 billion copies of a gene can be made. Valuable where only a minute amount of DNA is available, for example, at the scene of a crime. This can quickly be increased using PCR and so there is no loss of valuable time before forensic analysis and matching can take place. In vivo cloning would take many days or weeks to produce the same quantity.
-DOES NOT REQUIRE LIVING CELLS. All that is required is a base sequence of DNA that needs amplification. No complex culturing techniques, requiring time and effort are needed.
What are the advantages of in vivo cloning?
-PARTICULARLY USEFUL WHERE WE WISH TO INTRODUCE A GENE INTO ANOTHER ORGANISM. As it involves the use of vectors, once we have introduced the gene into a plasmid, this plasmid can be used to deliver the gene into another organism, such as a human being (i.e. it can transform other organisms). This is done by using a technique called gene therapy.
-IT INVOLVES ALMOST NO RISK OF CONTAMINATION. A gene that has been cut by the same restriction endonucleases can match the "sticky ends" of the opened up plasmid. Contaminant DNA will therefore not be taken up by the plasmid. In vitro cloning requires a very pure sample because any contaminant DNA will also be multiplied and could lead to a false result.
-IT IS VERY ACCURATE. The DNA copied has few, if any errors. However, any errors in copying DNA or any contaminants in the sample will also be copied into subsequent cycles.
-IT CUTS OUT SPECIFIC GENES. It is therefore a very precise procedure as the culturing of transformed bacteria produces many copies of a specific gene and not just copies of the whole DNA sample.
-IT PRODUCES TRANSFORMED BACTERIA THAT CAN BE USED TO PRODUCE LARGE QUANTITIES OF GENE PRODUCTS. The transformed bacteria can produce proteins for commercial or medical use.
How can the genetic make up of organisms now be altered?
By transferring genes between individuals of the same species or between organisms of different species.
How can genetic modification benefit humans?
-increasing the yield from animals or plant crops.
-improving the nutrient content of foods.
-introducing resistance to disease and pests.
-making crop plants tolerant to herbicides.
-developing tolerance to environmental conditions.
-producing medicines for treating disease.
Examples of substances produced by GM microorganisms (3).
-ANTIBIOTICS are produced naturally by bacteria. Although genetic engineering has not substantially improved the quality of antibiotics, it has produced bacteria that increases the quantity of the antibiotics produced and the rate at which they are made.
-HORMONES- insulin is needed daily by more than 2 million diabetics, in order for them to lead normal lives. Bacterial cells have the human insulin gene incorporated into them and so the insulin produced is identical to human insulin. Other hormones produced this way include, human growth hormone, cortisone and the sex hormones (oestrogen and testosterone).
-ENZYMES- many enzymes used in the food industry are manufactured by genetically modified bacteria. These include amylases used to break down starch during beer production, lipases used to improve the flavour of cheeses and proteases used to tenderise meat.
What are the advantages of using genetic modification to make insulin?
Has no adverse effects on the patient. Method avoids killing animals and the need to modify insulin before it is injected into humans.
Examples of GM plants. (5)
-GM TOMATOES have been developed using the insertion of a gene. This gene has a base sequence that is complementary to that of the gene producing the enzyme that causes the tomatoes to soften. The mRNA transcribed from this inserted gene is therefore complementary to the mRNA of the original gene. The two therefore combine to form a double-strand. This prevents the mRNA of the original gene from being translated. The softening enzyme is therefore not produced.
-HERBICIDE-RESISTANT CROPS have a gene introduced that makes them resistant to a specific herbicide. When the herbicide is sprayed on the crops, the weeds that are competing with the crop plants for water, light and minerals, are killed. The crop plants are resistant to the herbicide so are killed.
-DISEASE-RESISTANT CROPS have genes introduced that give resistance to specific diseases. GM rice, for example, can withstand infection by a particular virus.
-PEST-RESISTANT CROPS, e.g. maize, can have a gene added that allows the plant to make a toxin. This toxin kills insects that eat the maize, but is harmless to other animals including humans.
-PLANTS THAT PRODUCE PLASTICS are a possibility currently being explored. It is hoped that we can genetically engineer plants that have the metabolic pathways necessary to make the raw material for plastic production.
Examples of GM animals.
The transfer of genes from an animal that has natural resistance to a disease into a totally different animal. This second animal is then made resistant to that disease. In this way domesticated animals can be more economic to rear and hence help to reduce the price of food production.
Fast growing food animals such as sheep and fish that have a growth hormone gene added so that, in the case of salmon, they can grow 30X larger than normal and at 10X the rate.
Production of rare and expensive proteins for use in human medicine. Domesticated milk producing animals such as goats can be used. The gene for the required protein is inserted alongside the gene that codes for proteins in goats' milk. In this way the required protein is produced in the milk of the goat. the gene can be inserted into the fertilised egg of the goat, so that all the female offspring of that individual will be capable of producing the protein in their milk. One example of a protein made in this way is a protein that prevents blood from clotting (anticoagulant) called anti-thrombin.
Some individuals have an inherited disorder that affects one of the alleles that codes for the protein anti-thrombin. As a result, those affected are unable to produce sufficient quantities of anti-thrombin. These individuals are therefore at risk of blood clots. They are currently treated with drugs that thin the blood or are given anti-thrombin that has been extracted from donated blood.
Describe how anti-thrombin is produced in the milk of GM goats.
-Mature eggs are removed from female goats and fertilised by sperm.
-The normal gene for anti-thrombin production from a human is added to the fertilised eggs alongside the gene that codes for proteins in goats' milk.
-These genetically transformed eggs are implanted into female goats.
-Those resulting goats with the anti-thrombin gene are cross-bred, to give a herd in which goats produce milk rich in anti-thrombin.
-The anti-thrombin is extracted from the milk, purified and given to humans unable to manufacture their own anti-thrombin.
What is another means of producing drugs from genetically modified animals?
Domesticated chickens have had human genes for medicinal proteins added to their DNA. The eggs laid by these transgenic chickens contain the proteins in the white portion from which they can be easily extracted. The human genes are passed on from gen. to gen. Large flocks can therefore be formed that offer a potentially unlimited source of cheap medicinal proteins. Drugs that have so far been produced in this way include a form of interferon used to treat multiple sclerosis and an antibody with the potential to treat skin cancer and arthritis.
What are the agricultural benefits of GMO to humans?
-Higher yields and more nutritious crop, reduce risk of famine and malnutrition.
-Pest resistance so fewer pesticides used, reduces costs and fewer environmental problems.
What is an example of a GM crop? (Beneficial to agriculture)
GOLDEN RICE. It contains one gene from a maize plant and one gene from a soil bacterium, which together enable the rice to produce beta-carotene. The beta-carotene is used by our bodies to produce Vitamin A.
Being developed to reduce vitamin A deficiency in areas where there's a shortage of dietary vitamin A, e.g. South Asia, Africa. Vitamin A deficiency is a big problem in these areas, e.g. up to 500000 children per year go blind due to vit A deficiency.
What are the industrial benefits of GMO to humans?
-Industrial processes often use biological catalysts (enzymes). These enzymes can be produced from transformed organisms, so they can be produced in large quantities for less money, reducing crops.
What is an example of a GMO? (Beneficial in industry)
Chymosin (rennin) is an enzyme used in cheese making. It used to be made from rennet (a substance produced in the stomach of cows), but it can now be produced by GMOs. This means it can be made in large quantities, relatively cheaply and without killing any cows, making some cheese suitable for vegetarians.
What are the medicinal benefits of GMO to humans?
-Many drugs and vaccines are produced by transformed organisms, using recombinant DNA technology. They can be made quickly, cheaply and in large quantities using this method.
What are the agricultural concerns of the use of GMOs?
-Farmers might plant only one type of GM crop (monoculture). This could make the whole crop vulnerable to disease because the plants are genetically identical.
-Some people are concerned about the possibility of "superweeds"- weeds that are resistant to herbicides. These could occur if transformed crops interbreed with wild plants.
What are the industrial concerns with the use of GMOs?
-Without proper labelling some people think they won't have a choice about whether to consume food made using genetically engineered organisms.
-Some people are worried that the process used to purify proteins (from GMOs) could lead to the introduction of toxins into the food industry.
What are the medicinal concerns with the use of GMOs?
-Companies who own GM technologies may limit the use of technologies that could be saving lives.
-Some people worry this technology could be used unethically. e.g. to make designer babies. Currently illegal.
What is cystic fibrosis?
The most common genetic disorder among the white population of Europe and North America, with around 1 in every 20000 people having the disease.
What causes cystic fibrosis?
It is caused by a mutant recessive allele in which three DNA bases, AAA, are missing. It is therefore and example of a deletion mutation.
The normal gene, called the cystic fibrosis trans-membrane-conduction regulator (CFTR) gene, normally produces a protein of some 1480 amino acids. The deletion results in a single amino acid being left out of the protein.
This, however, is enough to make the protein unable to perform its role of transporting chloride ions across epithelial membranes. CFTR is a chloride ion channel protein that transports chloride ions out of epithelial cells, and water naturally follows, by the process of osmosis. In this way, epithelial membranes are kept moist.
In a patient with cystic fibrosis, the defective gene means that the protein is either not made or does not function normally. The epithelial membranes are therefore dry and the mucus they produce remains viscous and sticky.
What are the symptoms of cystic fibrosis?
-Mucus congestion in the lungs, leading to a much higher risk of infection because the mucus,which traps disease causing organisms, cannot be removed.
-Breathing difficulties and less efficient gaseous exchange.
-Accumulation of thick mucus in the pancreatic ducts, preventing pancreatic enzymes from reaching the duodenum and leading to the formation of fibrous cysts.
-Accumulation of thick mucus in the sperm ducts in males, possibly leading to infertility.
What are the two ways that gene therapy may be used to treat cystic fibrosis?
-GENE REPLACEMENT, in which the defective gene is replaced with a healthy gene.
-GENE SUPPLEMENTATION, in which one or more copies of the healthy gene are added alongside the defective gene. As the added genes have dominant alleles, the effects of the recessive alleles of the defective gene are masked.
What are the two different techniques of gene therapy?
-GERM-LINE GENE THERAPY involves replacing or supplementing the defective gene in the fertilised egg. This ensures that all cells of the organism will develop normally, as well as the cells of their offspring. This is therefore much more permanent solution, affecting future generations. Currently prohibited.
-SOMATIC-CELL GENE THERAPY targets just the affected tissues, such as the lungs, and the additional gene is therefore not present in the sperm or egg cells, and so is not passed on to future generations. As the cells of the lungs are continually dying and being replaced, the treatment needs to be repeated periodically, as often as every few days. At present, the treatment has limited success. Long term aim is to target undifferentiated stem cells that give rise to mature tissues.
What is the aim of somatic gene cell therapy?
To introduce cloned normal genes into the epithelial cells of the lungs.
What are the two ways that cloned normal genes are introduced into the epithelial cells of the lungs?
-Using a harmless virus.
-Wrapping the gene in lipid molecules.
Describe the process of using a harmless virus to introduce cloned normal genes into the epithelial cells of the lungs.
Adenoviruses make useful vectors for the transfer of the normal CTFR gene into the host cells. Process works as follows:
-The adenovirsues are made harmless by interfering with a gene involved in their replication
-These adenoviruses are then grown in epithelial cells in the laboratory along with plasmids that have has the normal CFTR gene inserted.
-The CFTR gene becomes incorporated into the DNA of the adenoviruses.
-These adenoviruses are isolated from the epithelial cells and purified.
-The adenovirsuses with the CFTR gene are introduced into the nostrils of the patients.
-The adenoviruses inject their DNA, which includes the normal CFTR gene, into the epithelial cells of the lungs.
Cause colds and other respiratory diseases by injecting their DNA into the epithelial cells of the lungs. Make useful vectors.
Why are genes wrapped in lipid molecules?
Lipid molecules can relatively easily pass through the phospholipid portion of the cell-surface membranes.
Describe the process of delivering the CFTR genes to their target cells. (wrapping genes in lipid molecules)
-CFTR genes are isolated from healthy human tissue and inserted into bacterial plasmid vectors.
-The plasmid vectors are reintroduced into their bacterial host cells and gene markers are used to detect which bacteria have successfully taken up the plasmids with the CFTR gene.
-These bacteria are cloned to produce multiple copies of the plasmids with the CFTR gene.
-The plasmids are extracted from the bacteria and wrapped in lipid molecules to form a liposome.
-The liposomes pass across the phospholipid portion of the cell-surface membrane of the lung epithelial cells.
Why are the forms of delivery to the lungs not always effective?
-Adenoviruses may cause infections.
-Patients may develop immunity to adenoviruses.
-The liposome aerosol may not be fine enough to pass through the tiny bronchioles in the lungs.
-Even where the CFTR gene is successfully delivered to the epithelial cells, very few are actually expressed.
What is severe combined immunodeficiency (SCID)?
Rare inherited disorder.
People who suffer from this condition do not show a cell-mediated immune response nor are they able to produce antibodies.
When does SCID arise?
When individuals inherit a defect in the gene that codes for the enzyme ADA (adenosine deaminase). This enzyme destroys toxins that would otherwise kill white blood cells.
What has survival of SCID relied on?
Patients being raised in the strictly sterile environment of an isolation tent, or "bubble", and giving them bone marrow transplants and/or injections of ADA.
How is SCID been attempted to be treated recently using gene therapy?
-The normal ADA gene is isolated from healthy human tissue using restriction endonucleases.
-The ADA gene is inserted into a retrovirus.
-The retroviruses are grown with the host cells in the laboratory to increase their number and hence the number of copies of the ADA gene.
-The retroviruses are mixed with the patients T-cells (a type of white blood cell)
-The retroviruses inject a copy of the normal ADA gene, into the T-cells.
-The T-cells are reintroduced into the patient's blood to provide the genetic code needed to make ADA.
Why is the effectiveness of the method of treating SCID by gene therapy limited?
T-cells live for only 6-12 months and so the treatment has to be repeated at intervals.
What is a more long term way of treating SCID? What is the drawbacks?
More recent treatment involves transforming bone marrow stem cells rather than T-cells. Because bone marrow stem cells divide to produce T-cells, there is a constant supply of the ADA gene and hence the enzyme ADA.
Although not totally effective because there is an increased risk of leukaemia.
What are the reasons for the limited success of gene therapy?
-THE EFFECT IS SHORT LIVED. Because the somatic cells, which have a cloned gene added, are not passed on to the daughter cells repeat treatments are necessary for the therapy to have any effect.
-IT CAN INDUCE AN IMMUNE RESPONSE. Both the gene that is being introduced and the structure used to deliver it (vector or liposome) can induce an immune response in the recipient. Means it is often rejected. This is made worse by the fact that the immune system typically responds to foreign material by making antibodies, some of which remain to initiate an even greater response to a future infection.
-USING VIRAL VECTORS TO DELIVER THE GENE PRESENTS PROBLEMS. Viruses can lead to toxic, inflammatory and immune responses in the recipients. There is also the fear that the disabled virus might recover the ability to cause disease once inside the patient.
-THE GENES ARE NOT ALWAYS EXPRESSED. Even if successfully delivered to their target cells, only a small proportion of the introduced genes are usually expressed.
-IT IS NOT EFFECTIVE IN TREATING CONDITIONS THAT ARISE IN MORE THAN ONE GENE. Gene therapy works best in disorders that are the result of a single mutation. However, many commonly occurring disorders, such as arthritis. heart disease and Alzehimer's disease, are the result of variations in a number of genes.
Why can knowing which specific mutation a type of cancer is usually caused by affect diagnosis? Example.
If the specific mutation is known then often more sensitive tests can be developed which can lead to earlier and more accurate diagnosis, improving the chances of recovery.
FOR EXAMPLE, there's a mutation in the RAS proto-oncogene in around half of all bowel cancers. Bowel cancer can be detected early by looking for RAS mutations in the DNA of bowel cells.
How does knowing which specific mutation the cancer is caused by affect treatment? Examples.
1. The treatment can be different for different mutations. FOR EXAMPLE, breast cancer caused by mutation of the HER2 proto-oncogene can be treated with a drug called Herceptin. This drug binds specifically to the altered HER2 protein receptor and suppresses cell division and tumour growth. Breast cancer caused by other mutations is not treated with this drug as it doesn't work.
2. The aggressiveness of the treatment can differ depending on the mutation. Different mutations produce different types of cancer, which affects the treatment. FOR EXAMPLE, if the mutation is known to cause an aggressive cancer, it may be treated with higher doses of radiotherapy or by removing larger areas of the tumour and surrounding tissue during surgery.
3. If the specific mutation is known, gene therapy may be able to treat it. FOR EXAMPLE, if you know it's caused by inactivated tumour suppressor genes, gene therapy could be used to provide working versions of the genes.
What can individuals with a family history of cancer do?
Have DNA analysed to see if they carry the specific mutation.
Avoid getting extra mutations by avoiding mutagenic agents.
If a mutation causes a very high risk of cancer what can a person do? Example.
Preventative surgery may be carried out- removing the organ the cancers is likely to affect before cancer develops.
FOR EXAMPLE, women with a mutation in BRCA1 sometimes choose to have a mastectomy to prevent breast cancer from developing.
Increased screening can lead to earlier detection and increased chances of recovery. FOR EXAMPLE, frequent colonoscopies for those with a mutated APC gene to diagnose hereditary colon cancer earlier.
A short, single stranded section of DNA that has some sort of label attached that makes it easily identifiable.
What are the two most commonly used probe?
-Radioactively labelled probes, which are made up of nucleotides with isotope 32P. The probe is identified using a photographic plate that is exposed by radioactivity.
-Fluorescently labelled probes, which emit light under certain conditions.
How are DNA probes used to identify a particular gene?
-A DNA probe is made that has bases that are complementary to the portion of the DNA sequence that makes up part of the gene whose position we want to find.
-The DNA that is being tested is treated to separate its two strands.
-The separated DNA strands are mixed with the probe, which binds to the complementary bases on one of the strands. This is known as DNA hybridisation.
-The site at which the probe binds can be identified by the radioactivity or fluorescence that the probe emits.
What needs to be done before a specific probe can be made?
Need to to know the sequence of nucleotides in the particular gene that we are trying to locate.
What is needed for the Sanger method?
Uses modified nucleotides that cannot attach to the next base in the sequence when they are being joined together. They therefore act as terminators, ending the synthesis of a DNA strand. Four different terminator nucleotides are used, each with one of four base A,T,G or C.
What is required in the four test tubes in the Sanger method
-Many single stranded fragments of the DNA to be sequenced. This acts as a template for the synthesis of its complementary strand.
-A mixture of nucleotides with the bases A, T, G and C.
-A small quantity of one of the four terminator nucleotides (test tube 1 - adenine terminator nucleotide, test tube 2- thymine terminator, etc.)
-A primer to start the process of DNA synthesis. This primer is radioactively labelled or labelled with a fluorescent dye.
-DNA polymerase to catalyse DNA synthesis.
Describe the Sanger method.
As the binding of nucleotides to the template is a random process, the addition of a normal nucleotide or template is a random process, the addition of normal nucleotide or a terminator nucleotide, is equally likely.
Depending upon exactly where the terminator nucleotide binds to the DNA template, DNA synthesis may be terminated after only a few nucleotides or after a long fragment of DNA has been synthesised.
As a result, the DNA fragments in each test tube will be of varying lengths.
What do all the DNA fragments in any of the test tubes have in common?
All the fragments of new DNA will all end with a nucleotide that has the same base; adenine in tube 1, thymine in tube 2, etc.
How are the DNA fragments identified?
Because the primer attached to the other end of the DNA section is labelled radioactively or with a fluorescent dye.
Describe gel electrophoresis.
The DNA fragments are placed on to an agar gel and a voltage is applied across it. The resistance of the gel means that the larger the fragments, the more slowly they move.
Therefore over a fixed period the smaller fragments move further than larger ones.
In this way DNA fragments of different lengths are separated.
A sheet of photographic film is then placed over the agar gel for several hours.
The radioactivity from each DNA fragment exposes the film and shows where it is situated on the gel.
How do you read the results of a DNA sequencing experiment?
Read from lightest to heaviest.
Lightest- one base long so goes to end, etc.
What needs to happen to genes larger than 500 base pairs?
cut into smaller pieces by restriction endonucleases and each fragment sequenced.
Have to be put back together- restriction mapping.
What does restriction mapping involve?
Cutting DNA with a series of different restriction endonucleases.
The fragments produced are then separated by gel electrophoresis.
The distance between the recognition sites can be determined by the patterns of fragments that are produced.
Describe the automation of DNA sequencing and restriction mapping.
In computerised systems, instead of radioactively labelling the DNA primer, the four types of terminators are labelled with fluorescent dye. Each type takes up a different colour: adenine (green), thymine (red), cytosine (blue) and guanine (yellow). The DNA synthesis takes place in a single test tube and is speeded up by using PCR cycles.
Electrophoresis is carried out in a single narrow capillary gel.
The results are scanned by lasers and interpreted by computer software, giving the DNA sequence in a fraction of the time taken by conventional methods.
Further automation includes the use of polymerase chain reaction machine to produce the DNA fragments required in these techniques.
What happens if a mutation results in a dominant allele?
all individuals will have the genetic disorders.
What happens if a mutation results in a recessive allele?
It will only be apparent in those individuals that have two recessive alleles. Homozygous recessive.
Individuals that are heterozygous recessive will be carriers. .
Why is it important to screen individuals who may carry a mutant allele?
Often have a family history of the disease.
Screening can determine the probabilities of a couple having offspring with a genetic disorder.
As a result, potential parents who are at risk can obtain advice from a genetic counsellor about the implications of having children, based on their family history and the results of genetic screening.
How is people's DNA screened for genetic diseases?
It is possible to fix hundred of different DNA probes in an array on the glass slide. By adding a sample of DNA to the array, any complementary DNA sequences in the donor DNA will bind to one or more probes. In this way it is possible to test simultaneously for many different genetic disorders.
Why is genetic screening valuable in the detection of oncogenes?
Oncogenes are responsible for cancer. Cancer may develop as a result of mutations that prevent the tumour suppressor genes inhibiting cell division.
Mutations of both alleles must be present to inactivate the tumour suppressor genes and to initiate the development of a tumour.
Some people inherit one mutated tumour suppressor gene. These individuals are at a greater risk of developing cancer.
If a mutated gene is detected by genetic screening, individuals who are at greater risk of cancer can then make informed decisions about their lifestyle and future treatment.
Check themselves more regularly.
What is genetic counselling?
Like a special form of social work, where advice and information are given that enable people to make personal decisions about themselves or their offspring.
What is an important aspect of genetic counselling?
To research the family history of an inherited disease and to advise parents on the likelihood of it arising in their children.
What can a genetic counsellor do?
The genetic counsellor can make the couple aware of how likely they are to pass disease to their offspring.
Can also inform the couple of the effects of the disease and it's emotional, physiological, medical, social and economic consequences.
Also make couple aware of any further tests that might give a more accurate prediction of whether their children will have the condition.
In the case of cancer, what can screening help to detect?
-Oncogene mutations, which can determine the type of cancer that the patient has and hence the most effective drug or radiotherapy to use.
-Gene changes that predict which patients are more likely to benefit from certain treatments and have the best chances of survival.
-a single cancer cell among millions of normal cells, thus identifying patients at risk of relapse from certain forms of leukaemia.
What is sickle cell anaemia?
The first human disease to be successfully understood at the molecular level.
Illustrates how the smallest mutations can significatnly influence the phenotype.
It is the result of a mutation in the gene producing haemoglobin.
What is the result of the mutation in the gene producing haemoglobin? (sickle cell anaemia)
In the DNA molecule that produces one of the amino acid chains in haemoglobin, the single nucleotide base adenine is substituted by the nucleotide thymine.
CTC -> CAC
mRNA produces a different code.
Code for amino acid valine rather than for glutamic acid.
This difference creates a molecule of haemoglobin (haemoglobin S) that has a sticky patch.
When the haemoglobin is not carrying oxygen they tend to adhere to one another by their sticky patches and become insoluble, forming long fibres within the red blood cells.
These fibres distort the red blood cells, making them inflexible and sickle (crescent) shaped. Sickle cells are unable to carry oxygen and may block smaller capillaries because their diameter is greater than that of the capillaries.
Why is sickle cell anaemia relatively common in some parts of Africa?
Sickle cell anaemia is the result of a gene that has two co-dominant alleles, Hb^A (normal) and Hb^S (sickled).
The malarial parasite, Plasmodium, is unable to exist in sickled red blood cells.
If homozygous for haemoglobin S- sickle cell anaemia outweighs advantage of being resistant to one form of malaria and so individuals are always selected against.
If homozygous for haemoglobin A, no sickle cell anaemia but susceptible to malaria.
If heterozygous, individuals have sickle cells trait, but are not badly affected, except when O2 conc. of blood is low. Sufferers may become more tired quickly but in general condition is symptomless. They have resistance to malaria and this advantage outweighs disadvantage.
Heterozygous individuals are selected for in areas without malaria.
What does genetic fingerprinting rely on?
The fact that the genome of any organism contains many repetitive, non coding bases of DNA.
95% of human DNA does not code for any characteristic.
These non coding bases are known as introns and they contain repetitive sequences of DNA called core sequences.
For every individual the number and length of core sequences has a unique pattern.
They are different in all individuals except identical twins, and the probability of two individuals having identical sequences of non-coding bases is extremely small.
However the more closely related two people are, the more similar the core sequences will be.
What are the 5 main stages of making a genetic fingerprint?
Describe the extraction stage of making a genetic fingerprint.
Even the tiniest sample of animal tissue, such as a drop of blood or a hair root, is enough to give a genetic fingerprint.
Whatever the sample, the first stage is to extract the DNA by separating it from the rest of the cell.
As the amount of DNA is usually small, its quantity can be increased by using PCR.
Describe the digestion stage of making a genetic fingerprint.
The DNA is then cut into fragments, using restriction endonucleases.
The endonucleases are chosen for their ability to cut close to, but not within, groups of core sequences.
Describe the separation stage of making a genetic fingerprint.
Gel electrophoresis under influence of an electrical voltage.
The gel is then immersed in an alkali in order to separate the double strands into single strands.
The single strands are then transferred on to a nylon membrane by a technique called Southern blotting.
-A thin nylon membrane is laid over the gel.
-The membrane is covered with several sheets of absorbent paper, which draws up the liquid containing the DNA by capillary action.
-This transfers the DNA fragments to the nylon membrane in precisely the same relative positions that they occupied on the gel.
-The DNA fragments are then fixed to the membrane using UV light.
Describe the hybridisation stage of making a genetic fingerprint.
Radioactive (or fluorescent) DNA probes are now used to bind with the core sequences. The probes have base sequences which are complementary to the core sequences, and bind to them under specific conditions, such as temperature and pH.
The process is carried out with different probes, each of which binds with a different core sequence.
Describe the development stage of making a genetic fingerprint.
Finally, an X-ray film is put over the nylon membrane. The film is exposed by the radiation from the radioactive probes.
Because these points correspond to the position of the DNA fragments as separated during electrophoresis, a series of bars is revealed.
The pattern of bands is unique for every individual except identical twins.
How are the results of DNA fingerprinting interpreted?
DNA fingerprints from two samples, e.g. from blood found at the scene of a crime and from a suspect, are visually checked.
If there appears to be a match, the pattern of bars of each fingerprint is passed through an automated scanning machine, which calculates the length of the DNA fragments from the bands.
It does this using data obtained by measuring the distances travelled during electrophoresis by known lengths of DNA. Finally, the odds are calculated of someone else having an identical fingerprint.
The closer the match between the two patterns, the greater the probability that the two sets of DNA have come from the same person.
What are the uses of DNA fingerprinting?
-Forensic science- indicate whether or not an individual is connected with a crime.
-Resolve questions of paternity- individuals inherit half of their genetic material from their mother and half from their father. Therefore each band on a DNA fingerprint of an individual should have a corresponding band in the parents' DNA fingerprint.
-Determining genetic variability within a population- the more closely two individuals are related the closer the resemblance of their genetic fingerprints. A population whose members have very similar genetic fingerprints has little genetic diversity.
A population whose members have a greater variety of genetic fingerprints has greater genetic diversity.
-Medical diagnosis, help diagnosing diseases such as Huntington's disease.
-Plant and animal breeding- used to prevent undesirable inbreeding during breeding programmes on farms or in zoos. Also to identify plants or animals that have a particular allele of a desirable gene. Also pedigree.