DNA technology Flashcards
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.
RECOMBINANT DNA
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.
- ISOLATION of the DNA fragments that have the gene for the desired protein.
- INSERTION of the DNA fragment into a vector.
- TRANSFORMATION, that is, the transfer of DNA into suitable host cells.
- IDENTIFICATION of the host cells that have successfully taken up the gene by use of gene markers.
- 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
RECOGNITION SITES
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?
Plasmid.
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?
- 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.
- 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.
- 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?
2 minutes.
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.
- making vaccines
- 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.
