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11.4 – Applications of Gene Technology Gene technology has important applications in many areas including biotechnology, medicinal drug production, gene therapy, agriculture and horticulture Genetic engineering can generate many potential ________________ for humans. But there are hazards too. The economic advantages may be outnumbered by environmental and ________________ drawbacks or dangers. The issues require balanced and informed judgements Principles of Genetic Engineering The first step in genetic engineering usually involves inserting a short piece of donor ____________ into the DNA of a recipient organism. The recipient organism is unrelated to the donor e.g. from human to bacterium. The recipient organism then acquires the ability to synthesise the _____________for which the donor DNA codes. Transferring a gene….. T.G. AS Module 2 – Genes and Genetic Engineering – 11.4 – Applications of Gene Technology – Student Notes 29/06/17 Page 1 The outcomes are new varieties of organisms – mostly _____________organisms Microoganisms are preferred because they are easier to modify To remove the DNA from a cell the cell membrane needs to be _______________ and the nucleus broken open. The method used depends on the type of cell. One common way to disrupt a cell is a ________________ called sodium dodecyl sulphate (SDS) which breaks down cell membranes (including the nuclear membrane) and cell walls The protein framework of the chromosomes is removed by incubation with a ______________ Once the DNA has been isolated from the rest of the cell, the part of the DNA molecule which contains the required gene has to be cut out and the rest of the DNA discarded. (There are on average 2200 genes on each human chromosome) It is important to know the base sequence of the gene required – so that either a gene ____________ can be used and/or the appropriate restriction endonuclease selected Genes are isolated using enzymes which cut across DNA molecules at particular positions – ______________________________ ENDONUCLEASE ENZYMES. Several __________________ types of restriction endonuclease exist Restriction endonuclease enzymes are found naturally in ________________ where their function is to chop up and destroy the DNA of any viruses that infect the cell. They are so named because the RESTRICT the multiplication of viruses A given restriction endonuclease cuts a bacterial plasmid open at a ________________ site which is determined by the base sequence in that region The same enzyme will cut donor DNA wherever an identical base sequence occurs It is characteristic of most restriction endonucleases that they cut the 2 strands of DNA at slightly different points – the result is that each end of the foreign DNA segment has a short row (4 bases long) of unpaired bases which match the complementary bases at each end of the opened up plasmid. These are called ___________________ _______________ Example of how a restriction endonuclease works: T.G. AS Module 2 – Genes and Genetic Engineering – 11.4 – Applications of Gene Technology – Student Notes 29/06/17 Page 2 Genetically Engineered Microorganisms The next stage is to insert the isolated gene into a _________________. A vector is a piece of DNA that can take the gene into the chosen microbe. A common vector is a small circular molecule of DNA called a ___________________. These occur naturally in bacteria in addition to the larger molecule of chromosomal DNA Plasmids are easily isolated from a bacterium and they can be re-introduced into another bacteria relatively easily too. As mentioned above, the ______________ restriction endonulclease is used to cut the plasmid DNA as was used for to isolate the gene from the donor DNA. This means that the sticky ends produced will fit together The donor gene is then inserted into the plasmid loop using the enzyme __________________. Ligase catalyses the LIGATION reaction which joins 2 sections of DNA together – the hydrogen bonds form between complementary bases. Ligase occurs naturally in nuclei where it ‘repairs’ DNA damaged in replication Inserting the gene into the plasmid: T.G. AS Module 2 – Genes and Genetic Engineering – 11.4 – Applications of Gene Technology – Student Notes 29/06/17 Page 3 The new DNA is called ________________________ DNA Once in position, the foreign DNA (donor gene) replicates along with the rest of the plasmid every time the bacterial cell divides Plasmids containing the donor gene must now be transferred into the ________________ A culture of the intended bacterial recipients is placed in cold calcium chloride solution for 30 mins This changes the membranes of the bacteria – making them more _______________ to the plasmids Plasmids with the recombinant DNA are added to the culture medium and warmed up for a short time. Some of the bacteria take up plasmids – those that do are said to have undergone _________________________________ This process is ____________ very efficient – only a small proportion of the bacteria are transformed Obtaining transformed bacteria T.G. AS Module 2 – Genes and Genetic Engineering – 11.4 – Applications of Gene Technology – Student Notes 29/06/17 Page 4 Genetic Markers The transformed cells must now be identified and isolated. One technique for doing this involves inserting a ___________________ gene into the plasmids as well as the donor gene This marker gene may make the bacteria ___________________ to a particular antibiotic If the culture containing the transformed bacteria is grown on a medium containing the antibiotic, bacteria with the plasmids that have the antibiotic resistance marker gene (and the donor gene) will __________________ and grow better than those without it Replica plating can the be done Replica Plating……….. This ___________________ colonies without the resistance gene (and therefore no donor gene) The donor gene also has to be made to start working once it has been transferred to the recipient bacteria Not all genes in a bacterium are switched on all the time It is possible to overcome this by using ___________________ GENES that control the expression of the main gene. They indicate where transcription should begin and ensure mRNA is translated by the ribosomes The promoter genes are inserted into the plasmid, with the donor gene and the marker gene _____________________ transformation T.G. AS Module 2 – Genes and Genetic Engineering – 11.4 – Applications of Gene Technology – Student Notes 29/06/17 Page 5 Large Scale Culturing Since the gene product is often needed in _________________ amounts the transformed bacteria are cultured on a large scale in an industrial fermenter The medium in it contain all the nutrients the bacteria need for rapid growth and reproduction and supplies the oxygen they need for respiration In favourable conditions the bacteria will divide as often as every _________ hour A huge clone of the bacteria can be produced within days As the bacteria grow, the gene within the plasmids synthesises its _________________ The product is extracted from the fermenter for commercial use Industrial Fermentation Useful substances produced by genetic engineering include antibiotics, ___________________(insulin, human growth hormone, factor VIII) and _________________(those in biological washing powders, produced for use in food industry) Genetic engineering is also used to make pest _________________ varieties of crops – genes producing chemicals which confer resistance to attack by fungi or insects can increase yields and reduce the amount of pesticide which needs to be used T.G. AS Module 2 – Genes and Genetic Engineering – 11.4 – Applications of Gene Technology – Student Notes 29/06/17 Page 6 The Polymerase Chain Reaction (PCR) PCR is like a nuclear chain reaction in that it proceeds at an ever – increasing rate PCR can amplify a ____________ amount of DNA into quantities large enough for scientific analysis (e.g. at the scene of a crime just a few white blood cells or a tiny sample of sperm may be found) It is the basis of genetic fingerprinting / DNA fingerprinting / genetic profiling (all same thing!) Other uses of PCR include amplifying DNA from samples of tissues from extinct animals (e.g. Tasmanian wolf) to establish their closest ______________and amplifying DNA from buried human bodies where some soft tissue has been preserved. This helps to understand the migration of early human populations PCR uses the enzyme DNA ___________________– this is the enzyme which occurs naturally in cells and catalyses the replication of DNA in the nucleus The first stage of PCR is to heat the DNA to 95oC – this makes the 2 polynucleotide strands separate DNA nucleotides are added and the mixture is cooled to 40oC. The DNA polymerase attaches the new nucleotides to each strand (as in normal replication) The process can then be repeated _____________ – producing 2,4,16,32 DNA molecules and so on A single PCR cycle: T.G. AS Module 2 – Genes and Genetic Engineering – 11.4 – Applications of Gene Technology – Student Notes 29/06/17 Page 7 Once a large enough sample has been generated using PCR it can be analysed and compared with samples from ________________sources This allows us to ________________ DNA from the same or different species. Forensic scientists often have to compare DNA found at the crime scene with DNA from suspects In each case the first stage is to cut the DNA into short lengths with ____________ enzymes This produces DNA fragments of _________________ lengths – because the sequence of bases where the enzymes cut occur at irregular intervals along the DNA molecules The lengths of these fragments will also be different in different individuals – since we all have a unique sequence of nucleotides in our DNA. The same restriction endonuclease will chop up our DNA in different places The DNA fragments are then separated using ____________________ The mixture of DNA fragments is placed at one end of a long piece of agar jelly in a trough containing a dilute solution of ionic salts Electrodes are placed in the solution at either end and a ______________________is applied The phosphate groups in the fragments of DNA give them a ________________ charge – so they are attracted through the gel towards the positive electrode. The smaller fragments move more _________________ than the larger ones – so the different sized fragments are separated in a similar way to molecules in chromatography The pattern of fragments is like a ‘fingerprint’ - this fingerprint is _____________ to each of us. The only exception is identical twins – they share the same pattern To be able to compare fingerprints they must be made visible. The pattern of DNA fragments in the gel is transferred to a nitrocellulose sheet T.G. AS Module 2 – Genes and Genetic Engineering – 11.4 – Applications of Gene Technology – Student Notes 29/06/17 Page 8 This is done overnight and produces an ‘imprint’ of the pattern of the fragments from the original gel onto the nitrocellulose sheet A ________________ labelled with radioactivity is then used to reveal the position of the bands on the sheet that contain the DNA sequence you are interested in. To be able to pick out a particular gene or genes using a proble you first need to know its ___________ _____________________ The gene probe can then be made which will seek out the particular gene. The gene probe may be labelled – often done by using a radioactive isotope of __________________ (32P) as a component of the phosphate groups A general probe can be used which make all the bands show up or a specific probe can be used. A specific probe is a piece of single stranded DNA that is complementary to the base sequence of the specific stretch of DNA The nitrocellulose sheet is incubated with the probe in a sealed plastic bag containing a buffer solution. The probes then bind (base pair) to the appropriate DNA fragments The sheet is removed and placed next to an unexposed piece of ___________________ film The radioactivity in the probe causes a band to show up on the film Analysing DNA samples Other examples of where the technique has been used: Paternity cases Relationships within families Social behaviour in animals Immigration cases T.G. AS Module 2 – Genes and Genetic Engineering – 11.4 – Applications of Gene Technology – Student Notes 29/06/17 Page 9 Gene Therapy This is the field of medicine that aims to treat people with genetic disorders by giving them a copy of a __________________gene to overcome the problems produced by the mutated gene There are 2 types of cells in our bodies 1. _________________ cells – give rise to eggs and sperm 2. _________________ cells – ‘normal’ body cells Genetic changes to somatic cells ___________________be passed onto future generations. Changes to germ line cells _______________ be passed onto future generations The genetic engineering of somatic cells (somatic gene therapy) is legal, but tightly regulated. The genetic engineering of germ-line cells (germ-line therapy) is _________permitted in any country Gene therapy is being developed to treat the genetic disease c__________ f____________ Common in white North Americans and Northern Europeans 1 in 25 carriers 1 in 2000 sufferers Most people have a gene that produces a protein called the CYSTIC FIBROSIS TRANSMEMBRANE REGULATOR (CFTR) This is a complex molecule made up of 1480 amino acids and it is one of the essential channel proteins in cell membranes The function of this particular protein is to transport _____________ ions through the cell membrane Cause is a recessive mutation of chromosome 7 In CF sufferers this protein does not function because there are _________ copies of the faulty gene Responsible gene was cloned in 1989 Usually the cause is a _________________mutation – 3 base pairs are missing so codon 508 in the mRNA is missing As a result, the amino acid phenylalanine (F) is not inserted at position 508 in the 1480 aa protein The mutation is ^F508 (^ = d for deletion) There are at least 400 other mutations of this protein but this is the most common This mutation affects the __________ shape of the channel protein so that it can no longer transport chloride ions through a membrane T.G. AS Module 2 – Genes and Genetic Engineering – 11.4 – Applications of Gene Technology – Student Notes 29/06/17 Page 10 Symptoms Normal epithelial cells form mucus glands which secrete mucus In CF patients this mucus is unusually _____________ and sticky In CF the normal outward flow of Cl- ions from the cell is prevented So, chloride ions build up, a negative charge builds up inside the cell so more sodium ions (+’ve) move in to balance the –‘ve charge The high ion concentration in the cell prevents ______________ leaving the cell by osmosis and so mucus is abnormally thick and sticky Transport of ions by CFTR: T.G. AS Module 2 – Genes and Genetic Engineering – 11.4 – Applications of Gene Technology – Student Notes 29/06/17 Page 11 The lungs, ________________ and liver are the most affected organs Sweat produced is unusually salty The thick mucus _____________ up airways in the lungs, branches of the pancreatic duct and bile duct from the liver and so there are repeated lung infections and digestive problems 50% of sufferers die by age 20, but average life expectancy has now increased to about 20-30 Treatment Vigorous __________________ to dislodge the mucus from the lungs _________ supplements to aid digestion ______________ to fight infections (in respiratory and digestive systems) Heart lung transplants for severe cases Clinical trials are underway for gene therapy, attempting to insert CFTR gene into lung / tracheal epithelial cells to replace the defective genes 2 techniques are being tested 1. wrapping the CFTR genes in lipids that can be absorbed through the csm 2. inserting CFTR genes into harmless viruses that are then allowed to ‘infect’ the cells 1. wrapping the CFTR genes in lipids that can be absorbed through the csm This method uses tiny lipid droplets called ____________________ These can fuse with the phospholipid molecules that make up the csm and so carry genes into the target cells CFTR genes are isolated from healthy _______________ cells, cloned and inserted into plasmids using recombinant DNA technology The genes are inserted into liposomes ___________ sprays are used to get the liposomes into the lungs – like inhalers for asthma sufferers In the lungs, the liposomes fuse with the csm of _____________ cells and the genes are transported into the cell One problem with the technique is developing a __________ enough spray to get the liposome through the narrow bronchioles in the lungs and into the alveoli Also, only a ______________ proportion of the genes that are absorbed into cells are actually expressed These are problems which must be overcome before this form of gene therapy becomes widely available as a treatment for CF T.G. AS Module 2 – Genes and Genetic Engineering – 11.4 – Applications of Gene Technology – Student Notes 29/06/17 Page 12 2. inserting CFTR genes into harmless viruses that are then allowed to ‘infect’ the cells In this method viruses called _____________________ are used These viruses reproduce themselves by injecting their DNA into host cells The viral DNA uses the cell’s enzymes and ribosomes to replicate and produce copies – which then reconstruct the rest of the virus before being released from the cell Adenoviruses infect the cells of the airways in the ____________, and they are adapted for replication in the epithelial cells They normally cause colds and other respiratory diseases The adenoviruses used for gene therapy have been modified so they infect the cells but _______________cause the diseases The modification is that the genes which allow the virus to replicate are __________________ The modified viruses are cultured in epithelial cells grown in the laboratory and exposed to plasmids that have the CFTR gene The CFTR gene is incorporated into the viral DNA The adenoviruses containing the CFTR gene are then extracted from the epithelial cells, purified and sprayed into the lungs Here they infect the epithelial cells, taking the CFTR gene into the cells The CFTR channel protein is synthesised as normal – but as the viruses cannot replicate – they do not damage the cells Problems include not completely inactivating the genes controlling viral replication and the patients becoming ill. Also, patients treated repeatedly may develop _______________ than make them immune the virus and resistant to the treatment Perfecting Gene Therapy Techniques Neither of the above methods provides a permanent cure for CF but both have the potential to alleviate some of the symptoms Before using either method on people, research has to be done to ensure gene therapy is safe The first tests, carried out on laboratory animals like mice aimed to find out the following: 1. The best way of getting the gene into cells 2. Check that the gene was expressed in the target cells 3. Detect any ill effects of the therapy itself The next step was to start testing the delivery systems on _______________ T.G. AS Module 2 – Genes and Genetic Engineering – 11.4 – Applications of Gene Technology – Student Notes 29/06/17 Page 13 This was initially done by giving small doses of liposomes containing the CFTR gene applied to the lining of the nose to volunteers – (since it is easier to ________ the cells here than deep in the lungs) The next stage – which is currently ongoing – involve clinical trials with CF patients Many trials must be done before gene therapy becomes an officially approved treatment for CF The Future of Gene Therapy Progress is proceeding at a very _____________rate Soon it will be possible to 1. Incorporate a healthy gene into one of the chromosomes before putting it into a cell – the chromosomes would be replicated as the cell divides – providing a longer lasting treatment 2. Treat rapidly dividing cancer cells with genes that kill them 3. Provide protection for viral infections by inserting genes that interfere with virus replication into cells More effective ways of getting genes _________ _______________ will make treatment of genetic diseases more effective The 2 methods described for CF have to be repeated at regular intervals because copies of the gene are not passed form cell to cell during mitosis Epithelial cells divide ________________ so this is a major limitation If however the healthy gene was inserted into a chromosome then it would be replicated each time the divided One problem here though is inserting the gene into the DNA in a chromosome may damage other genes Gene therapy could help in the fight against c________________ – There are genes which cause cells death – so if they could be inserted into cancer cells – tumours cold be destroyed Reproduction of viruses in cells could also be prevented by inserting genes which produce mRNA that is complementary to the viral RNA This would inactivate the virus The most effective treatment for genetic disorders is to replace the defective genes completely This could be done using in vitro fertilisation (similar to method for producing transgenic animals) The dangers of damaging the embryo though means that this technique is not suitable for humans Such a genetic change would affect the ______________________ T.G. AS Module 2 – Genes and Genetic Engineering – 11.4 – Applications of Gene Technology – Student Notes 29/06/17 Page 14 This means the genes of sex cells would be altered and the genetic change would be passed on to future generations One other proposed method is to create an __________________ chromosome – containing only healthy extra genes and the promoter genes needed to express them Once added to the nucleus this chromosome would be replicated and take part in mitosis in the normal way There is less chance it will cause danger to other chromosomes and genes Once the full human genome is known further developments will be able to be made At present there are trials using somatic gene therapy for 100’s of diseases including cancers, Duchenne muscular dystrophy and haemophilia Some of the possibilities raise difficult moral and ethical questions Balancing the benefits for the emerging technology against the potential for its abuse will take many years of consultation and consideration T.G. AS Module 2 – Genes and Genetic Engineering – 11.4 – Applications of Gene Technology – Student Notes 29/06/17 Page 15 Genetically Modified Animals Animals which have been given genes from another species are called _______________________ organisms Transgenic mammals are useful because they can express _________________ genes It is not always possible for bacteria to express human genes since prokaryotic bacterial cells do not have the ribosomes and other cellular machinery necessary to make complex mammalian proteins Tracey is the name of a famous transgenic sheep When she was an ________________, human genes were transferred into her cells T.G. AS Module 2 – Genes and Genetic Engineering – 11.4 – Applications of Gene Technology – Student Notes 29/06/17 Page 16 The human gene to make the human protein alpha – 1- antitrypsin (____________) was isolated from a culture of human cells The gene was then combined with a promoter sequence that that allows the gene to be expressed in sheep mammary glands Mature egg cells were removed from the ovary of a sheep and fertilised in vitro. The AAT gene and its promoter sequence were injected into the nucleus of the fertilised egg cells Once the zygote had divided to form a small embryo – this was placed in the uterus of a sheep _____________________ mother When the sheep grew to adulthood she was able to produce the protein human alpha – 1- antitrypsin (AAT) in her milk. This could then be collected easily and used AAT is used to treat ___________________and cystic fibrosis The normal function of the ATT glycoprotein is to inhibit an enzyme called elastase that is produced by some types of white blood cell Elastase, if not inhibited, breaks down the elastic tissue in the lungs, causing emphysema In emphysema, the walls of the alveoli disintegrate, so there is less SA available for the absorption of oxygen and fluid leaks into the air spaces The fluid disrupts normal functioning of the lungs and can cause infections Emphysema can be caused by a number of factors. Having the Z allele for AAT is rare. More common causes are smoking and working with fine dust particles There are many different alleles of the AAT gene About 3% of the population have the Z allele – this codes for a version of AAT that differs from the normal form by one amino acid. This is a defective form and patients often develop emphysema Affected people can be treated with an aerosol spray containing AAT When inhaled this stops the breakdown of the alveoli and helps breathing But it is not a permanent cure – patients must have treatment for the whole of their lives The blood clotting protein Factor VIII is also produced in this way and is used to treat haemophilia T.G. AS Module 2 – Genes and Genetic Engineering – 11.4 – Applications of Gene Technology – Student Notes 29/06/17 Page 17 Evaluation of Genetic Engineering Rapid developments in genetic engineering are making it possible to alter living organisms in a variety of ways It is in theory possible to transfer genes from almost ____________organism to _________ another There are many ways in which DNA recombinant technology can be used in food production and medicine However, many people are suspicious of these developments and are concerned about the possible ways in which the technology might go wrong There are concerns about the release of genetically engineered organisms into the environment. Once released, these organisms cannot be recalled – No one can be sure of what effect they may have on other organisms in the environment Genetically modified crop plants have also caused concern e.g. potatoes have been produced containing a gene which makes them resistant to a herbicide This herbicide can be sprayed onto the crop and kills weeds but not the potatoes Might this gene somehow spread to wild plants and affect the balance of the ecosystem in some way – creating ‘superweeds’. There are concerns GM foods may be harmful to health There are concerns genetically modifying crops will INCREASE our current use of pesticides Will a gene, added to a genome behave in an unforeseen manner? Could it trigger a disease? Should the money used to fund research into GE by developed countries be used to provide basic things like food and clean water in developing countries? Is DNA fingerprinting reliable? What about contamination of samples used in evidence? (OJ Simpson trial) Are we interfering with nature? For gene therapy, most people agree that it could be used to relieve the symptoms of a genetic disease. But the possibility of inserting the ‘correct’ genes into someone’s cells opens up all sorts of possibilities In theory, any gene could be inserted into the human egg. What types of gene should be inserted? Designer babies? At the moment there is a complete ban on performing any genetic engineering on sex cells. This is likely to continue for the foreseeable future T.G. AS Module 2 – Genes and Genetic Engineering – 11.4 – Applications of Gene Technology – Student Notes 29/06/17 Page 18 T.G. AS Module 2 – Genes and Genetic Engineering – 11.4 – Applications of Gene Technology – Student Notes 29/06/17 Page 19