Chapter 21- Genome Projects and Recombinant DNA technology Flashcards

Question

What is the process of producing a gene using reverse transcriptase.

Answer 1

* A cell that readily produces the protein is selected e.g. B-cells from the islets of Langerhans from the pancreas are used to produce insulin. * These cells will have high levels of the relevant mRNA. * mRNA is first isolated from cells. Any DNA in the sample is hydrolysed by enzymes before the sample is added to the reaction mixture- **removes DNA present so that its’ not amplified. ** * mRNA is mixed with free DNA nucleotides and reverse transcriptase. * Reverse transcriptase uses the mRNA as a template to catalyse the synthesis of a single-stranded complementary DNA- cDNA- with complementary nucleotides to the mRNA. * Single stranded cDNA is isolated by hydrolysis of the mRNA with an enzyme. * To make the other strand of DNA, DNA polymerase is used to build up complementary nucleotides on the cDNA template. * This produces a double stranded piece of DNA which is the required gene.

Answer 2

* Technology- developed so fragments of DNA can be synthesised without the need for a pre-existing DNA template. * Can now use a gene machine to manufacture genes in a laboratory. * A database contains all the necessary information to produce the DNA fragment- means that the DNA sequence doesn’t have to exist naturally- any sequence can be made.

Answer 3

* The desired sequence of nucleotide bases of a gene is determined from the desired protein- the amino acid sequence of the protein is determined- mRNA codons are looked up and the complementary DNA triplets are worked out. * The desired sequence of nucleotide bases for the gene is fed into the computer. * The sequence is checked for biosafety and biosecurity to ensure it meets international standards and ethical requirements. * The computer designs a series of small, overlapping single strands of nucleotides- oligonucleotides- can be assembled into the desired gene. * The first nucleotide in the sequence is fixed to a support e.g. a bead. * Nucleotides are added step by step in the correct order in a cycle of processes including adding protecting groups which make sure the nucleotides are joined to the right points to prevent unwanted branching. * Short sections of DNA called oligonucleotides- roughly 20 nucleotides long- produced. Once complete they are broken off from the support and the protecting groups are removed. * Automated process- each of the oligonucleotides is assembled by adding one nucleotide at a time to the required sequence. * Oligonucleotides are then joined together to make a gene- doesn’t have introns or other non-coding DNA. * The gene is replicated using the polymerase chain reaction which also constructs the complementary strand of nucleotides that make the required double-stranded gene. It then multiplies the gene many times to give numerous copies. * Genes are checked using standard sequencing techniques and those with errors are rejected. * This process enables any sequence of nucleotides to be produced in a short time with high accuracy. * The artificial genes are free of introns as other non-coding DNA so can be transcribed and translated by prokaryotic cell.

Answer 4

* It is necessary to find the fragment that has the required gene after cutting DNA into fragments. This is done using a DNA probe. * Once the fragment with the gene has been obtained, it is cloned so there is sufficient quantity for medical or commercial use. * Gene cloning involves making identical copies of a gene- amplification.

Answer 5

* In vivo cloning- where the gene copies are made within a living organism by transferring fragments to a host cell using a vector. As the organism grows and divides, it replicates the DNA, creating multiple copies of the gene. * In vitro cloning- gene copies are made outside of a living organism using the polymerase chain reaction (PCR).

Answer 6

* In vivo gene cloning involves inserting the gene using vectors. * Once a DNA fragment containing the target gene has been obtained it can be used in in vivo cloning. * A culture of transformed host cells is used as an in vivo method to amplify DNA fragments. * These transformed cells can be used in transgenic organisms to produce desired proteins/ characteristics.

Answer 7

* Sticky ends left on DNA by restriction endonucleases are important for DNA cloning. * Restriction endonucleases cut sequences of DNA at specific recognition sites. * If the recognition site is cut in a staggered fashion, the cut ends of the DNA double strand are left with a single strand- few nucleotides base long. * Nucleotides on the single strand at one side of the cut are complementary with those on the other side because they were previously paired together. * If the same restriction endonuclease is used to cut DNA, all the fragments produced will have ends complementary to each other. * The single-stranded end of any one fragment can be joined to the single-stranded end of any other fragment- the ends are sticky. * Sticky ends- important as if the same restriction endonucleases are used, they can combine the DNA of one organism with the DNA of another organism. * Once the complementary bases of two sticky ends are paired up, DNA ligase is used to form phosphodiester bonds on the two sections of DNA and join them together.

Answer 8

1. Preparing the gene for cloning- the addition of promoter and terminator regions to the fragments of DNA. 2. Inserting the gene into a vector- the use of restriction endonucleases and ligases to insert fragments of DNA into vectors. 3. Transforming the host cell using these vectors. 4. Identifying cells with the gene- The use of marker genes to detect genetically modified (GM) cells or organisms. (Students will not be required to recall specific marker genes in a written paper.)

Answer 9

* Once an appropriate fragment of DNA has been cut from the rest of the DNA, extra lengths of the DNA called the promoter and terminator regions are added, preparing the DNA fragments for insertion. * If you want the transformed host cells to produce the protein coded for by the DNA fragment, you need to make sure the vector contains specific promoter and terminator regions. * Promoter and terminator regions may be present in the vector DNA or they may have to be added in along with the fragments.

Answer 10

* The sequence of DNA which is a binding site where RNA polymerase must attach near a gene to start transcription and produce mRNA. * Nucleotide bases of the promoter attach both RNA polymerase and transcription factors and begin the process of transcription. * If the DNA fragment is to be transcribed into mRNA to make a protein it is essential to attach the promoter region to start the protein. * Without the right promotor region, the DNA fragment won’t be transcribed by the host cell and a protein won’t be made. * Promoter genes ensure that genes are only expressed within target cells to make the protein- see later notes on transformed animals.

Answer 11

* Releases RNA polymerase and end transcription. * It is important to add a terminator region to the other end of the DNA fragment to stop transcription at the appropriate point.

Answer 12

* After the promoter and terminator regions are added, the next step is to insert the DNA fragment into a vector’s DNA. * Vector- used as a carrying unit to transport the DNA into the host cell. * Vectors can be plasmids (small, circular molecules of DNA in bacteria) or bacteriophages (viruses that infect bacteria).

Answer 13

* The vector DNA is isolated. * The **vector DNA** is cut open using the same **restriction endonuclease** that was used to isolate the DNA fragment containing the target gene. The sticky ends of the vector DNA are complementary to the sticky ends of the DNA fragment containing the gene. * The vector DNA and DNA fragment are mixed together with DNA ligase. * **DNA ligase joins the sticky ends of the DNA fragment of the gene to the sticky ends of the vector** DNA in a process called ligation. * The new combination of bases in the DNA, vector DNA and DNA fragment is called recombinant DNA. * Restriction endonucleases and DNA ligase are used to stick the DNA fragment and vector DNA together.

Answer 14

* Most commonly used vectors are plasmids- circular lengths of DNA found in bacteria, separate from main bacterial DNA. * Plasmids almost always contain genes for antibiotic resistance and restriction endonucleases are used at one of these antibiotics -resistance genes to break the loop. * Restriction endonucleases cut the plasmid to produce complementary ‘sticky ends’. * Restriction endonuclease used is the same as the one that cut out the DNA fragment- ensures the sticky ends of the plasmid are complementary to the sticky ends of the DNA fragment. * When the DNA fragments are mixed with open plasmids, they may become incorporated. * Where they become incorporated, DNA ligase joins the gene and plasmid at complementary sticky ends- plasmids now have recombinant DNA. * Once DNA has been incorporated into some of the plasmids, they are reintroduced into bacterial cells.

Answer 15

* The vector with the recombinant DNA is used to transfer the gene into cells- host cells. * Host cells that take up the vectors containing the gene of interest are transformed.

Answer 16

* Transformation- reintroduction of recombinant plasmids into bacterial cells. * Host cells have to be persuaded to take in the plasmid vector * Involves the plasmids and bacterial cells being mixed together in a medium containing calcium ions- usually ice-cold calcium chloride solution to make their cell walls more permeable. * When the plasmids are added, the mixture is heat-shocked (heated to 42℃) which encourages the cell to take up the plasmids. * Calcium ions and changes in temperature make the bacterial membrane permeable, allowing the plasmids to pass through the cell-surface membrane into the cytoplasm.

Answer 17

* With a bacteriophage vector, the bacteriophage will infect he host bacterium by injecting its DNA into it. * The phage DNA with the target gene in it then integrates into the bacterial DNA

Answer 18

* Only a few bacterial cells take up plasmids when the two are mixed together. * Some plasmids close up again without incorporating the DNA fragment. * Sometimes the DNA fragment ends join together to form its own plasmids.

Answer 19

* Only around 5% of host cells will take up the vector and its DNA, it’s important to be able to identify which cells have been transformed so that energy isn’t wasted culturing them and the untransformed cells don’t outcompete the transformed ones. * Identification can be used to identify which bacteria have taken up the plasmid.

Answer 20

* Marker genes are used as **not all of the target cells (specify e.g. bacteria, eggs) will successfully take up the plasmid. ** * Marker genes** can be used to identify the transformed cells quickly using** either **UV light** or X-rays in autoradiography. * With UV light, **organisms that have taken up the gene will glow. ** * Marker genes can be inserted into vectors at the same time as the gene is cloned. This means any transformed host cells contain the gene to be cloned and the marker gene. * Host cells are grown on agar plates and each cell divides and replicates its DNA, creating a colony of cloned cells. * Transformed cells produce colonies where all the cells contain the cloned gene and the marker gene.

Answer 21

* The marker gene can code for antibiotic resistance- host cells are grown on agar plates containing the specific antibiotic, only transformed cells that have the marker gene will survive and grow. * The marker gene can also code for proteins which produce fluorescence when the agar plate is placed under UV light, only transformed cells will fluoresce. * The marker gene can also code for enzymes which cause a colour change. * You are not required to recall specific marker genes, but you may need to interpret information on them.

Answer 22

* Bacteria can resist antibiotics by producing an enzyme that breaks down the antibiotic before it can destroy the bacteria. * Genes for the production of enzymes are found in the plasmids. * Some plasmids carry genes for resistance to more than one antibiotic- e.g. the R-plasmid which carries genes for resistance to two antibiotics and tetracycline. * Using a gene for antibiotic resistance in plasmids can help to find which bacterial cell has taken up the plasmids. * Not favourable compared to the other methods as relies on antibiotic resistance which is problematic for human health and involves killing bacteria, which results in it taking longer to identify transformed cells.

Answer 23

* The plasmid used for the insertion contains a gene for antibiotic resistance. * Bacteria can resist antibiotics by producing an enzyme that breaks down the antibiotic before it can destroy the bacteria. * Genes for the production of enzymes are found in the plasmids. * Some plasmids carry genes for resistance to more than one antibiotic- e.g. the R-plasmid which carries genes for resistance to two antibiotics and tetracycline. * Using a gene for antibiotic resistance in plasmids can help to find which bacterial cell has taken up the plasmids. * * While this method is good in showing which bacterial cells have taken up the plasmids, it doesn’t show which bacterial cells that have taken up plasmids that haven’t incorporated the new gene. * To identify the cells without the new gene and eliminate them, a second marker genes is used. * Marker genes involve using a second, separate gene on the plasmid. * The second gene is identifiable usually because it is either resistant to another antibiotic, makes an easily seen florescent protein, or produces an enzyme whose action is easily identified.

Answer 24

* To identify cells with plasmids that have taken up the new gene replica plating is used. * This process uses the other antibiotic resistance gene on the plasmid- which was cut to incorporate the required gene. * As the gene is cut permanently when it acquires the new gene, it no longer is resistant to the antibiotic as it doesn’t produce the necessary bacteria. * The bacteria can then be grown using replica plating to copy the culture and apply the antibiotic to identify colonies of bacteria containing the required gene.

Answer 25

* Transfer of a gene from a jellyfish into the plasmid- produces a green fluorescent protein (GFP). * The gene to be cloned is transplanted into the centre of the fluorescent gene * Any bacterial cell that has taken up the plasmid with the gene will not be able to produce the fluorescent protein. * Bacterial cells that have not taken up the gene will continue to produce will continue to produce GFP. * As the bacterial cells with the desired gene are not killed there isn’t a need for replica plating. * Results can be obtained by viewing cells under a microscope and retaining those that do not fluoresce, making the process more rapid.

Answer 26

* Use genes that produce the enzyme lactase. * Lactase turns a particular colourless substrate blue. * The required gene is transplanted is not the gene that makes lactase. * If a plasmid with the required gene is present in a bacterial cell, the colonies will not produce lactase and change the colourless substrate to blue. * When the gene has not been taken up by the bacteria, they won’t turn the substrate blue- these bacteria aren’t cultured.

Answer 27

* Identified transformed cells are allowed to grow more, producing lots of copies of the cloned genes. * Alongside genes, the transformed cells can produce the desired protein due to the organism being recombinant (see notes on using transformed organisms).

Answer 28

DNA fragments are amplified outside of a living organism using **the polymerase chain reaction (PCR).**

Answer 29

* PCR- method of copying fragments of DNA- automated- rapid and efficient- makes millions of copies of fragments of DNA in few hours. * PCR- several stages and is repeated over and over to make lots of copies. * Not the same as semi-conservative DNA replication.

Answer 30

* PCR **requires DNA fragment, DNA polymerase, DNA nucleotides and primers. ** * The DNA fragment which will be copied. * DNA polymerase- enzyme which catalyses the condensation reaction w**hich joins together nucleotides to produce DNA**. Taq polymerase, obtained from bacteria in hot springs is often used as it is tolerant to heat and doesn’t’ denature when high temperatures are used as part of the process. * Primers- short sequences of nucleotides that have sets of bases complementary to those at the start of each of the DNA fragments. A variety of primers can be used if there are** a variety of DNA fragments as the base sequences differ so different complementary primers are required. ** * Free DNA nucleotides- contain each of the 4 bases found in DNA. * Thermocycler- computer-controlled machine- varies the temperatures over a period of time.

Answer 31

1. Separation of the DNA strand: * The target DNA fragments, free DNA nucleotides, primers and DNA polymerase are placed in a reaction mixture in the thermocycler. * The mixture is **heated is to 95℃ to break the hydrogen bonds between the two strands of the DNA fragments to separate them. ** 2. Annealing of the primers: * Temperature of the mixture r**educed to 55℃ causing the primers to bind** (anneal) to their complementary bases at the end of the **DNA** fragment. * The primers provide the starting sequences for DNA polymerase to being DNA copying as DNA polymerase can only attach nucleotides to the end of an existing chain. * Primers also prevent the two separate DNA strands from rejoining. 3. Synthesis of DNA: * **Temperature is increased to 72℃** - optimum temperature for **the DNA polymerase** line up and **join** free floating complementary DNA **nucleotides** along each separated template DNA strand, forming phosphodiester bonds. * DNA polymerase begins at the primer on both strands and adds the nucleotides in sequence until it reaches the end of the chain. * Specific base pairing means new complementary strands are formed. * 2 new identical copies of the fragment of DNA are formed and one cycle of PCR is complete. * The PCR cycle starts again, with the mixture being heated to 95℃ and all the 4 strands (two original and two new are used as templates).

Answer 32

* Both separated strands are copied simultaneously, resulting in two copies of the original fragments. * Once the two DNA strands are completed, the process is repeated using the temperature cycle again, resulting in four strands. * Each PCR cycle doubles the amount of DNA- so each cycle is 2n+1, where n is the cycle number. * E.g. one double helix produces 2x2- 4 DNA fragments, second cycle- 4x2= 8 and so on. * Over a million copies of DNA can be made in only 25 temperature cycles * The temperature cycle takes around two minutes so billions of copes of DNA can be manufactured in a few hours. * **Initially numbers of doubling is low but the doubling each cycle produces an exponential increase. The amount of PCR/ DNA replication occurring eventually plateaus or stops as there are no more/ limited numbers of nucleotides/ primers. **

Answer 33

PCR has enabled small samples of DNA to be multiplied to allow forensic examination and accurate cross matching.

Answer 34

* Rapid- 100 billion copies can be made within hours. Important when there is a limited time frame e.g. for forensic analysis. * Only needs a small amount of DNA available e.g. at the scene of the crime. * Doesn’t require living cells- only requires a base sequence of DNA that need simplification. There are no complex culturing techniques requiring time, effort and biological risks.

Answer 35

* High risk of contamination- will rapidly copy contaminating DNA. Requires a very pure sample because contaminant DNA will also be multiplied leading to a false result. * Likely to have errors- while modern techniques have improved accuracy, any errors in copying DNA or contaminants in the sample will be copied in subsequent cycles.

Answer 36

* Useful to introduce a gene into another organism- uses vectors. Once the gene has been introduced into a plasmid, the plasmid can be used to deliver the gene to another organism to transform it e.g. humans, through gene therapy. * Little risk of contamination- only a gene that has been cut by the same restriction endonuclease can match the sticky sends of the open plasmid. Contaminant NDA will to be taken up by the plasmid. * Very accurate- few errors- mutations are rare. * Precise- cuts out specific genes- the culturing of transformed bacteria produced many copies of a specific gene rather than copies of the whole DNA sample. * Produces transformed bacteria that can be used to produce gene products- proteins for commercial or medical use e.g. insulin.

Answer 37

* Slow- In vivo cloning would take days or weeks to produce the same amount of DNA that in vitro cloning can make in hours. * Complex culturing techniques requiring time, effort and biological risks.

Answer 38

* Both use DNA polymerase to form phosphodiester bons. * Both semi-conservative. * Both use free DNA nucleotides.

Answer 39

* DNA primers are used in PCR, whereas RNA primers synthesised by primase are used to initiate DNA replication- provide a staring point for the addition of nucleotides by DNA polymerase. Once the primer is in place, DNA polymerase can start adding nucleotides to the template strand, building a new complementary strand. * Speed- PCR is faster. * Temperature is used to break hydrogen bonds instead of DNA helicase. * DNA replication occurs in a living cell whereas PCR occurs in a thermocycler.

Answer 40

* Gel electrophoresis- used in the analysis of DNA. * Electric current- used to separate the DNA molecules according to their size/ mass of the net charge. * Separation occurs as DNA is negatively charges and moves towards the positive electrode. * Different sized molecules move through the gel at different speeds. Small= faster. * When a sample of DNA is digested by restriction enzymes, restriction fragments of different lengths are produced- number and size of these restriction fragments can be found using gel electrophoresis. * The restriction fragments move through the gel at different speeds due to their different lengths, bands are formed * DNA can be seen using a stain, radioactive marker or fluorescent probes. * Restriction enzymes- highly specific- always produce the same number and sizes of restriction fragments if the same initial sample of extracted DNA is used. * An unknown restriction enzyme can easily be identified by comparing restriction fragments it produces to those of a known restriction enzyme- two sets of bands produced by electrophoresis should be exactly the same.

Answer 41

* You may need to interpret information relating to the use of recombinant DNA technology. * Microorganisms, plants and animals can all be transformed using recombinant DNA technology- genetic engineering.

Answer 42

* Made using the same technology as in vivo cloning. * Once a recombinant plasmid is added to a micro-organism and the transformed bacteria is identified, the protein produced from the cloned bacteria are extracted and purified.

Answer 43

* A gene that codes for a desirable protein is inserted into a plasmid. * Plasmid- added to a bacterium and the bacterium is used as a vector to get the gene into the plant cells. * If the right promoter region has been added along with the gene the transformed cells will be able to produce the desired protein.

Answer 44

* A gene that codes for a desirable protein can be inserted into an early animal embryo or into the egg cells of a female. * If the gene is inserted into a very early embryo- all the body cells of the resulting transformed animal will contain that gene. * If the gene is injected into an egg of an organism- **gene gets into all cells so it gets into the target cells that make the protein. ** * Inserting it into the egg cells means when the female reproduces, all the cells of her offspring will contain the gene. * If the gene is inserted too late into an organism- its’ offspring may not present the characteristic- **cell division will have occurred before the gene is added so cells producing gametes don’t receive the gene.** * Promoter regions that are only activated in specific cell types can be used to control which of an animals body cells the protein is produced in. * If the protein is only produced in certain cells, **it can be harvested more easily.** * Producing the protein in the **wrong cells could harm the organism.**

Answer 45

* Genetic research has many important benefits in curing disease and malnutrition, but it also has risks to do with transformed organisms. * You need to be able to evaluate the ethical, financial and social issues associated with the use and ownership of recombinant DNA technology in agriculture, in industry and in medicine. * It is important to develop safeguards and ethical guidelines to allow recombinant DNA technology to be used in a safe and effective manner and mitigate risks. * Need to understand both sides of the debate- balance humanitarian benefits of recombinant DNA technology with opposing views from environmentalists and anti-globalisation activists. * Evaluation- should always involve looking at the positives and negatives- benefits and risks of an issue.

Answer 46

* Ethics- set of standards followed by a particular group of individuals and are designed to regulate behaviour. Determine what is acceptable in pursuing the aims of the group. * Social issues- relate to human society and organisation- relationships, interdependence and cooperation of humans. ## Footnote What

Answer 47

* Genetically modified crops- engineered to have financial and environmental advantages. * Crops can be transformed to give higher yields. * Can also be transformed to be more nutritious e.g. golden rice helps produce vitamin A. * Crops can also be transformed to have resistance to environmental extremes or pests. * Crops to be grown where they usually can’t be grown. E.g. salt-tolerant tomatoes in the UK, makes lots of land more productive. Drought- resistance crops can survive in areas with little water. * Pest-resistant crops need fewer pesticides, which reduces cost and any environmental problems associated with chemicals. * Plants can be used to reduce the risk of famine and malnutrition.

Answer 48

* Industrial processes often use enzymes. * Enzymes- can be produced form transformed organisms- can be produced in large quantities for less money, reducing costs. * E.g. Biological laundry detergent. * Micro-organisms- used to control pollution- breakdown oil slicks, destroy harmful gases. Care needs to be taken to release the bacteria in the right place so they don’t destroy things- suicide genes can be incorporated which causes the bacteria to destroy themselves once they digest the oil slick. * Genetic fingerprinting- used in forensics.

Answer 49

* Drugs and vaccines used to combat disease are produced by transformed organisms using recombinant DNA technology. * Drugs made using recombinant DNA technology can be produced quickly, cheaply and in large quantities * Micro-organisms modified to produce range of substances- e.g. antibiotic, hormones, enzymes- used to treat diseases and disorders. * Genetically modified plants- transformed to produce specific substances in organs- organs harvested and substances extracted- can manufacture antibodies to pathogens and their toxins, and antigens which when injected into humans induce natural antibody production. * GM animals- produce drugs, antibiotics, hormones and enzymes cheaply. * Makes drugs more affordable and available to more people. * E.g. human insulin can be produced to treat type 1 diabetes. * Gene therapy- replacing defective genes- cure certain genetic disorders e.g. SCID, cystic fibrosis.

Answer 50

* Ethical, financial and social concerns about the use of recombinant DNA technology. * Anti-globalisation activists have concerns- growth of large multinational companies at the expense of smaller ones.

Answer 51

* Environmentalists- concerned about the possible environmental effects. * Hard to predict ecological consequences, could cause irreversible damage to habitats. * Recombinant genes- could passed from the original organisms to other ones with unknown effect on the environment- uncontrolled spread of recombinant DNA with unknown consequences on the environment which could also impact human health. * Suicide genes can be inserted into organisms engineered so they can only survive with a supplement. * Monocultures reduce biodiversity, damaging the environment. * Long-term consequences for evolution- artificial selection could reduce variety for evolution- should we be modifying genes instead of using nature.

Answer 52

* Manipulation of DNA- could have consequences to metabolic pathway of the cell- unforeseen by-products of the change might be produced- could create new diseases, cancers and metabolic malfunctions. * Mutation- could turn organism into a pathogen without control. * Genetically modified bacteria also have antibiotic resistance marker genes- added- might spread resistance to harmful bacteria. * People may not have a choice on whether to consume food/ other products using genetically engineered organisms without proper labelling. * Some people are concerned processes used to purify proteins form genetically engineered organisms could lead to the introduction of toxins into the food industry.

Answer 53

* Concerns of ‘superweeds’ development- viruses and pollen could transfer herbicide resistance in recombinant crops to wild plants- uncontrollable weeds. * Farmers might plant monocultures of transformed crops making the whole crop vulnerable to the same disease because the plants are genetically identical.

Answer 54

* Companies owning genetic engineering technologies may limit the use of technology to save lives. * Technology could be used unethically e.g. to make designer babies. * Replacing defective genes could go too far- adding genes for cosmetics, intelligence, and muscular bodies- risk of eugenics. * Financial cost- would be better if used to fight hunger and poverty. More sophisticated expensive treatments may be put before everyday treatments. Treatments may also only reach the better off. * Testing/ genetically modifying animals could cause them discomfort. * Concern technology could be used in ways other than for medical treatment- such as for treating the cosmetic effects of aging. * There’s the potential to do more harm than good by using the technology for gene expression- e.g. risk of overexpression- too much protein, the wrong cells producing the protein, and cancer. * Genetic screening for disease may lead to discrimination by insurance companies and employers if people are known to have high risk of developing a condition.

Answer 55

* Small number of large corporations own patents to particular seeds. Can charge prices and require farmers to purchase seeds each year. If non-GM crops are contaminated, farmers can be sued for breaching patent law. * Financial implications in new region- e.g. growing bananas in Britain which could be grown in tropical countries. * Small numbers of large biotechnology companies control genetic engineering. * As use of the technology increases- companies get bigger and more powerful- causes smaller companies to become out of business and causes power imbalances and inequalities- concern of anti-globalisation activists. * Could be used in the wrong hands to achieve political control. * Genetic fingerprinting- ability to identify DNA accurately- could be used wrongfully- exchanging DNA samples leading to wrongful convictions.

Answer 56

* Individuals or companies can patent and own genes. * Debate over who owns genetic material from humans once it is removed from the body- the donor or the researcher. * Some argue that the individual holds the right to their own genetic information. However, others argue its value is created by the researcher who uses it to develop a medicine or in diagnosis.

Answer 57

* Differences in DNA between individuals of the same species can be exploited for identification and diagnosis of heritable conditions. * Many diseases have a genetic origin often as a result of a gene mutation. * Recombinant DNA technology- enables diagnosis and treatment of genetic disorders. * It is often necessary to know exactly where a particular DNA sequence is located so methods to locate genes are used including DNA probes and DNA hybridisation. * Labelled DNA probes and DNA hybridisation can locate a specific allele of a gene, for example an allele which causes a genetic disorder.

Answer 58

* Identify and isolate the base sequence of the allele to be located. * A fragment of DNA complementary to the target allele is produced that has a sequence of bases. * Multiple copies of the DNA are formed using the polymerase chain reaction. * A DNA probe is made by attaching a marker, e.g. fluorescent dye, to the DNA fragment. * **DNA from the organism investigated is extracted and a restriction endonuclease is added to split the DNA into fragments. ** * **The fragments are separated using electrophoresis.** and transferred to a nylon membrane. * **DNA** from the person investigated for the target allele is heated or **treated to** separate the two strands into **single strands and expose the bases so that the DNA probe can anneal.** * The separated strands are cooled in a mixture containing DNA probes. * If the DNA contains the target allele, **the probes bind and hybridise with it** because the probe has base sequences that are exactly complementary to those on the mutant allele- DNA becomes hybridised. * DNA is washed clean of unattached probes. * Remaining hybridised DNA will now be fluorescently or radioactively labelled. * The labelling is detected using UV light to cause the dye to fluoresce under a microscope or using X-rays for radioactive probes in **autoradiography to show the bound probe. **

Answer 59

DNA hybridisation- occurs when a section of DNA or RNA is combined with a single-stranded section of DNA which has complementary bases.

Answer 60

* Denaturation- double strands of DNA molecule are heated so they separate into two complementary single strands. * The donor’s DNA must be made single-stranded to allow the use of DNA probes and hybridisation because both depend on the formation of complementary base sequences. * Means a DNA probe will bind- hybridise to the target allele if its present in a sample of DNA. * When the DNA is cooled, the complementary bases on each strand anneal with each other to reform the original double strand. * With enough time, all strands in a mixture of DNA will pair up with their partners. * If other complementary sections of DNA are present in the mixture as the DNA cools, they are also likely to anneal with one of the separated DNA strands- hybridisation

Answer 61

* DNA probe- **short, single-stranded** length of **DNA** with a **complementary base sequence** to part of target allele or DNA to be located. * DNA probes are labelled to make easily identifiable. * DNA probes can be used to locate specific alleles of genes e.g. on chromosomes or to see if a person’s DNA contains a mutated allele that causes a genetic disorder.

Answer 62

* Radioactively labelled probes- make up of nucleotides with isotope 32P- identified using X-ray film exposed by radioactivity. * Fluorescently labelled probes- emit light under certain conditions e.g. when the probe has bound to the target DNA sequence.

Answer 63

* To make a specific probe, the base sequence of the particular allele the probe aims to locate needs to be known. * The nucleotide bases of the target allele are sequenced. * To produce a DNA probe, you need to isolate the sequence of the allele that you want to screen for. * Different methods are used to isolate the sequence- exact order of bases in the target length of DNA. * Due to extensive genetic libraries, the base sequences of most genetic diseases are known, so these libraries are often used to obtain the sequence which can be produced in a gene machine. * You can use PCR to produce multiple complementary copies of part of the allele- form basis of the probes. * The fragments are combined with labels to produce DNA probes.

Answer 64

* **Single-stranded DNA probe is made that has a specific base sequence which is complementary** to part of the base sequence of the DNA that makes up the allele of the gene we want to find/ the part of DNA. * The double-stranded DNA that is being tested is broken down into smaller fragments using **restriction endonucleases to break phosphodiester bonds and cut DNA at a specific base sequence at the recognition/ restriction site. ** * The DNA fragments are separated by electrophoresis and transferred to a nylon membrane. * The DNA fragments are treated to form single strands and expose the bases so that the **DNA probe can anneal.** * Single-stranded DNA probes are added to separated DNA strands. * DNA probes binds to the complementary base sequence on one of the strands- DNA hybridisation. * The site where the probe binds can be identified by the radioactivity or **fluorescence** the probe emits, allowing specific base sequences of DNA to be detected.

Answer 65

* DNA probe- label attached so it can be detected. * Most common labels are radioactive labels detected using X-ray film in autoradiography or fluorescent label detected using UV light.

Answer 66

* A sample of DNA is digested into fragments using restriction enzymes and separated using electrophoresis. * The separated DNA fragments are then transferred to a nylon membrane and incubated with a fluorescently labelled DNA probe. If the allele is present, the DNA probe will binds – hybridise- to it. * Membrane is then exposed to UV light and if the gene is present there will be a fluorescent.

Answer 67

* It is possible to fix hundreds of DNA probes on a glass slide in an array- pattern. * DNA probe can be used as part of a DNA microarray which can screen for lots of different genes at the same time- useful to find genetic disorders quickly. * It is possible to test simultaneously for many different genetic disorders by detecting fluorescence that occurs where binding has taken place in the array.

Answer 68

* DNA microarray is a glass slide with microscopic spots of different DNA probes attached to it in rows of pores. * Sample of fluorescently labelled human DNA is washed over the array. * If the labelled human DNA contains any DNA sequences that are complementary to any of the probes it sticks to the array in the pores. * The array is washed to remove any fluorescently labelled DNA that hasn’t bound to it and then visualised under UV light. * Any labelled DNA attached to a probe will fluoresce. * Any spots that fluoresce mean that the person’s DNA contains that specific allele. If the probe is for a mutated allele that causes a genetic disorder this person has the allele.

Answer 69

Labelled DNA probes can be used to screen patients for heritable conditions, drug responses or health risks.

Answer 70

* Genetic disorders- often the result of gene mutations. * Gene mutations- arise if one or more nucleotide bases in DNA are changed. * If the mutation results in a dominant allele- all individuals with the allele will have the genetic disorder. * If the allele is recessive, only those individuals who are homozygous recessive will have the phenotype for the disease. * Individuals who are heterozygous for the recessive allele won’t display symptoms of the disease but are carriers of the mutant allele, meaning they can pass the disease onto their offspring if the other parent is heterozygous or homozygous recessive.

Answer 71

* Screening individuals who may carry a mutant allele can determine the probability of a couple having offspring with a genetic disorder. * Screening often occurs when individuals have a family history of a disease, so potential parents who are at risk can obtain advice from a genetic counsellor about having children based on their family history and genetic screening.

Answer 72

* Genetic screening can detect oncogenes, responsible for cancer, or mutations in tumour suppressor genes. * Two mutated alleles must be present to inactivate the tumour suppressor gene, but some people inherit open mutated tumour suppressor gene, these individuals are at greater risk of developing cancer. * Individuals at risk can be detected by genetic screening so they can make decisions about their lifestyle (giving up smoking, losing weight, eating healthily and avoiding mutagens), be screened for early diagnosis and treatment, and be prepared for treatment.

Answer 73

* Identifying inherited conditions so treatment/ preparation can start e.g. Huntington’s, cystic fibrosis. * Used to help determine how a patient will respond to specific drugs e.g. drugs in response to specific cancer-causing genes such as the HER2 proto-oncogene. * It can also be used to help identify health risks. E.g. inheriting particular mutated alleles increases risk of developing cancer- people may make choices to reduce risk. * Identify if parents are carriers to assess the probability of their offspring having a hereditary disease if there is a family history of it. * Identifying the pathogen a patient is infected with- **to check if the strain is resistant to antibiotics and prescribe the right antibiotic, to see if there is a vaccine against the pathogen or to produce a vaccine to vaccinate potential contacts to stop spread, test other individuals to see who has caught to disease to allow the control of the spread of the disease so people don’t get the disease- make sure answer with two points that relate to each other rather than two separate points.**

Answer 74

* Gene therapy involves altering the defective genes- mutated alleles inside cells to treat genetic disorders and cancer- can use the same technology as recombinant DNA technology. * Both processes involve inserting in a DNA fragment into the person’s original DNA. * Like in recombinant DNA technology, a vector is needed to get the DNA into the cell. * Range of different vectors can be used e.g. altered viruses, plasmids or liposomes (spheres made of lipids).

Answer 75

* Treatment depends on the alleles the disorder is caused by. * A mutated dominant allele- can silence the dominant allele e.g. by sticking a bit of DNA in the middle of the allele so it doesn’t work anymore. * Two mutated recessive alleles- add working dominant alleles to make up for them- supplement the faulty ones.

Answer 76

* Somatic therapy- involves altering the alleles in body cells particularly the cells that are most affected by the disorder. Somatic therapy doesn’t affect the individual’s sex cells (sperm and eggs) so any offspring could still inherit the disease. E.g. somatic therapy for epithelial cells lining the lungs. * Germ line therapy- altering the alleles in the sex cells. Every cell of any offspring produced from these cells will be affected by the gene therapy and they won’t suffer from the disease. Currently illegal in humans.

Answer 77

* Results of genetic screening can be used in personalised medicine- tailored to an individual’s genes. * Genes- determine response to drugs. * Doctors can provide advice and healthcare based on an individual’s genotypes. * Different people respond to the same drug in different ways- some people’s genes mean that a particular drug may be more or less effective in treating a condition. * If doctors have genetic information, they can use it to predict response to different drugs and only prescribe ones most effective to an individual. * By genetically screening patients, doctors and pharmacists can determine the correct dose of drug, which can save money otherwise wasted on overprescribing the drug. * Genetic screening can help avoid medications that could cause harm or raise false hopes. * E.g. painkillers- need specific enzymes to activate them- some of the population have genes that alter the function of the enzyme- screening for these genes allows the dosage to be adjusted to compensate for how genes affect an individual’s metabolism of the painkiller- ensures their sue is safe and effective.

Answer 78

* The results of genetic screening can be used for genetic counselling. * Genetic counselling involves explaining the results of a screening and advising patients and relatives about risks of genetic disorders. * Expert advice and information provided by genetic councillors helps individuals to understand the results and implications of screening so they can make appropriate personal decisions about themselves and their offspring. * Like a genetic councillor, you should be able to evaluate information relating to screening individuals for genetically determined conditions and drug responses.

Answer 79

* Genetic councillors- research family history of a mutated allele for an inherited disease and advise parents on the likelihood of it arising in their children. * Genetic councillors can make couples aware of the chances their children will be affected by the disease and be carriers of the disease. * Genetic councillors can also provide information on the consequences of genetic conditions, enabling couples to make informed decisions about having children. * Counselling can also make people aware of further medical tests giving them a more accurate predication of whether their children will have the condition e.g. IVF screening of embryos.

Answer 80

* Genetic screening results provide genetic counsellors with the basis for informed discussion. * Screening can help identify if someone is the carrier of a mutated allele, the type of mutated allele the person is carrying, and indicate the type of genetic disorder or cancer the person has and the most effective treatment. * If a person is positive for a mutation after screening, genetic counselling can be used to advise patients on the options of prevention or treatment available. * E.g. Sickle-cell anaemia- recessive genetic disorder caused by mutation in the haemoglobin gene. Couple susceptible might test and undergo genetic counselling to understand the chances of having a child with sickle-cel anaemia. Genetic counselling- provides advice on the possibility of IVF and screening embryos for the allele. Embryos without the mutation are implanted in the womb. The councillor could also provide information on the help and drugs available to deal with sickle-cell anaemia.

Answer 81

* Oncogene mutations- determine the type of cancer the patient has and 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 chance of survival. * Single cancer cells among millions of normal cells- identifying patients at risk of relapse. * A person with a family history of breast cancer may be screened for known mutations that lead to breast cancer e.g. BRCA1 mutation. * If the results come back positive, genetic councillors might explain the chances of developing cancer, refer the patient for further monitoring, and advise on treatments, lifestyles and surgical steps (mastectomy) to reduce the risk of breast cancer. * Information can help a counsellor discuss with the patient the best treatment and their prospects of survival.

Answer 82

* Genetic fingerprinting is a diagnostic tool used in forensic science, plant and animal breeding and medical diagnosis. * Based on how DNA of every individual, except identical twins is unique.

Answer 83

* Technique relies on how the genome of most eukaryotic organisms contains many repetitive, non-coding bases on DNA. * Non coding DNA- doesn’t code for amino acid/ tRNA/ rRNA. * Not all of an organism’s genome codes for proteins. 95% of human DNA is currently not known to code for any characteristic. * An organism’s genome contains many variable number tandem repeats (VNTRs) - base sequences that don’t code for proteins and repeat next to each other e.g. CATGCATG. * Most of an organisms genome consists of variable number tandem repeats (VNTRs) * For every individual, the number and length of VNTRs has a unique pattern- different in all individuals except identical twins. * The probability of two individuals having the same VNTRs is very low. * The number of times these sequences are repeated differs in each person so the length of these sequences in nucleotides differs too. * The repeated sequences occur in lots of places in the genome. * The number of times a sequence is repeated at different places in the genome can be compared between individuals using genetic fingerprinting. * The probability of two individuals having the same genetic fingerprint is low because the chance of two individuals having the same number of VNTRs at each place they’re found in DNA is very low. * The genetic fingerprints can therefore be compared between different individuals. * The more closely related two individuals are, the mere similar the VNTRs are.

Answer 84

* The technique of genetic fingerprinting is used to analyse DNA fragments that have been cloned by PCR. * Separation of DNA fragments by gel electrophoresis. * Hybridisation. * Analysis of genetic fingerprints.

Answer 85

* A sample of DNA is obtained e.g. from blood, saliva etc. * A small sample can used to give a genetic fingerprint. The DNA is extracted, separating it from the rest of the cell. * The amount of DNA is usually small so its quantity can be increased using the polymerase chain reaction * PCR is used to make many copies of the areas of DNA that contain the VNTRs. * Primers are used that bind to either side of those repeats so the whole repeat is amplified. * Different primers are used for each position under investigation. * The result is DNA fragments where the length in nucleotides corresponds to the number of repeats the person has at each specific position. * A fluorescent or radioactive tag is added to all the DNA fragments (usually to the primers) so they can be viewed under UV light or X-rays. * Only DNA fragments up to 500 bases long can be sequenced. * Larger genes and whole genomes must be cut into smaller fragments by restriction endonucleases. * DNA is cut into fragments, using the same restriction endonucleases. Endonucleases are chosen for their ability to cut close to, but not within the target DNA.

Answer 86

* Gel electrophoresis is used to separate DNA fragments by size. * The DNA fragments are placed in wells in agar gel with an alkaline buffer solution which separates the double strands into single strands and conducts electricity when a voltage is applied. * DNA fragments are negatively charged and move towards the positive electrode at the far end of the gel. * The resistance of the gel means the larger the fragment, the more slowly they move. * Over a fixed period, smaller fragments move further and faster than larger ones, so DNA fragments of different lengths are separated. * This produces a pattern of bands.

Answer 87

* Radioactive or fluorescent DNA probes are used to bind with VNTRs. * The probes have base sequences which are complementary to the base sequences of VNTRs, and bind to them under specific conditions such as temperature and pH. * The process occurs with different probes, which bind to different target DNA sequences.

Answer 88

After the gel has been running long enough, the equipment is turned off and the gel is analysed either under X-rays or UV light.

Answer 89

* Under the UV light the DNA fragments can be seen as bands. * The locations on the probes are located visually.

Answer 90

* An X-ray film is put over the nylon membrane. The film is exposed by the radiation from the radioactive probes. * The final position in the gel can be determined by placing X-ray film over the agar gel for several hours. * The radioactivity from each DNA fragment exposes eth film and shows where fragments are situated on the gel.

Answer 91

* Bands- make up the genetic fingerprints. * Locations of bands correspond to the position of the DNA fragments as separated during electrophoresis- a series of bars is revealed. The pattern of bands is unique to every individual except identical twins. * DNA ladder- added to one well- mixture of DNA fragments of **known lengths** allowing you to **compare the position of fragments** and work out the length of other bands on the gel. * Genetic fingerprints from different samples can be compared. * If both fingerprints have a band in the same location on the gel it means they have the same number of nucleotides and so the same number of VNTRs at that place- it’s a match. * If there appears to be a match after the fingerprints are visually compared, 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. * The machines use data obtained by measuring the distances travelled during electrophoresis and known lengths of DNA. * The probabilities are calculated of someone else having an identical fingerprint. * The closer the match between two patterns, the greater the probability that two sets of DNA have come from the same person.

Answer 92

* Gel electrophoresis can be used to separate DNA, RNA and proteins. * The charges, mass and size of molecules is used to separate them using the cathode (negative pole) and anode (positive pole). Different sized molecules move at different rates and different charges move towards different poles. Pores in gel result installer molecules moving quickly while larger molecules move slowly. * Food dyes are attracted to the positive current but have different levels of speed of movement depending on their molecule size. This helps determine the size and charge of the molecule. * Gel electrophoresis can also be used to separate proteins- **charge, different R groups, and number of amino acids in the polypeptide** affect the distance they travel, enabling them to separate.

Answer 93

* You may need to compare or locate DNA fragments to determine relations/ matches in a gel electrophoresis plate of genetic fingerprints- look for the variables and label them in order of shortest to largest depending on which travel further, look for alignment and matches. * Thicker bands in a genetic fingerprint mean there is more of that gene and therefore more o fht proteins it produces. * You may need to explain the biological principles that underpin genetic fingerprinting techniques * You may need to interpret data showing the results of gel electrophoresis to separate DNA fragments. * You may need to interpret flow charts/ diagrams about the processes discussed in this topic, read the boxes and arrows carefully and break down the logical order of events, and be careful of inhibition vs. stimulation.

Answer 94

You may need to explain why scientists might use genetic fingerprinting in the fields of forensic science, medical diagnosis, animal and plant breeding.

Answer 95

* Used to determine the biological father of a child by comparing genetic fingerprints. * If lots of bands match then the person is most probably the father. * The higher the number of places in the genome compared, the more accurate the test result. * Roughly half the bands will match the paternity test as half the DNA is inherited from each parent. * Each band on a DNA fingerprint of an individual should have a corresponding band on one of the parent’s DNA fingerprints.

Answer 96

* VNTR base sequences are inherited from parents. * Half of VNTR sequences come from each parent. * More bands on a genetic fingerprint that match, the more closely related two people are.

Answer 97

* Used to determine the biological father of a child by comparing genetic fingerprints. * If lots of bands match then the person is most probably the father. * The higher the number of places in the genome compared, the more accurate the test result. * Roughly half the bands will match the paternity test as half the DNA is inherited from each parent. * Each band on a DNA fingerprint of an individual should have a corresponding band on one of the parent’s DNA fingerprints.

Answer 98

* The male and female line of descent can be traced using mitochondria. * Female line of decent is found in mitochondria because in humans and most other organisms mitochondrial DNA (mtDNA) is only inherited from the mother. The Y chromosome can be explored to look at inheritance from the father.

Answer 99

* Genetic fingerprinting can be used to look at wider genetic relationships and evolution- phylogenetics- the more bands populations have in common, the more closely related they are. * Genetic fingerprinting is useful in determining genetic variability within a population. * The more closely two individuals are related the closer the resemblance of their genetic fingerprints. * Greater the number of bands that don’t match on a genetic fingerprint- more genetically different individuals are. * Can compare the number of repeats at several places in the genome for a population to find out how genetically varied a population is. * The more the number of repeats varies at several places, the greater the genetic variability within the population. * A population whose member have a greater variety of genetic fingerprints has greater genetic diversity, and vice versa for low genetic diversity. * To determine whether species are closely related- **use genetic fingerprinting, the polymerase chain reaction, electrophoresis, DNA/ genome sequencing. **

Answer 100

* Compare samples of DNA collected from crime scenes (e.g. blood, semen, skin cells, saliva, hair etc.) to samples of DNA from possible suspects which could link them to the crime scenes. * Genetic fingerprinting can establish whether a person is likely to have been present at the crime scene.

Answer 101

* Doesn’t prove the person did the crime- other possible explanations need to be investigated including: * DNA being left on another innocent occasion. * DNA belongs to a close relative. * DNA sample may have been contaminated after the crime, by the suspect or by chemicals that have affected the action of the restriction endonucleases used to prepare the fingerprint.

Answer 102

* The DNA is isolated from all the collected samples- crime scene and suspects. * Each sample is replicated using PCR. * The PCR products are run on an electrophoresis gel and he genetic fingerprints produced are compared to see if they match. * If the bands from the samples match, it links the person to the crime scene.

Answer 103

* The probability that someone else’s DNA might match the suspect has to be calculated. * The calculation is based on the assumption that the DNA which produces the banding patterns is randomly distributed in community. * In religious or ethnic groups where people have partners from within a small community the calculation may not be random.

Answer 104

* Genetic fingerprints can help in diagnosing diseases. * Unique pattern of several alleles can be used in medical diagnosis to diagnose genetic disorders and cancer. * In theory, the inheritance of VNTRs doesn’t have any influence on the phenotype of an organism. * However, the fingerprint can be matched with fingerprints of people with various forms of the disease and those without the disease, so the probability of developing the symptoms and when can be determined. * Useful when the specific mutation isn’t known or where several mutations could cause the disorder as it identifies broader altered gene patterns. * Genetic fingerprints can also be used to identify the nature of a microbial infection by comparing the fingerprint of the microbe found in patients with that of known pathogens.

Answer 105

* Preimplantation genetic haplotyping. * Screens embryos created by IVF for genetic disorders before they’re implanted into the uterus. * The faulty regions of the parent’s DNA are used to produce genetic fingerprints which are compared to the genetic fingerprint of the embryo. * If the fingerprints match, the embryo inherited the disorder so it is not implanted. Can be used to screen for cystic fibrosis, Huntington’s etc.

Answer 106

* Genetic fingerprinting can be used to diagnose tumours which cannot easily be identified using conventional methods which only show the physical differences between tumours. * Genetic fingerprint of known tumours can be compared to the genetic fingerprint of the patient’s tumour. * The patient’s tumour can then be specifically diagnosed and the treatment targeted to the specific type.

Answer 107

* Nervous system disease. * Huntington’s results from a three-base sequence at one end of a gene on a chromosome being repeated. People with fewer than 30 repeats are unlikely to get the disease while those with more than 38 repeats are likely to. A person with over 50 repeats will often express the disease earlier. * DNA from a person with the allele for Huntington’s disease can be cut with restriction endonucleases and a DNA fingerprint prepared.

Answer 108

* Genetic fingerprinting- used on animals and plants to prevent inbreeding in farms and zoos, which decreases the gene pool- number of different alleles in a population. * Inbreeding- leads to an increased risk of genetic disorder- health, productivity and reproductive problems. * Genetic fingerprinting- can be used to identify the least related individuals in a population so they can be breed together. * Also used to identify plants or animals that have a particular allele for a desirable gene. * Individuals with the desirable allele can be selected for breeding in order to increase the probability of their offspring having the characteristic. * Genetic fingerprinting- used by animal breeders to prove pedigree (the animals’ parents)- animals with a good pedigree sell for more money.

Chapter 21- Genome Projects and Recombinant DNA technology Flashcards

3.8.3, 3.8.4 (132 cards)