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Topic 4.4 notes Genetic engineering and Biotechnology 4.41 Outline the use of polymerase chain reaction ( PCR) to copy and amplify minute quantities of DNA. ( Details of methods are not required) See page 382 of text for more detail. The piece of DNA is multiplied by adding a promoter sequence and a heat resistant DNA Polymerase. The DNA strands are separated by heating… which does not denature the enzyme. Of course, the 4 nucleotides ( A,T,G,C ) also have to be added to the sample vial. The sample doubles every cycle, which takes only 3 minutes. Millions of copies can be made easily. Note that samples can be contaminated if careful procedures are not followed. The machine copies all DNA in the sample, so any extraneous DNA in the sample will also be copied. The PCR process is what has expanded the use of DNA evidence in crime scenes, and in a myriad of scientific investigations such as reading the DNA code of Neanderthals. 4.4.2 State that, in gel electrophoresis, fragments of DNA move in an electric field and are separated according to their size. The negative charged DNA moves to the positive end, but smaller pieces move faster and further. This results in a separation according to size. One can also remove segments of DNA from the gel for further analysis once they are sorted by size. Restriction enzymes are used to cut DNA in specific places. These enzymes were discovered years ago in bacteria which use them to protect themselves against invading viruses by cutting up their DNA. We know of and use dozens of different restriction enzymes. They are one the most important tools of modern gene technology. Each restriction enzyme recognizes and cuts the DNA at a specific nucleotide sequence. ( eg: AAGCGCTT) Restriction enzymes can leave " sticky ends", so that other sets of DNA can be inserted at that point. Other restriction enzymes cut off both strands at the same place, ( blunt ends) Example: To compare a crime scene set of DNA with a possible suspect's, you first amplify both sets of DNA using a PCR machine. You then cut both sets of DNA with the same restriction enzyme, and put through gel electrophoresis. If the DNA is the same, it will be cut in the same places and into the same length segments. The gel patterns will match. 4.4.3 State that gel electrophoresis of DNA can be used in DNA profiling 4.4.4 Describe the application of DNA profiling to determine paternity and also in forensic investigations [There is a variety of social implications stemming from DNA profiling, such as identity issues for a child who learns unexpectedly who his or her biological father is, self-esteem problems in relationships where the male partner learns that he did not father a child, but also relief for crime victims when those responsible for the crime are identified and convicted, sometimes decades later.] [TOK: Comparison between using blood groups and DNA profiling for paternity: Blood group analysis can only prove one is not the father, not that one is the father. Even if the child has a similar blood type it does not mean that the potential father with the same blood type is the father.] 4.4.5 Analyse DNA profiles to draw conclusions about paternity or forensic investigations ( see Campbell page 396) [The outcomes of this analysis could include knowledge of the number of human genes, the location of specific genes, discovery of proteins and their functions, and evolutionary relationships. We can either emphasize the large shared content of the human genome, which is common to all of us and should give us a sense of unity, or we can emphasize the small but significant allelic differences that create biodiversity within our species, which should be treasured. Differences in the success of human races in coping with the modern world and the threat to some small human tribes could be mentioned. It is important to stress parity of esteem of all humans, whatever their genome.] 4.4.6 Outline three outcomes of the sequencing of the complete human genome The outcomes include the knowledge of how many genes code for humans. We thought there could be as many as 100,000 genes, turns out to be around 25,000 genes. The complete genome allows for evolutionary comparisons of humans to other organisms. The project included mapping the genomes of other species such as E. coli, yeast, a nematode worm, Drosophila fly, and the mouse. The genome map allows scientists to locate specific genes on specific chromosomes. This opens up the potential of gene manipulation. Experiments can be done on genes that are in flies and mice that we know have direct application to humans. Gene expression control is critical. We can now detect and measure which genes are turned on at which time. 4.4.7 State that, when genes are transferred between species, the amino acid sequence of polypeptides translated from them is unchanged because the genetic code is universal. [ There is an ethical or moral question here: whether it is right to change the genetic integrity of a species by transferring to it from another species. The discussion should include the wider question of selective breeding of animals, and whether this is distinctively different and always acceptable. The possibility of animals suffering as a result of genetic modification could be considered] The genetic integrity of many species has already been compromised. Look at Monsanto's Round-up® resistant corn, and the Bt toxin added to crop plants. Corn is pollinated by wind, the engineered pollen has escaped the original engineered corn fields. 4.4.8 Outline a basic technique used for gene transfer involving plasmids, a host cell ( bacterium, yeast or other cell), restriction enzymes and DNA ligase. [ The use of E. coli in gene technology is well documented. Most of its DNA is in one circular chromosome, but it also has plasmids ( smaller circles of DNA). These plasmids can be removed and cleaved by restriction enzymes at target sequences. DNA fragments from another organism can also be cleaved by the same restriction enzyme, and these pieces can be added to the open plasmid and spliced together by ligase. The recombinant plasmids formed can be inserted into new host cells and cloned.] The important part is using the same restriction enzymes on both the plasmid and the target DNA fragment. The resultant sticky ends of the DNA can complementary bond and the ligase connects the phosphate/sugar backbone. 4.4.9 State two examples of the current uses of genetically modified crops or animals. [ Examples include salt tolerance in tomato plants, synthesis of beta carotene ( vitamin A precurser) in golden rice, herbacide resistance in crop plants, and factor IX ( human blood clotting) in sheep milk.] Amgen earns billions of dollars profit each year by inserting the gene to make the hormone that stimulates red blood cell production into E. coli. (Erythropoetin) Cancer patients recovering from chemotherapy need to re-stimulate blood cell production. This is also a drug abused by certain world class athletes… more red blood cells, more oxygen to muscles. 4.4.10 Discuss the potential benefits and possible harmful effects of one example of gene modification. [There are ethical questions here about how far it is acceptable for humans to change other species, as well as other ecosystems, in order to benefit humans. This is an opportunity to discuss how we can assess whether risks are great enough to justify banning techniques and how the scientific community can inform communities generally about potential risks. Informed decisions need to be made but irrational fears should not be propagated. Consideration could be given to the paradox that careful research is needed to assess the risks, but performing this research in itself could be risky. Do protester who destroy trials of GM crops make the world safer?] I think the best example of a genetically modified crop that has obvious benefits and drawbacks is the Round-up® resistant crop. Yes you get a greater yield per acre of land, but the entire point of it is to sell and use more of the herbacide Round-up®. 4.4.11 Define clone A group of genetically identical organism or a group of cells derived from a single parent cell. 4.4.12 Outline a technique for cloning using differentiated animal cells. (See page 408 of Campbell )Dolly, the sheep, was created by removing the nucleus of a sheep egg, then fusing the egg with a mammary cell of the donor sheep. The mammary cell had been given low levels of nutrients in a Petri dish, which de-differentiated it. An electrical charge fused the cells and stimulated the zygote to start dividing. The resultant zygote was implanted in a surrogate mother who gave birth to the clone of the donor sheep. 4.4.13 Discuss the ethical issues of theraputic cloning in humans. ( see page 409 of Campbell)The point of stem cells is that they are undifferentiated. They can turn into any tissue in the body. This holds great promise for a host of human diseases. Embryonic stem cells are the most pluripotent, but we have discovered ways to cause cells to revert to stem cell status. We seem to have solved the ethical problem of using embryos to supply stem cells.