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Biotechnology and Genetic Engineering - What is it? - electrophoresis - PCR - the Human Genome Project DNA sequencing - Cloning (genes), - Gene therapy - Cloning (cells, and multicellular organisms) - Agricultural applications Refer to chapter 14 in text What is it? biotechnology noun: “the use of living organisms or other biological systems in the manufacture of drugs or other products or for environmental management, as in waste recycling: includes the use of bioreactors in manufacturing, microorganisms to degrade oil slicks or organic waste, genetically engineered bacteria to produce human hormones, and monoclonal antibodies to identify antigens.” Random House Unabridged Dictionary There is WAY more included than the examples listed here. 1 genetic engineering . 1. the development and application of scientific methods, procedures, and technologies that permit direct manipulation of genetic material in order to alter the hereditary traits of a cell, organism, or population. 2. a technique that produces unlimited amounts of otherwise unavailable or scarce biological product by introducing DNA isolated from animals or plants into bacteria and then harvesting the product from a bacterial colony, as human insulin produced in bacteria by the human insulin gene. - Mixtures of fragments are separated by size due to resistance to passage through a gel when pulled by an electric charge. - Can be used (with different matrices) to separate proteins and other molecules as well as DNA. → - May be used for diagnosis, paternity testing, criminal forensics, to purify a section of DNA needed for further study. a32.lehman.cuny.edu/molbio_course/agarose1.htm gel electrophoresis of DNA restriction fragments (in case you have forgotten….) → http://porpax.bio.miami.edu/~cmallery/150/protein/electrophoresis.jpg electrophoresis (This is an application from the DNA restriction digest/ electrophoresis lab analysis….) Fluorescent dyes↑ make bands visible under UV light. Southern blot to identify specific fragments. - Gel is pressed onto a special membrane. - DNA bands transfer. - A radio-labeled DNA fragment base-pairs with the target fragment. - This fragment can be excised for further study. (Named for Edwin Southern who pioneered this procedure, subsequent variations are called northern and western blots. Really.) askabiologist.asu.edu/.../southern/southern.html application: DNA fingerprinting (Here, paternity testing) A selected segment of (usually) “junk” DNA is isolated ← STR: variable # of short tandem repeats e.g… ACGGACGGACGGACGG… between restriction sites results in RFLP: restriction fragment length polymorphism (“riflips”). ←The RFLP pattern is established for the persons of interest…. and compared to the child’s pattern: He had to get those restriction sites from his parents. → http://www.thetech.org/genetics/ask.php?id=20 Consequences of DNA fingerprinting (DNA profile): What are the implications for paternity testing? There are historical applications, too. How might it be medically important? In forensics, the evidence is much stronger than eye witnesses, cold cases are often resolved in the alleged prep’s favor… CODIS (Combined DNA Index System) files: pros and cons… as it were. In 1999 180,000 rape kits were unprocessed.1 In 2009 11,000 rape kits in an abandoned Detroit police warehouse.2 1 for more info, NIJ report 7/2002:http://www.ncjrs.gov/pdffiles1/nij/194197.pdf 2 Alter, Charlotte TIME Magazine, July 28, 2014, p 28ff PCR: polymerase chain reaction -used to amplify DNA samples for fingerprinting, sequencing, or further genetic manipulation. - isolate segment of interest - warm to denature - cool to anneal primer - allow polymerase to proceed -“Taq” polymerase* - Each cycle doubles the DNA. DNA from as few as 20 cells can be enough to use PCR and catch a criminal. This technology was an essential starting point for …. * from Thermus aquaticus (more heat tolerant). the Human Genome Project Started in 1990, finished in 2003 (way ahead of schedule, due to technology and competition issues), for disease diagnosis, genetic research, evolution studies, possible drug therapies, identify the 20,000- 30,000 genes… It was a publicly funded effort of global cooperation between companies, universities, and nations. From the start a portion of the funds went to ethical research. Why? - Began with linkage mapping to show relative order of genes, - continued with ordering of restriction fragments to determine physical distances, - and ended with sequencing of the DNA. Since then many other organisms’ genomes have been sequenced. HGP, under auspices of department of energy: http://genomics.energy.gov/ Sanger method of DNA sequencing: uses ddNTPs to stop replication of a target strand. Products then sorted by size… The Campbell illustration adds a very cool touch: The ddNTPs are each labeled with a distinctly colored fluorescent tag, the gel is run in a tube, and the colors are read by a laser detector. p. 397 genetic engineering Genes can be transferred from one organism to another, and the gene products produced even if the organisms are very different, because of the universality of the genetic code. (Prokaryotes have the same genetic language as do you.) We will discuss some of what can be done, and what is being pursued. Whether things should be done is an ethical issue: Is it OK to change the genome of a corn plant? Of a human? What if it alleviates suffering? What is the risk of genetically engineering cows? - risk to cows, cows’ neighbors, cows’ consumers? (Science) Is it justified by the possible benefit? (Ethics) transgenic technology - Subject gene isolated restriction digest, electrophoresis, - amplified PCR, - inserted into plasmid restriction enzymes, DNA ligase, transformation, and used to either generate the subject gene’s product (right), or to alter a new host’s genome (left). (E. coli is most common host, but yeasts or other cell types work.) cloning (genes) (IB doesn’t include this as cloning.) Recall that prokaryotes don’t have mRNA processing like eukaryotes: In order to get a useful product, human (e.g.) DNA is transcribed, pre- mRNA is processed, mRNA is isolated, and viral reverse transcriptase is used to make a cDNA (copy DNA), and then a complementary strand is built. (Sticky ends can be added for direct insertion) NOW it is ready for the snipping, sorting, and inserting on the previous slide, so the bacterium can generate the protein. The cloned gene is not of much use until you can screen for/select which bacteria have picked up the plasmid. This is part of an AP lab on transformation you will do shortly. Gene therapy Somatic cell line therapy: changes made in the DNA of somatic cells. This can be done in adults, but has to be redone periodically. e.g. SCID is the lack of ADA (adenosine deaminase) in white blood cells. - cells are removed from the patient - a virus engineered to carry the ADA gene infects the cells - the gene enters the cell, allowing for the manufacture of ADA. - The gene is NOT in the stem cells, so the process is not self propagating. - These therapies are mostly experimental still. - vector here is viral, liposomes (phospholipid droplets) can be used. Germ line therapy: changes made in the DNA of gametes. This would be in all cells of the resulting person. At this point this approach is theoretical as a human therapy. There are practical and ethical issues. It is done in lab animals as a research tool, and used in agriculture. cloning (cells) For single-celled organisms, their simple mitosis or binary fission is creating identical progeny. In a lab, they just need to be isolated and cultured. For cells from multicellular organisms it is trickier: - Some isolated cells may grow as sheets in culture (such as skin used in burn treatment). - Therapeutic cloning, (the creation of an embryo to supply embryonic stem cells for medical use) is only allowed to the blastocyst stage in most places: uses? pros and cons? - The race is on to reprogram differentiated somatic cells into stem cells, for in-vitro tissue and organ growth. http://newsimg.bbc.co.uk/media/images/44251000/gif/_44251016_stem_cells_416.gif cloning (multicellular organisms) You have a lab for an on-line whole organism cloning activity. You provide the answers to confirm participation in the exercise, followed by questions on the ethical considerations. www.sheep101.info/interestingfacts.html So how is all of this used? - β-carotene, (pro-vitamin A, needed for good vision), made by rice plant, but not in endosperm… - PSY gene from daffodils and bacterial CRTI gene inserted into rice. - Rice-dependent people experience significant drop in blindness. http://www.goldenrice.org/Content2-How/how1_sci.html Agricultural applications “Crop production is limited by salinity on 40 percent of the world's irrigated land and on 25 percent of irrigated land in the United States”. Salt is sequestered in leaf vacuoles, not in fruit, and may draw salt out of soil. “… Blumwald and Zhang genetically engineered tomato plants that produce higher levels of …protein known as a "transport protein." The gene that controls increased production of the transport protein was taken from Arabidopsis, a relative of the cabbage …. The transport protein uses energy available in the cells to move salt – in the form of sodium ions -- into compartments within the cells called vacuoles. Once the salt is stashed inside the vacuoles it is isolated from the rest of the cell and unable to interfere with the plant's normal biochemical activity”. http://www.news.ucdavis.edu/search/news_detail.lasso?id=5840 Specific example of GMO battle in ’16 IB syllabus. Recall…. Potatoes make two starches: amylose (little branching, less soluble, slow digesting, poor for gels) amylopectin (opposite of the above) http://franziska-brantner.eu/blog/wp-content/uploads/2010/05/basf-500x334.jpg Industry prefers amylopectin. Amflora is genetically modified to not have an enzyme needed to make amylose. It was OKed by the EU in 2010, but the OK was defeated in 2012. https://thescienceofnutrition.wordpress.com/tag/amylopectin/ Amflora Pharming (producing medically useful products from genetically modified plants and animals) This method is hit or miss (insertion site is random and often deleterious), but if desired human protein is linked to a milk production promoter, and a gene for antibiotic resistance, eggs or embryos can be selected before implantation. Current applications: spider silk in goat milk to make “Biosteel”, human serum albumin in cow’s milk to expand blood in trauma cases, factor IX (to treat hemophilia) made in sheep’s milk. Bt (Bacillus thuringiensis), a highly specific parasitic bacterium that produces endospores and crystals. The crystals punch holes in the gut of pests, the spores exit gut, germinate, re-enter gut, and exit the now dead host. http://www1.umn.edu/news/prod/groups http://muou.sc.mahidol.ac.th/images/mechanism_bt.jpg Bt crops: The genes for the crystals are introduced into crop genomes, so the nibbling pests die, without needing chemicals or the actual bacterial culture. Analysis of risks to monarch butterflies of Bt crops. argument counter argument http://www.news.cornell.edu/stories/1999/0 4/toxic-pollen-bt-corn-can-kill-monarchhttp://www.ars.usda.gov/is/br/btcorn/index.html# bt11 butterflies Concerns: - ethics of engineering animals (and plants…) - unidentified affects of specific gene insertions - unintended dispersal of gene in broader environment - ability to choose whether or not to consume such products - loss of income to previous providers - lack of understanding/information to public Advantages: - cheaper/more accessible protein products - availability of more nutritious/cheaper foods - reduced reliance on pesticides/herbicides - serious money to the developers/new industries (Discuss pros and cons of any one example of genetic modification.) You have an unidentified hunk of DNA in a test tube. Describe a line of research that would investigate this find, and perhaps put it to some good use. Employ as many of the techniques explored here as possible. Pick a technology, such as gene therapy: How is it done? When should it be applied? To whom should it be available? biotechnology Sanger method genetic engineering ddNTP electrophoresis transgenic Southern blot plasmid DNA fingerprinting reverse transcriptase paternity testing somatic line therapy RFLP vector STR liposome DNA profile germ line therapy forensics Therapeutic cloning CODIS binary fission PCR: in-vitro Human Genome Project pharming Bt crops