Today: Biotechnology Exam #2 W 7/9 in class Comparative genomics also has been used to identify recently mobilized transposons in genetically diverse humans. For example, over 600 recent transposon insertions were identified by examining DNA resequencing traces from 36 genetically diverse humans. Tbl 1 Which transposable elements are active in the human genome? (2007) Ryan E. Mills et al. Trends in Genetics 23: DNA fingerprinting using RFLPs Visualizing differences in DNA sequence by using restriction enzymes Sequence 1 Sequence 2 Restriction Enzymes cut DNA at specific sequences Fig 18.1 Examples of some restriction enzymes tbl 18.3 Visualizing differences in DNA sequence by using restriction enzymes Sequence 1 Sequence 2 Fig Separating DNA on a gel by size Fig 20.6 Gel electrophoresis Fig 24.21 Visualizing differences in DNA sequence by RFLPs Fig 22.23 DNA fingerprinting Can DNA be obtained from hair? How can DNA be obtained from such a small sample? The inventor of PCR Polymerase Chain Reaction: amplifying DNA Fig 18.6 Polymerase Chain Reaction Fig 18.6 Polymerase Chain Reaction: Primers allow specific regions to be amplified. Fig 18.6 The inventor of PCR PCR animation Areas of DNA from very small samples can be amplified by PCR, and then cut with restriction enzymes for RFLP analysis. Genetic Engineering: Direct manipulation of DNA Fig 18.2 Bacteria can be modified or serve as intermediates Fig 18.2 a typical bacteria Bacterial DNA plasmid DNA A typical bacterial plasmid used for genetic engineering tbl 18.2 Moving a gene into bacteria via a plasmid Fig 18.2 Bacterial DNA plasmid DNA What problems exist for expressing eukaryotic gene in bacteria? Reverse transcriptase can be used to obtain coding regions without introns. Fig 18.4 After RT, PCR will amplify the gene or DNA Fig 18.6 Moving a gene into bacteria via a plasmid RT and PCR Fig 18.2 Restriction Enzymes cut DNA at specific sequences Fig 18.1 Restriction enzymes cut DNA at a specific sequence Fig 18.1 Cutting the plasmid and insert with the same restriction enzyme makes matching sticky ends Fig 18.1 A typical bacterial plasmid used for genetic engineering Using sticky ends to add DNA to a bacterial plasmid Fig 18.1 If the same restriction enzyme is used for both sides, the plasmid is likely to religate to itself. Fig 18.1 The plasmid is treated with phosphatase to remove the 5-P, preventing self- ligation Fig 18.1 Transformation of bacteria can happen via several different methods. tbl 6.1 Bacteria can take up DNA from the environment Fig 9.2 Tbl 6.1 Transformation of bacteria can happen via several different methods all involving perturbing the bacterial membrane: Electroporation Heat shock Osmotic Stress How can you know which bacteria have been transformed, and whether they have the insert? Fig 18.1 Resistance genes allow bacteria with the plasmid to be selected. Bacteria with the resistance gene will survive when grown in the presence of antibiotic Fig 20.5 Is the insert present? Plasmids with the MCS in the lacZ gene can be used for blue/white screening Fig 18.1 A typical bacterial plasmid used for genetic engineering Intact lacZ makes a blue color when expressed and provided X-galactose When the lacZ gene is disrupted, the bacteria appear white Blue/white screening: Transformed bacteria plated on antibiotic and X- gal plates. Each colony represents millions of clones of one transformed cell. Fig 18.1 Successful transformation will grow a colony of genetically modified bacteria Fig 18.1 Inserting a gene into a bacterial plasmid RT and/or PCR Fig 18.1 Millions of Hectares Texas = 70 ha Bacteria can be used to transform plants Global area planted with GM crops Agrobacterium infect plants, inserting their plasmid DNA into the plants genome. Fig 19.15b Fig Agrobacterium infect plants, inserting their plasmid DNA into the plants genome. By replacing the gall forming genes with other DNA when the Agrobacterium infect a plant, it will insert that DNA into the plant. Fig 19.16 The generation of a transgenic plant Grown on herbicide How do you know whether the gene you want to express has the correct sequence? DNA sequencing *Not covered in class; will NOT be on the exam; check it out if you are interested.* The structure of 2 ,3 -dideoxynucleotides Fig *Not covered in class; will NOT be on the exam; check it out if you are interested.* Fig The dideoxy sequencing method *Not covered in class; will NOT be on the exam; check it out if you are interested.* Fig The dideoxy sequencing method *Not covered in class; will NOT be on the exam; check it out if you are interested.* Gel produced by the dideoxy sequencing method *Not covered in class; will NOT be on the exam; check it out if you are interested.* Fig Computerized sequencers use a similar method *Not covered in class; will NOT be on the exam; check it out if you are interested.* now enjoy making Frakencritters. What is the largest scale experiment that is being currently performed? Genetically Modified Organisms Human resource production and use Worldwide Grain Production per Person World Grain Stocks as Days of Consumption ~60 percent of the world grain harvest is consumed as food, ~36 percent as feed, and ~3 percent as fuel. While the use of grain for food and feed grows by roughly 1 percent per year, that used for fuel is growing by over 20 percent per year. Wheat prices Corn prices late Dec 2007 Increasing oil prices drive up potential profit, and cost of food crops Cost of oil----Profitable cost of corn for ethanol $ $4/bushel $ $7/bushel $ $10/bushel Current corn price: ~$8/bushel Whereas previous dramatic rises in world grain prices were weather-induced, this one is policy-induced and can be dealt with by policy adjustments. If the entire U.S. grain harvest were converted into ethanol, it would satisfy scarcely 18 percent of our automotive fuel needs. Land use in USA, 1997 Hunter- Gatherer Agricultural Industrial Artificial Selection (breeding) Modern cows were bred from earlier bovine species. from TeosinteMaize By artificial selection that began ~10,000 years ago. Different foods from one species of plant. Created by artificial selection. Wild member of Brassica oleracea: small side buds Brussels sprouts: extremely large side buds Artificial selection Number of Individuals Size of buds (cm) Select individuals that have the largest side buds and breed them. 2. Of the offspring, select individuals that have the largest side buds and breed them. 3. Of the offspring, select individuals that have the largest side buds and breed them. 4. After several generations, bud size increases dramatically. Hunter- Gatherer Agricultural Industrial increased technology and understanding of molecular biology Genetic Engineering: Direct manipulation of DNA Fig 18.1 Millions of Hectares Texas = 70 ha Global area planted with GM crops Why Change a Plants DNA? Can change plant so that it has new or different characteristics Produce needed protein Insect resistance (Bt toxin) Herbicide resistance (Round-up) Drought or other stress resistance Tbl 19.5 % of Total US Acres Common GM Crops in the U.S.ce/2007/09/mon santo-is- hap.html Herbicide Tolerance; Insect Resistance; Viral Resistance Types of Genetically Modified Crops % of Total US Acres Common GM Crops in the U.S.ce/2007/09/mon santo-is- hap.html Millions of Hectares Texas = 70 ha Is changing an organsisms DNA a good idea? Is Changing an Organisms DNA a Good or Bad Idea? Genes flow within species and between species. Are GM foods safe for the environment? Are GM foods profitable for: Consumers, Farmers, and Companies? AAL Pollution in Gulf of Mexico from run-off in the Mississippi river Pollen is easily transferred from plant to plant, and to related species. GM crops illegal GM crops found in Oaxaca and other MX states Genetic information moves. GM crops illegal GM crops found in Oaxaca and other MX states Genetic information moves, and is self-replicating. Many Questions, but Few Answers? Are herbicide or insect resistant plants safe to eat? Are GM foods safe for the environment? Are GM foods profitable for: Consumers, Farmers, and Companies? Herbicide Tolerance; Insect Resistance; Viral Resistance Types of Genetically Modified Crops Patents give 17 year monopoly for inventor Examples of current patents: Atryn- antithrombin produced in transgenic goats (in milk) has anti-clotting properties Examples of current patents: Atryn- antithrombin produced in transgenic goats (in milk) has anti-clotting properties Evolutec has patents on proteins in tick saliva for use as anti-inflammatory Examples of current patents: Atryn- antithrombin produced in transgenic goats (in milk) has anti-clotting properties Evolutec has patents on proteins in tick saliva for use as anti-inflammator GTG in Australia has patents on non-coding human DNA for detecting risk of various diseases Examples of current patents: Atryn- antithrombin produced in transgenic goats (in milk) has anti-clotting properties Evolutec has patents on proteins in tick saliva for use as anti-inflammatory GTG in Australia has patents on non-coding human DNA for detecting risk af various diseases Harvard patented cancer susceptible mouse Examples of current patents: Atryn- antithrombin produced in transgenic goats (in milk) has anti-clotting properties Evolutec has patents on proteins in tick saliva for use as anti-inflammatory GTG in Australia has patents on non-coding human DNA for detecting risk af various diseases Harvard patented cancer susceptible mouse Over 70 U.S. patents for use of Indian neem tree Neem, say the women, helps babies sleep, keeps flies away, is a cosmetic, a disinfectant and a pesticide. Its leaves make good cattle fodder, its twigs are good for teeth and gums. It is used, they say, for snake bites, malaria, hysteria, high blood pressure, pain relief, skin diseases and a host of other ailments. Over 70 U.S. patents for use of Indian neem tree Neem, say the women, helps babies sleep, keeps flies away, is a cosmetic, a disinfectant and a pesticide. Its leaves make good cattle fodder, its twigs are good for teeth and gums. It is used, they say, for snake bites, malaria, hysteria, high blood pressure, pain relief, skin diseases and a host of other ailments. Over 70 U.S. patents for use of Indian neem tree One was rejected due to previous use in indigenous Indian culture Concerns of developing nations vs. industrialized countries Bioprospecting Lack of research on third world problems. (tuberculosis, malaria, cholera, dysentery Not profitable. Where does the information about genetically modified foods come from? Millions of Hectares Texas = 70 ha Global area planted with GM crops Today: Biotechnology Exam #2 W 7/9 in class