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DNA TECHNOLOGY. Genetic engineering and all that stuff. Glowing mice. Goals of Recombinant DNA Technology. Eliminate undesirable phenotypic traits in humans, animals, plants, and microbes Combine beneficial traits of two or more organisms to create valuable new organisms
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DNA TECHNOLOGY Genetic engineering and all that stuff
Goals of Recombinant DNA Technology • Eliminate undesirable phenotypic traits in humans, animals, plants, and microbes • Combine beneficial traits of two or more organisms to create valuable new organisms • Create organisms that synthesize products humans need
Overview of recombinant DNA technology Figure 8.1.1
Overview of recombinant DNA technology Figure 8.1.2
Plasmid Vectors Example plasmid (pBR322)
Tools and Techniques • Restriction Enzymes Cut DNA at precise locations based upon a specific target sequence of nucleotides • DNA Ligase Enzyme that “seals” or ligates DNA pieces together
Restriction Endonucleases • Eco RI --GAATTC-- --G AATTC-- --CTTAAG-- --CTTAA G-- • SmaI --CCCGGG-- --CCC GGG-- --GGGCCC-- --GGG CCC-- Recognition sequenceResults of cutting
Using a restriction enzyme and DNA ligase to make recombinant DNA
Cloning Steps • Isolate gene of interest (cut out of genome with a Restriction Enzyme) • Cut Vector with same R.E. to get compatible ends • Mix 2 DNAs together and Ligate(DNA Ligase) • Transform • Screen for successful event • Celebrate
Inserting DNA into Cells • Goal of DNA technology is insertion of DNA into cell (transformation) • Can use vectors and natural methods of transformation, transduction, and conjugation • Artificial methods • Electroporation • Protoplast fusion • Injection – gene gun and microinjection
Tools and Techniques • Gel Electrophoresis
Gel Electrophoresis Figure 8.7a
Using restriction fragment patterns to distinguish DNA from different alleles RFLPs restriction fragment length polymorphisms
DNA Fingerprinting M = mother F = Father C = children both children show some bands with each parent Figure 8.13
Tools and Techniques • Reverse transcriptase
Tools and Techniques • DNA synthesis • DNA sequencing
Tools and Techniques • PCR (Polymerase Chain Reaction) • Large number of identical molecules of DNA are produced in vitro • Allows any piece of DNA to be rapidly amplified ! • Only minute amounts of DNA are needed !
PCR procedure • Heat to ~ 95 degrees to denature the DNA strands • Cool and elongate with heat tolerant DNA Polymerase (e.g. Taq polymerase from Thermus aquaticus) • Repeat cycle … Repeat cycle …Repeat cycle … Repeat cycle … Repeat cycle …
Animation: Polymerase Chain Reaction PLAY
Applications • Diagnosis of Disease • Gene Therapy • Vaccines and other Pharmaceuticals • Forensic use • Environmental use (bioremediation) • Agricultural use * Transgenic animals * Transgenic plants
PHARM animals Transgenic Sheep Carrying a human gene for a human protein which they secrete in their milk. The protein is then purified from their milk
“Golden” rice contrasted with ordinary rice Transgenic Golden Rice (High levels of Beta-carotene –used to make Vitamin A – give the rice a golden color.
Using the Ti plasmid as a vector for genetic engineering in plants TRANSGENIC PLANT
Safety and Ethics GMO (genetically modified organism) food controversy
BT Corn basics • BT protein is produced by the bacterium Bacillus thuringiensis. Toxic to some insects, nontoxic to all other life forms. • BT corn has the gene inserted so that the corn plant makes the BT protein. • Ingestion of BT protein by the larvae of the European Corn Borer kills the larvae • The corn plant now has it’s own defense
Pluses and minuses • Reduced need for pesticides • Greater Yield, etc. etc. • Potential impact on other species None of any consequence noted…. …but still the GREAT UNKNOWN.
The Biotech Floodgate 2008 Stats 90% of all soybeans 75% of all corn 65% of all foods on grocery store shelves (soy and corn syrup are in all sorts of stuff!)
What’s Next • Bananas/potatoes that produce human vaccines • Fish that mature more quickly • Plants that produce plastics • Fruit/nut trees that yield years earlier • Crops that grow where they could not before • Human milk from cows • Etc. etc. etc. etc. etc. Two featherless chickens peck around in some grass at the Hebrew University in Rehovot. Israeli scientists at the Agriculture department of the university have genetically engineered bare-skinned chickens as part of a research project to develop succulent, low fat poultry that is environmentally friendly. (AP)