2. Biotechnology 2007-2008

3. Recombinant DNA DNA produced by combining DNA from different sources

4. Applications of Recombinant DNA Technology found in industry, food production, human and veterinary medicine, agriculture, and bioengineering. EXAMPLES: Recombinant chymosin, is an enzyme required to manufacture cheese. Today about 60% of U.S. hard cheese is made with genetically engineered chymosin. Recombinant human insulin almost completely replaced insulin obtained from animal sources (e.g. pigs and cattle) for the treatment of insulin- dependent diabetes. Herbicide-resistant crops commercial varieties of important agricultural crops (including soy, maize/corn, sorghum, canola, alfalfa and cotton) have been developed that incorporate a recombinant gene that results in resistance to the herbicide Roundup

5. Restriction enzymes discovered in 1960s evolved in bacteria to cut up foreign DNA Action of enzyme cut DNA at specific sequences restriction site produces protruding ends sticky ends will bind to any complementary DNA “restrict” the action of the attacking organism 

6. Sticky ends help glue genes together TTGTAACGAATTCTACGAATGGTTACATCGCCGAATTCACGCTT AACATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGTGCGAA gene you wantcut sites AATGGTTACTTGTAACG AATTCTACGATCGCCGATTCAACGCTT TTACCAATGAACATTGCTTAA GATGCTAGCGGCTAAGTTGCGAA chromosome want to add gene tocut sites AATTCTACGAATGGTTACATCGCCG GATGCTTACCAATGTAGCGGCTTAA isolated gene sticky ends chromosome with new gene added TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATC CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC sticky ends stick together DNA ligase joins the strands Recombinant DNA molecule

7. Why mix genes together? Gene produces protein in different organism or different individual TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATC CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC aa “new” protein from organism ex: human insulin from bacteria human insulin gene in bacteria bacteriahuman insulin How can bacteria read human DNA?

8. Plasmids Accessory ring of DNA found in bacteria Can replicate on quickly and easily in the bacterial cell Used as a vector (mechanism that transports the gene of interest) in genetic engineering

9. Grow bacteria…make more grow bacteria harvest (purify) protein transformed bacteria plasmid gene from other organism + recombinant plasmid vector

10. insert recombinant plasmid into bacteria grow recombinant bacteria in agar cultures bacteria make lots of copies of plasmid (accessory DNA in bacteria) “cloning” the plasmid production of many copies of inserted gene production of “new” protein transformed phenotype DNA  RNA  protein  trait Transformation

11. Genetically Modified Organisms (GMO) The universal nature of the genetic code makes it possible to construct organisms that are transgenic, containing genes from other species. GM Crops Larger crops Resistant to herbicides and insecticides More nutritious foods 92% of soy beans 86% of cotton 80% of corn GM Animals Cows – more milk production Salmon – growth hormones = larger Pigs – produce more lean meat

13. Desirable Traits Increased yields, more nutritious, quality, etc., More resistant to pestilence, weeds, water and nutrient deprivations, Ability to withstand marginal growth conditions, and thrive in new environmental ranges, Profit!!!!!!!!!!!

14. Wild tomato Genome Era Traditional Breeding Cultivar w/ 1 wild gene replacement

15. Transgenic Plants based on DNA technology, single genes/traits can be transferred, species boundaries are not limiting.

16. How are GMOs generated? insert into plant …via biolistics - or - Agrobacterium tumefaceins...uses tools of molecular genetics, - i.e. applied bacteria and virus genetics.

17. Agrobacterium Plant Cells Nature Ti-PlasmidTransfer-DNA Hormone genes Opines genes Lab Selectable Markers, etc Any Gene Out: Ti genes, opine genes, In: DNA of choice. T-DNA Ti: tumor inducing Plasmid: extrachromosomal DNA evolved for genetic transfer.

18. Construct T-DNA infect plant, select for plants with T-DNA T-DNA (Transfer DNA) transform, select for agro with T-DNA Agrobacterium Plant chromosome with T-DNA insert. …with gene of interest,  carotene, - herbicide resistance, etc..

19. Many uses of restriction enzymes… Now that we can cut DNA with restriction enzymes… we can cut up DNA from different people… or different organisms… and compare it Genetic Fingerprinting forensics medical diagnostics paternity evolutionary relationships and more…

20. Comparing cut up DNA How do we compare DNA fragments? separate fragments by size How do we separate DNA fragments? run it through a gelatin agarose made from algae gel electrophoresis

21. Gel electrophoresis A method of separating DNA in a gelatin-like material using an electrical field DNA is negatively charged when it’s in an electrical field it moves toward the positive side + – DNA         “swimming through Jello”

22. DNA moves in an electrical field… so how does that help you compare DNA fragments? size of DNA fragment affects how far it travels small pieces travel farther large pieces travel slower & lag behind Gel electrophoresis + – DNA        “swimming through Jello”

23. Gel Electrophoresis longer fragments shorter fragments power source completed gel gel DNA & restriction enzyme wells - +

24. Running a gel 12 cut DNA with restriction enzymes fragments of DNA separate out based on size 3 Stain DNA ethidium bromide binds to DNA fluoresces under UV light

25. How is DNA fingerprinting done? 1 Make the agarose gel 2Extract DNA and Cut DNA with a restriction enzyme to cut DNA into fragments * 3Insert DNA into wells of gel 4Run a gel by electrophoresis to separate the pieces of DNA fragments by length 5Stain the Gel 6Interpret the banding patterns for each person * Explain why restriction enzymes cut different sized pieces of DNA for different individuals.

26. Uses: Medical diagnostic Comparing normal allele to disease allele chromosome with disease-causing allele 2 chromosome with normal allele 1 – + allele 1 allele 2 DNA  Example: test for Huntington’s disease

27. Uses: Forensics Comparing DNA sample from crime scene with suspects & victim – + S1 DNA  S2S3V suspects crime scene sample

28. Differences at the DNA level Why is each person’s DNA pattern different? sections of “junk” DNA doesn’t code for proteins made up of repeated patterns CAT, GCC, and others each person may have different number of repeats many sites on our 23 chromosomes with different repeat patterns GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTT CGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA GCTTGTAACGGCATCATCATCATCATCATCCGGCCTACGC TT CGAACATTGCCGTAGTAGTAGTAGTAGTAGGCCGGATGC GAA

29. Allele 1 GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTT CGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA repeats DNA patterns for DNA fingerprints cut sites GCTTGTAACG GCCTCATCATCATCGCCG GCCTACGCTT CGAACATTGCCG GAGTAGTAGTAGCGGCCG GATGCGAA 123 DNA  –+ allele 1 Cut the DNA

30. DNA fingerprints Comparing blood samples on defendant’s clothing to determine if it belongs to victim DNA fingerprinting comparing DNA banding pattern between different individuals ~unique patterns

31. Allele 1 GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTT CGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA Differences between people cut sites DNA  –+ allele 1 Allele 2: more repeats GCTTGTAACGGCCTCATCATCATCATCATCATCCGGCCTACCGAACATTG CCGGAGTAGTAGTAGTAGTAGTAGGCCGG DNA fingerprint allele 2 123

32. RFLPs Restriction Fragment Length Polymorphism differences in DNA between individuals  change in DNA sequence affects restriction enzyme “cut” site  creates different fragment sizes & different band pattern Alec Jeffries 1984

33. RFLP / electrophoresis use in forensics 1st case successfully using DNA evidence 1987 rape case convicting Tommie Lee Andrews “standard” semen sample from rapist blood sample from suspect How can you compare DNA from blood & from semen? RBC?

34. Electrophoresis use in forensics Evidence from murder trial Do you think suspect is guilty? “standard” blood sample 3 from crime scene “standard” blood sample 1 from crime scene blood sample 2 from crime scene blood sample from victim 2 blood sample from victim 1 blood sample from suspect OJ Simpson N Brown R Goldman

35. Uses: Paternity Who’s the father? + DNA  childMomF1F2 –

36. Uses: Evolutionary relationships Comparing DNA samples from different organisms to measure evolutionary relationships – + DNA  13245 12345 turtlesnakeratsquirrelfruitfly

37. 1.Get out notes sheet from yesterday 2. Read DNA Goes to the Races article 3. Make an outline of the steps of gel electrophoresis on your notes sheet