1. © life_edu Lecture 6 Some More Techniques in Biotechnology Issues in Biotechnology: The Way We Work With Life Dr. Albert P. Kausch life edu.us The Mechanics of DNA

2. Issues in Biotechnology: Biotechnology, Our Society and Our Future OnCampus Live BCH 190, MIC 190, AFS 190, NRS 190, PLS 190 OnLine BCH 190 A Sweeping General Survey on Life and Biotechnology A Public Access College Course The University of Rhode Island Kimberly Nelson Issues in Biotechnology: The Way We Work With Life Dr. Albert P. Kausch life edu.us

3. © life_edu Section I. The Mechanics of Life and General Biotechnology Section II. The Applications of Biotechnology A Sweeping General Survey on Life and Biotechnology A Public Access College Course The University of Rhode Island BCH 190 life edu.us Kimberly Nelson Issues in Biotechnology: The Way We Work With Life Dr. Albert P. Kausch

4. © life_edu 5. Trends, Patterns and Relationships in Biology 6. Some More Techniques in Biotechnology A Sweeping General Survey on Life and Biotechnology A Public Access College Course The University of Rhode Island Issues in Biotechnology: The Way We Work With Life Dr. Albert P. Kausch life edu.us The Mechanics of DNA

5. © life_edu Lecture 6 Some More Techniques in Biotechnology Issues in Biotechnology: The Way We Work With Life Dr. Albert P. Kausch life edu.us The Mechanics of DNA

6. © life_edu Are there any foods or ingredients that you have avoided or eaten less of? (A) yes (B) no (C) do know (D) refuse to answer

7. © life_edu Half of consumers are avoiding some food/ingredient… Are there any foods or ingredients that you have avoided or eaten less of?

8. © life_edu If yes, what foods or ingredients did you avoid or eat less of? Foods/ingredients avoided (A) sugar / carbohydrates (B) fats / cholesterol (C) animal products (D) salt / spices (E) biotechnology products

9. © life_edu …it’s not biotech! If yes, what foods or ingredients did you avoid or eat less of? (Open-ended; Multiple responses allowed, n = 478) IFIC 2005 Foods/ingredients avoided3/05 Sugar/Carbohydrates58% Fats/Cholesterol37% Animal Products34% Salt/Spices14% Snack Foods11% Biotechnology < ½ %

10. © life_edu FDA requires special labeling when a food is produced under certain conditions: when biotechnology’s use introduces an allergen or when it substantially changes the food’s nutritional content... Otherwise special labeling is not required. Would you say that you support or oppose this policy of FDA? (A) support (B) oppose (C) neither support or oppose (D) don’t know (E) refuse to answer

11. Current FDA labeling policy supported by majority FDA requires special labeling when a food is produced under certain conditions: when biotechnology’s use introduces an allergen or when it substantially changes the food’s nutritional content... Otherwise special labeling is not required. Would you say that you support or oppose this policy of FDA?

12. © life_edu It is now possible to clone any gene from any organism and move it into plants

13. © life_edu Plasmids are circular pieces of DNA found in some bacteria Many copies per cell Antibiotic resistance gene Plasmids can be cut and pasted back together Foreign genes can be inserted

14. Enzymes were discovered that cut DNA at specific sequences And subsequently, enzymes were discovered that paste DNA together The ability to cut and paste DNA allowed gene cloning Boyer, Cohen, and Berg, 1972

15. How is a gene cloned? Foreign DNA (gene) is inserted into a plasmid that has a gene for antibiotic resistance The plasmid is introduced into a bacterial cell and grown on the antibiotic Only bacteria with the plasmid grow…the inserted gene is copied many times

16. © life_edu Promoter Coding Sequence Terminator Cell specificity Developmental specificity Start transcription Protein coding sequence Stop transcription Message stability Gene constructs can be moved into plants and the gene is expressed driven by the promoter sequence Anatomy of a Transgene

17. © life_edu Agricultural Biotechnology Panacea Or Pandora’s Box? Genetically Modified Foods:

18. © life_edu Genetically Modified Organisms GMO How is it done? It is now possible to clone any gene from any organism and move it into plants Agricultural Biotechnology

19. A Success Story: Genetically Modified Salmon Constitutive expression of rainbow trout growth factor hormone All salmon shown are fourteen months old, those at the bottom are the controls

20. Transgenic Atlantic salmon produced by Aqua Bounty Inc. Transgenic Atlantic salmon produced by Aqua Bounty Inc. Growth rate, not ultimate Growth rate, not ultimate size is enhanced. Commercial production of Aqua Bounty salmon is being reviewed by FDA. First transgenic meat product.

21. Would you order genetically modified salmon at a restaurant if you also had a choice of wild salmon? (A) yes (B) no (C) doesn’t matter (D) undecided

22. Tools and Techniques used in Biotechnology

23. Gene transfer from one organism to another is not new Image of two species of bacteria transferring viral phage particles Bacteria transfer genes to other bacteria and plants Now in nature there is another organism capable of Transferring DNA: we call that organism a human being

24. Electro refers to the energy of electricity. Phoresis, from the Greek verb phoros, means “to carry across.” Thus, gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. Gel Electrophoresis: the separation of molecules, DNA, RNA and proteins by charge and size

25. Applications of Gel Electrophoresis DNA Fingerprinting DNA Recombinant Technology Forensics The Human Genome Project

26. DNA carries a net negative charge; it is negatively charged because the phosphates (red circles) that form the sugar-phosphate backbone of a DNA molecule have a negative charge.

27. As the separation process continues, the separation between the larger and smaller fragments increases.

28. Molecular weight markers are often electrophoresed with DNA. Molecular weight markers are usually a mixture of DNAs with known molecular weights. Molecular weight markers are used to estimate the sizes of DNA fragments in a DNA sample.

29. The Techniques of Molecular Biotechnology Technology has created new Fields DNA detection Genomics DNA synthesisBioinformatics DNA sequencingPharmacogenomics DNA cloning Transgenics Expression cassette Computational constructionBiology RNA detection Population Genetics Protein detection Proteomics

30. Techniques of Molecular Biotechnology Molecular Biotechnology Polymerase Chain Reaction Southern Blot Analysis Northern Blot Analysis Western Blot Analysis cDNA Library Construction DNA Sequencing Gene Isolation Gene Vector Construction

31. PCR

32. PCR The Polymerase Chain Reaction

33. PCR You Leave a Piece of You Behind

34. PCR was used on the “BLUE DRESS” and showed President Clinton's association with Monica Lewinsky.

35. PCR is Amplification Nobel laureate in Chemistry, 1994 Polymerase Chain Reaction

36.  THERE ARE MILLIONS OF DIFFERENT GENES OR SEQUENCES WITHIN ANY DNA SAMPLE (BLOOD, TISSUE, PLANT, ETC.).  A SPECIFIC SEQUENCE IS SELECTED TO BE AMPLIFIED (RED ABOVE). THIS SEQUENCE CAN BE ANY GENE OF INTEREST OR A NON-CODING MARKER REGION OF DNA.

37. IN ORDER TO COPY THE SEQUENCE OR GENE, A SHORT SEQUENCE ON EITHER SIDE OF THE SECTION MUST BE KNOWN. THIS REGION (BLUE ABOVE) WILL SERVE AS A PRIMER ATTACHMENT SITE TO COPY THE DNA TARGET SEGMENT.

38. IN ORDER TO AMPLIFY A SPECIFIC FRAGMENT OF DNA, SEVERAL THINGS ARE NEEDED, INCLUDING PRIMERS AND DNA POLYMERASE. AN ENZYME WHICH COPIES DNA, PRIMERS ARE SHORT PIECES OF DNA OR RNA DESIGNED TO PAIR WITH GENOMIC DNA AT A SPECIFIC ATTACHMENT SITE FOR THE MAIN PURPOSE OF HELPING THE DNA POLYMERASE BIND AT THE DESIRED SECTION.

39. NUCLEOSIDE TRIPHOSPHATES, THE BUILDING BLOCKS OF DNA ARE ALSO NEEDED. EACH NUCLEOSIDE TRIPHOSPHATE CONSISTS OF:  A BASE (ADENINE, THYMINE, CYTOSINE OR GUANINE).  A SUGAR AND THREE PHOSPHATES.

40. WITHOUT A SHORT PIECE OF DNA(OR RNA) TO ATTACH TO, DNA POLYMERASE CAN NOT COPY A DNA STRAND.

41. PCR REQUIRES SEVERAL CYCLES OF AMPLIFICATION. EACH CYCLE CONSISTS OF THREE TEMPERATURE CHANGES.  THE STARTING TEMPERATURE (95 C) SEPARATES THE DNA STRANDS.  A LOWERED TEMPERATURE (50-60 C) ALLOWS PRIMERS TO BIND TO COMPLEMENTARY SEQUENCES IN THE DNA.  A SLIGHTLY HIGHER TEMPERATURE (72 C) ALLOWS DNA POLYMERASE TO ATTACH TO THE PRIMERS AND COPY THE DNA STRANDS (EXTENSION).

42. DNA STRANDS ARE SEPARATED BY HEATING @ 94 o C.

43. THE TEMPERATURE IS LOWERED TO 54 o C TO ALLOW PRIMERS TO PAIR WITH COMPLEMENTARY DNA SEQUENCES.

44. MAKING NEW DNA MOLECULES:  DNA POLYMERASE ATTACHES TO THE PRIMERS @ 72 C.  DNA POLYMERASE ADDS NUCLEOSIDE TRIPHOSPHATES TO THE PRIMERS TO COPY THE DNA STRANDS.

45. COPYING IS COMPLETED FOR EACH STRAND.

46. THE PROCESS IS REPEATED IN THE NEXT CYCLE. THE TEMPERATURE IS RAISED AGAIN TO SEPARATE THE DNA STRANDS.

47. THE TEMPERATURE IS LOWERED TO ALLOW PRIMERS TO ANNEAL. DNA POLYMERASE ATTACHES TO THE PRIMERS AND DNA IS COPIED TO MAKE 4 STRANDS OF DNA.

48. The stability of TAQ Polymerase allows for this amplification process through the three temperature changes.

49. THE PROCESS OF COPYING DNA STRANDS IS REPEATED 32-35 TIMES. WITH EACH AMPLIFICATION CYCLE, THE NUMBER OF COPIES OF THE DNA SEQUENCE IS DOUBLED UNTIL MILLIONS OF COPIES HAVE BEEN MADE.

50. Issues in Biotechnology A method used to copy small amounts of DNA many times over was invented by Dr. Kary Mullis in the 1980s and is called PCR. PCR stands for: (A) Protein Chromosomal Replication (B) Polymerase Chain Reaction (C) Pipetman California Reaction (D) Peptide Catalytic Reactors (E) Polysaccharide Catalyst Repair

51. Forensic applications of DNA based technologies Fingerprinting OJ Simpson Identification Paternity Crime Solving World wide data base

52. Forensic Identification: Basic Principles  Each of us is genetically unique  If enough genetic variation is tested, each of us can be uniquely identified  DNA is found in nearly all cells (blood, semen, hair, etc.)  DNA from an evidentiary sample can be matched with DNA from a suspect to implicate or exonerate

53. ...atatatacaacttactaccatata ccgattacgatcgaattataccgcgga cgtagtaatgacgatgaagtaactata tatatatatatatatatatatatatatatatatatat atatatatatatatatatatatatatatatatatat atatatatatatatatatatatatatatatatatat atatatatatatatatatatatatatatatatatat atatatatatatatatatatatatatatatatatat atatatat atactacctaccagggaggagata... “Short Tandem Repeat sequence” STRs Are Used in Identity Testing

54. Laboratory PCR Instrument INPUT: Sample DNA, PCR enzymes, primers, individual nucleotide building blocks (and maybe fluorescent labels) OUTPUT: Specific DNA fragments amplified millions of times for easy visualization With sizes that vary between individuals

55. After PCR, DNA Fragments are Separated on a Gel PCR products for each sample - + TIME 5 45 15 10 40 20 25 30 35 Minutes Fragment Size 100 180 120 110 170 130 140 150 160 DNA fragments move through gel based on their size

56. Analysis of four different sections of the DNA Identity Testing Using PCR Possible conclusions: A. Suspect 1 DNA was at the scene B. Suspect 2 DNA was at the scene C. Suspect 3 DNA was at the scene D. None were at the scene E. Multiple suspects were at the scene F. Data is inconclusive S = size standards V = victim’s DNA 1 = suspect #1 blood 2 = suspect #2 blood 3 = suspect #3 blood E = evidence #1 S = size standards

57. 50 450 150 100 400 200 250 300 350 S V 1 2 3 E S A complete match! S = size standards V = victim’s DNA 1 = suspect #1 blood 2 = suspect #2 blood 3 = suspect #3 blood E = evidence #1 S = size standards

58. PCR was used on the “BLUE DRESS” and showed President Clinton's association with Monica Lewinsky.

59. Issues in Biotechnology DNA tests exclude Martin from gun grip in Zimmerman case September 19, 2012 Another round of evidence was released Wednesday in the second-degree murder case against George Zimmerman, including forensic tests that show Zimmerman’s DNA was the only one that could be identified on the grip of the gun used to shoot 17-year-old Trayvon Martin. Zimmerman’s DNA also was identified on the gun’s holster. The tests were inconclusive as to whether Martin’s DNA was on the gun’s holster.

60. Congratulations!!!!! You have $100,000.00!!!!!!! To Invest in Biotech.

61. Issues in Biotechnology Stock Project The idea is to select five biotechnology companies and invest $100,000 (fictitiously, of course)

62. Issues in Biotechnology Stock Project You can spread your money evenly across five companies (i.e. $20,000 each) or not. For example, if a company is trading at $20/share you can purchase 1,000 shares for $20, 000. Look up companies on Google (e.g.: type Monsanto stock) and record their stock ticker designation (e.g.. MON of the New York Stock Exchange, NYSE). Observe their performance over the past year. Record current trading price per share and calculate how many shares you can buy. You must choose your companies and the number of shares. Submit a single page summary on these results. Toward the end of the course you will look up these same companies and determine the cost per share at that time. Calculate your losses or gains for each company and your total losses and gains. This project will be summarized at the end of the course in a one page summary of losses and gains.

63. Biotechnology Stocks Project Congratulations!!!!! You have $100,000.00!!!!!!! To Invest in Biotech. 1. Select and Research five Biotech companies. 2. Print and submit the current stock quote and for each company. 3. Invest chosen amounts in each. Calculate the number of shares in each (Submit a One page summary). 4. Contact company to receive investors information (Optional). 5. Monitor Stock during the course. 6. Calculate gains and losses. Submit report with final exam.

64. Biotechnology and Industry “In retrospect, recombinant-DNA may rank as the safest revolutionary technology ever developed.” - James D. Watson, Nobel Prize Winner, 1953

65. The Development of Biotechnology is directly linked with Industry The application of the biological sciences has moved from academia to the private sector Driven by profits and the promise of profits The distinction between Basic and Applied Science is blurred Basic science is immediately applied in today’s biotech

66. Biotech Industry Red vs Green companies pharmaceuticals vs agriculture (blood vs chlorophyll) Information Sciences Genomics Proteomics Phenomics Bioinformatics Population Genetics Clinical trials

67. The Role of Companies in the Development of Biotechnology Technology Development Patents Markets and Products Market-driven Innovation

68. Genomics Companies Celera Incyte Genome Therapeutics Corp Millenium Paradigm Genetics DuPont Genaissance Monsanto

69. Technology Companies Invitrogen Stratagene Qiagen Packard PE Biosystems Kairos BioRobotics

70. Biotechnology Industry Advertisement of services in international journals NATURE Science Nature Biotechnology “Picks and shovels for the Gold Rush”

71. Innovative technologies become biotech products Big money in licenses Commercialization of biotech products requires Freedom to Operate (FTO) with all the technologies used And look at the ads!

73. Home Who We Are Our Pledge Our Locations Company Leadership Company History Contact Us Our Products Leading Brands Seeds & Traits Agricultural Productivity Product Pipeline Benefits of Our Products Technical & Safety Info News & Media News Releases RSS & Email Subscriptions Monsanto in the News Key Contacts Calendar of Events Press Kit & Multimedia Executive Forum Monsanto is an agricultural company. We apply innovation and technology to help farmers around the world be successful, produce healthier foods, better animal feeds and more fiber, while also reducing agriculture's impact on our environment. » View Biotechnology VideosView Biotechnology Videos Roundup RReady2Yield™ Soybeans — Another Step Closer to Farmers' Fields Read More Read More 2007 Farm Progress Show — “Road to Success” — Monsanto Technology Showcase Read More Read More Delta & Pine Land — Monsanto Company Begins Combining Delta & Pine Land Business Read More Read More Featured Story $21 Million Data Center Completed High-tech center will provide global IT support for Monsanto's growing data analysis needs Read More Read More View All Featured Stories Recent News Mon, 17 Sep 2007 — Monsanto Increases Ongoing Earnings Per Share Guidance, Provides Free Cash Flow Guidance For Fiscal Year 2007Monsanto Increases Ongoing Earnings Per Share Guidance, Provides Free Cash Flow Guidance For Fiscal Year 2007 Fri, 14 Sep 2007 — Monsanto, Dow Agreement Paves the Way for Industry's First-Ever, Eight- Gene Stacked Offering in CornMonsanto, Dow Agreement Paves the Way for Industry's First-Ever, Eight- Gene Stacked Offering in Corn Thu, 13 Sep 2007 — Monsanto to Provide Information to Iowa Attorney General Concerning Seed, Trait and Chemistry BusinessMonsanto to Provide Information to Iowa Attorney General Concerning Seed, Trait and Chemistry Business View All News Job Opportunities Monsanto Fund Scholarships News Releases Executive Speeches Financial Reports Our Locations Monsanto Gift Shop DEKALB WingWear MSDS & Labels Biotech Resources Biotech Knowledge Center Our Values, described in the Monsanto Pledge, guide our business decisions.Monsanto Pledge Copyright © 2004-07 Monsanto Company Contact UsContact Us | Sitemap | Legal Notice | Privacy PolicySitemapLegal NoticePrivacy Policy BioBelt Corporate Responsibility Our Pledge Business Conduct Corporate Governance Stewardship Corporate Giving Youth and Education Diversity Investors Corporate Profile Stock Performance Presentations & Financial Reports Calendar of Events Contact & Shareholder Info Careers Getting Started U.S. Job Opportunities International Job Opportunities Benefits & Rewards Career Development Diversity

74. » View Biotechnology VideosView Biotechnology Videos Roundup RReady2Yield™ Soybeans — Another Step Closer to Farmers’ Fields Read More Read More 2007 Farm Progress Show — “Road to Success” — Monsanto Technology Showcase Read More Read More Delta & Pine Land — Monsanto Company Begins Combining Delta & Pine Land Business Read More Read More Featured Story $21 Million Data Center Completed High-tech center will provide global IT support for Monsanto’s growing data analysis needs Read More Read More View All Featured Stories Recent News Mon, 17 Sep 2007 — Monsanto Increases Ongoing Earnings Per Share Guidance, Provides Free Cash Flow Guidance For Fiscal Year 2007Monsanto Increases Ongoing Earnings Per Share Guidance, Provides Free Cash Flow Guidance For Fiscal Year 2007 Fri, 14 Sep 2007 — Monsanto, Dow Agreement Paves the Way for Industry’s First-Ever, Eight-Gene Stacked Offering in CornMonsanto, Dow Agreement Paves the Way for Industry’s First-Ever, Eight-Gene Stacked Offering in Corn Thu, 13 Sep 2007 — Monsanto to Provide Information to Iowa Attorney General Concerning Seed, Trait and Chemistry BusinessMonsanto to Provide Information to Iowa Attorney General Concerning Seed, Trait and Chemistry Business

75. Stock Price Monsanto (MON) 89.79-0.66 Dow Jones (DJIA) 13,451.41 -6.14Stock Chart | Annual ReportStock Chart Annual Report Stock Price Stock Chart Stock Chart | Annual ReportAnnual Report Agricultural Biotechnology

76. Stock Price Stock Chart Stock Chart | Annual ReportAnnual Report Stock Quote at: Sep 26 2012 12:22PM Change0.09Vol12,802,922 Day High24.9952 Wk. High25.15 Day Low24.7952 Wk. Low17.05 Open24.91Mkt. Cap(Bil)186.21 Prev Close24.84 Stock Symbol: PFEStock Exchange: NYSE NYSE: PFE 24.93 0.09 Pharmaceutical Biotechnology

77. Biotechnology Stock Project Example Report 1 Henrietta Lack Student’s Name PLS 190 Issues in Biotechnology Professor Albert Kausch Due Date 10-03-2012 Biotechnology Company 1. Intel Corporation (INTC) $22.05 per share / 900 shares = $19,845 2. Amgen (AGN)$55.66 per share / 350 shares = $19,481 3. Pfizer (PFE) $14.78 per share / 1,500 shares = $22,170 4. Apple Inc. (AAPL) $413.81 per share / $18,621 5. General Electric (GE) $15.38 per share / 1,292 shares = $19,870.96 Total = $99,987.96

78. Biotechnology Stock Project Example Report 1 Student’s Name Jack Straw BCH 190 Issues in Biotechnology Professor Albert Kausch Date Due October 3, 2012 Biotechnology Company Amgem Inc. AMGN: 56.04 per share 356 shares for $20,000 Pfizer Inc PFE: 17.84 per shares 1121 shares for $20,000 Monsanto Co MON: 65.86 per share 303 shares for $20,000 Bio Rad Laboratories Inc BIO: 89.50 per share 223 shares for $20,000 Syngenta AG SVJ: 205.30 Euros per share 71 shares for 14567.49 Euro or $20,000 Total; $100,000

79. Biotechnology Companies: AstraZeneca Active Motif Aventis Adventa Celera PE Incyte Invitrogen Pfizer Merck Smith Kline Beecham Novartis Monsanto Qiagen Roche Paradigm Genetics Chemicon International Trevigen Avigen Metabasis Therapeutics Pintex Pharmaceuticals BioGen Garst Pioneer DuPont Pharmacia Bristol-Myers Squibb Eli Lilly Cistem Molecular Corp. Operon Technologies

80. Biotechnology Companies: DNX Transgenic Sciences Chemdex MWGAG BIOTECH Double Twist Larova Biochemie Greiner Labortechnik Sungene Molecular Devices Corp. Evotec BioSystems AG BIACORE TIB MOLBIOL PE Biosystems Bruker Daltonics Inc. Eppendorf Scientific Curagen Cargill Dow Agri Sciences Dow Chemical Co. AmGen Bayer Dynal Noxxon Pharma Schering Boehringer Rhein Biotech Hyseq Genome Therapeutics

81. O sweet spontaneous earth how often have the doting fingers of purient philosophers pinched and poked thee, has the naughty thumb of science prodded thy beauty how often have religions taken thee upon their scraggy knees squeezing and buffeting thee that thou mightest conceive gods (but true to the incomparable couch of death thy rhythmic lover thou answerest them only with spring) e.e. cummings

82. For those who are interested in taking this course for college credit through the University of Rhode Island; For those who are interested in taking this course for college credit through the University of Rhode Island; For more information please contact: Issuesinbiotechnology190@gmail.com

83. Credits Credits Lectures by: Edited by: Video Produced by: Thank You to The University of Rhode Island Thank You to The University of Rhode Island and all of the students of Issues in and all of the students of Issues in Biotechnology over the years Biotechnology over the years Dr. Albert Kausch and Kimberly Nelson Thaddeus Weaver