1. Biotechnology 1

2. Recombinant DNA Discovery of restriction enzymes revolutionized molecular biology. Restriction enzymes cut DNA at specific sites –Used by bacteria against viruses –Allow a form of gene mapping that was previously impossible –Allow the creation of recombinant DNA molecules (from two different sources) 2

3. 3 Types of Restriction Enzymes Type I and III cleave with less precision and are not used in manipulating DNA Type II –Recognize specific DNA sequences –Cleave at specific site within sequence –Can lead to “sticky ends” that can be joined Blunt ends can also be joined 3

4. 4 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. C CG GT A T A A T A T C CG GT A T A A T A T C G AATT C G AATT C G TTAA C G TTAA C G AATT C G AA T T C G AATT Sticky ends Restriction sitesEcoRI Sticky ends DNA duplex Restriction endonuclease cleaves the DNA Restriction endonuclease cleaves the DNA DNA from another source cut with the same restriction endonuclease is added. DNA ligase joins the strands. Recombinant DNA molecule

5. 5 DNA ligase –Joins the two fragments forming a stable DNA molecule –Catalyzes formation of a phosphodiester bond between adjacent pieces of DNA –Same enzyme joins Okazaki fragments on lagging strand in replication

6. Gel Electrophoresis Separate DNA fragments by size Gel made of agarose or polyacrylamide Submersed in a solution that can carry electric current Negatively-charged DNA (due to phosphates), migrates towards the positive pole Larger fragments move slower, smaller move faster DNA is visualized using fluorescent dyes 6

7. 7 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Long segment Medium segment Gel Lane Anode + Cathode – Long segment Reaction 2 Reaction 1 Reaction 3 a.b. Restriction Enzyme Digestion DNA samples are cut with restriction enzymes in three different reactions producing different patterns off ragments. Restriction endonuclease 1 cut site Short segment Restriction endonuclease 2 cut site Restriction endonuclease 3 Short segment Mixture of DNA fragments of different sizes in solution placed at the top of “lanes” in the gel Reaction 3 Reaction 2 Reaction 1 Power source Buffer Gel Electrophoresis Samples from the restriction enzyme digests are introduced into the gel. Electric current is applied causing fragments to migrate through the gel.

8. 8 Visualizing Stained Gel c.d. Gel is stained with a dye to allow the fragments to be visualized. Electrophoresis in the Laboratory Longer fragments Shorter fragments d: Courtesy of Biorad Laboratories Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

9. Transformation Introduction of foreign DNA from an outside source into a cell Natural process in many species Transgenic organisms are all or part transformed cells 9

10. Molecular Cloning Clone – genetically identical copy Molecular cloning – isolation of a specific DNA sequence (usually protein-encoding exon) –Sometimes called gene cloning The most flexible and common host for cloning is E. coli –Vector – carries DNA in host and can replicate in the host –Each host–vector system has specific uses 10

11. Types of Vectors Plasmids –Small, circular chromosomes –Used for cloning small pieces of DNA 11 lacZ gene A Plasmid Vector Foreign DNA and DNA ligase are added Restriction enzymes cuts within the laxZ gene Ampicillin resistance gene Restriction endonuclease Foreign DNA inserted No DNA inserted Transform Medium contains ampicillin and X-gal Active lacZ gene produces blue colonies Inactive lacZ gene produces white colonies

12. Artificial chromosomes –Useful because plasmids have limited insert size –Yeast artificial chromosomes (YACs) –Bacterial artificial chromosomes (BACs) –Allow for larger insert for large-scale analysis of genomes 12 Types of Vectors

13. Plant genetic engineering 13 Agrobacterium Plasmid Plant nucleus 1. Plasmid is removed and cut open with restriction endonuclease. 2. A gene of interest is isolated from the DNA of another organism and inserted into the plasmid. The plasmid is put back into the Agrobacterium. 3. When used to infect plant cells, Agrobacterium duplicates part of the plasmid and transfers the new gene into a chromosome of the plant cell. 4. The plant cell divides, and each daughter cell receives the new gene. These cultured cells can be used to grow a new plant with the introduced gene. Gene of interest

14. Allows the reproduction of a small DNA fragment Each PCR cycle involves three steps: 1. Denaturation (high temperature) 2. Annealing of primers (low temperature) 3. DNA synthesis (intermediate temperature) 14 Polymerase chain reaction (PCR)

15. 15 Primers anneal to DNA 5´ 3´ 5´ 3´ 5´ 1. Sample is first heated to denature DNA. 2. DNA is cooled to a lower temperature to allow annealing of primers. DNA segment to be amplified 5´ 3´ 5´ 3´ DNA is denatured into single strands PCR machine

16. 16 After 20 cycles, a single fragment produces over one million (2 20 ) copies! 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3. DNA is heated to 72°C, the optimal temperature for Taq DNA polymerase to extend primers Cycle 3: 8 copies Cycle 2: 4 copies Taq DNA polymerase Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

17. Applications of PCR –Allows the investigation of minute samples of DNA –Forensics – drop of blood, cells at base of a hair –Detection of genetic defects in embryos by analyzing a single cell –Analysis of mitochondrial DNA from early human species 17

18. RFLP analysis –Restriction fragment length polymorphisms –Generated by point mutations or sequence duplications –Restriction enzyme fragments are often not identical in different individuals 18 DNA Fingerprinting

19. 19 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Sequence duplication + + + + + – Original Sequence of Restriction Sites (no mutations) Point Mutations Change the Sequence of Restriction Sites Sequence Repetitions Can Occur Between Restriction Sites restriction endonuclease cutting sites Larger fragments Smaller fragments Single base-pair change a. Three different DNA duplexes b. Cut DNAc. Gel electrophoresis of restriction fragments – –

20. DNA fingerprinting –Identification technique used to detect differences in the DNA of individuals –Short tandem repeats (STRs) Typically 2–4 nt long Not part of coding or regulatory regions –Population is polymorphic for these markers –Using several probes, probability of identity can be calculated or identity can be ruled out –Also used to identify remains 20

21. 21 STR analysis Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Data provided by Dr. L. Roewer, DNA Laboratory of the Institute for Forensic Medicine of Charité, Berlin 1 234567 bp STRs and DNA fingerprinting Control Ladder DYS19 STR Y chromosome D12S66 STR Chromosome 12 202 absent 148 absent 160 168 178 absent 172 186 190 194 198 156 152 absent

22. Medical Applications of Biotechnology Medically important proteins can be produced in bacteria –Human insulin –Vaccines –Problem has been purification of desired proteins from other bacterial proteins 22

23. 23 Genetically engineering E. coli to make human insulin Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 108 amino acids Translation Exon Transcription PromoterExon In Humans IntronExonIntronExon Cut Achain Bchain Disulfide bond Disulfide bonds form Posttranslational modification Preproinsulin Cut In Bacterial Culture β-gal Bacterial promoter Insulin B chain minus introns and other “extra” sequence B chain Active insulin Disulfide bond COOH NH 2 A chain Purify A and Bchains Purify β-gal-insulin fusion proteins Culture cells Transform into E.coli β-gal Bacterial promoter Amp R Insulin A chain minus introns and other “extra” sequence a.a.b.b. Cut

24. Vaccines –Subunit vaccines Genes encoding a part of the protein coat are spliced into a fragment of the vaccinia (cowpox) genome Injection of harmless recombinant virus leads to immunity –DNA vaccines Depend on the cellular immune response (not antibodies) 24

25. 25 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Herpes simplex virus 1. DNA is extracted. 2. Herpes simplex gene is isolated. Gene specifying herpes simplex surface protein 3. Vaccinia DNA is extracted and cleaved. 4. Fragment containing surface gene combines with cleaved vaccinia DNA. 5. Harmless engineered virus (the vaccine) with surface like herpes simplex is injected into the human body. 6. Antibodies directed against herpes simplex viral coat are made. Harmless vaccinia (cowpox) virus Human immune response

26. Gene therapy –Adding a functional copy of a gene to correct a hereditary disorder –Severe combined immunodeficiency disease (SCID) illustrates both the potential and the problems On the positive side, 15 children treated successfully are still alive On the negative side, three other children treated have developed leukemia (due to therapy) 26

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28. Herbicide resistance –Broadleaf plants have been engineered to be resistant to the herbicide glyphosate –Benefits Crop resistant to glyphosate would not have to be weeded Single herbicide instead of many types Glyphosate breaks down in environment –In the United States, 90% of soy currently grown is GM soy 28 Agricultural Applications

29. Bt crops –Insecticidal proteins have been transferred into crop plants to make them pest-resistant –Bt toxin from Bacillus thuringiensis –Use of Bt maize is the second most common GM crop globally Stacked crops –Both glyphosate-resistant and Bt-producing 29

30. Golden rice –Rice that has been genetically modified to produce  -carotene (provitamin A) –Converted in the body to vitamin A –Interesting for 2 reasons Introduces a new biochemical pathway in tissue of the transgenic plants Could not have been done by conventional breeding as no rice cultivar known produces these enzymes in endosperm 30

31. 31 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Daffodil phytoene synthase gene (psy ) Bacterial carotene desaturase gene (crtI ) Daffodil lycopene β-cyclase gene (lcy ) Genes introduced Into rice genome Rice chromosome Rice chromosome psy lcy crtI Expression In endosperm GGPP Phytoene synthase Phytoene Carotene desaturase Lycopeneβ-Carotene (Provitamin A) β-Cyclase

32. Marker Assisted Breeding (MAB) Combines classic plant breeding with molecular biology DNA extracted from leaf tissue of young seedlings Screen for agriculturally important traits Screening uses DNA fingerprinting Seedlings with desired traits raised to maturity 32

33. Adoption of genetically modified (GM) crops has been resisted in some areas because of questions –Crop safety for human consumption –Movement of genes into wild relatives No evidence so far but it is not impossible 33

34. Transgenic animal technology has not been as successful as that in plants Molecular techniques combined with the ability to clone domestic animals could produce improved animals for economically desirable traits Main use thus far has been engineering animals to produce pharmaceuticals in milk (biopharming) 34

35. Transgenic animals 0 0 Days (from first feeding) Weight (g) 6000 5000 4000 3000 2000 1000 100200300400500600700800900 Transgenic salmon Wild salmon AquAdvantage salmon is a genetically modified (GM) Atlantic salmon developed by AquaBounty Technologies. A growth hormone-regulating gene from a Pacific Chinook salmon and a promoter from an ocean pout were added to the Atlantic's 40,000 genes. These genes enable it to grow year-round instead of only during spring and summer. The purpose of the modifications is to increase the speed at which the fish grows, without affecting its ultimate size or other qualities. The fish grows to market size in 16 to 18 months rather than three years.