4.4 Biotechnological Tools and Techniques

1. 4.4 Biotechnological Tools and Techniques Recombinant DNA & Gel electrophoresis

2. Recombinant DNA • Cutting DNA fragments from different sources and recombining them together • Cutting DNA fragments from different sources and recombining them together • 4.4.7: State that, when genes are transferred between species, the amino acid sequence of polypeptides translated from them is unchanged because the genetic code is universal. [Obj. 1] • Purpose • To investigate genetic disorders • Production of drugs (ie. insulin)

3. What complications do you foresee? • Consider: • The size of DNA • Where to cut? • How to put back together? • 4.4.8: Outline a basic technique used for gene transfer involving plasmids a host cell (bacterium, yeast or other cell), restriction enzymes (endonucleases) and DNA ligase. [Obj. 2]

4. 1. Restriction Endonucleases • Also known as restriction enzymes • Essentially are molecular scissors • Recognize a specific DNA sequence and cuts the strands at a particular position or “recognition site” • Isolated and purified only from bacteria • Name reflects which bacteria the enzyme originates • ie. EcoRI  Escherichia coli, strain R, 1st r.e. isolated HindII  Haemophilus influenzae, strain Rd, 2nd r.e.

5. 1. Restriction Endonucleases: Recognition site • Each restriction endonuclease recognizes its own specific recognition site (specific DNA sequence) • Usually 4-8 base pairs long, characterized by a complementary palindromic sequence

6. 1. Restriction Endonucleases: Function • Scans DNA and binds to its specific recognition sequence • Disrupts the phosphodiester bonds between particular nucleotides through a hydrolysis reaction • Hydrogen bonds of the complementary base pairs in between the cuts are disrupted • Result: 2 DNA fragments http://www.scq.ubc.ca/?p=249

7. 1. Restriction Endonucleases: DNA Fragment Ends • Different DNA fragment ends are produced after digestion by different restriction enzymes • Sticky ends: DNA fragment ends with short single-stranded overhangs (ie. EcoRI, HindIII) • Blunt ends: DNA fragment ends are fully base paired (ie. AluI)

8. http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf 1. Restriction Endonucleases: DNA Fragment Ends (continued) Restriction site Animation Palindrome Fragment 2 Fragment 1 http://www.bio-rad.com/LifeScience/docs/Official_Crime_Scene_PowerPoint_Spring_2005_rev_B.ppt

9. How do we control the snips? • Consider: • What about the organisms own DNA? • Frequency of recognition sequences within the DNA sequence

10. http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/bio38.swf::Early Genetic Engineering Experiment 1. Restriction Endonucleases: Length of recognition sites • Longer recognition sites result in lower frequency of cuts • EcoRI  5’-GAATTC-3’ = ¼×¼×¼×¼×¼×¼ = 1/4096 • AluI  5’-AGCT-3’ = ¼ ×¼ ×¼ ×¼ = 1/256 • Higher frequency of cuts – may cut gene into several fragments • Lower frequency of cuts – may produce large fragments than desired

11. 1. Restriction Endonucleases: Methylases • Enzymes that add a methyl group to a nucleotide in a recognition site to prevent restriction endonuclease from cutting DNA • Distinguishing between foreign (viral) DNA and bacteria’s own DNA

12. 1. Restriction Endonucleases: DNA Ligase • Enzyme that rejoins cut strands of DNA together by reforming a phosphodiester bond • DNA ligase joins sticky ends • T4 DNA ligase (from T4 bacteriophage) joins blunt ends

13. How do we sort out the DNA • DNA is chopped into many pieces • How to differentiate one piece from other

14. 2. Gel Electrophoresis • Technique used to separate charged molecules based on their size • 4.4.3: State that gel electrophoresis of DNA is used in DNA profiling [Obj. 1] • Acts like a molecular sieve http://www.biotech.iastate.edu/ppt_presentations/html/Fingerprinting/StudentInstruction-gel/images/image08.jpg http://www.solve.csiro.au/1105/img/sieve-bloke.jpg

15. DNA Profiling • 4.4.4: Describe the application of DNA profiling to determine paternity and also in forensic investigations. [Obj. 2] • A process of using DNA fragments to identify a person, or other organism. The DNA fragments have distinct bands separated by spaces and these band patterns are so distinct that they can be used like fingerprints.

16. 2. Gel Electrophoresis: DNA Preparation • Restriction enzymes digest DNA into smaller fragments of different lengths • Different DNA samples are loaded into wells of the gel (agarose or polyacrylamide) http://www.oceanexplorer.noaa.gov/explorations/03bio/background/molecular/media/gel_plate_600.jpg

17. 2. Gel Electrophoresis: Attraction Migration • Negatively charged electrode at the end where wells are located • Positively charged electrode at opposite end • Negatively charged DNA migrate towards positive end due to attraction

18. 2. Gel Electrophoresis: Rate of Migration • 4.4.2: State that, in gel electrophoresis, fragments of DNA move in an electric field and are separated according to their size. [Obj. 1] • Shorter/smaller DNA fragments migrate through gel faster since they can move through the pores in the gel more easily • Longer/larger DNA fragments migrate through gel slower • Rate of migration = 1/log(size) • Different DNA fragment lengths are separated A B C D E A = kilobase DNA ladder B = uncut plasmid DNA C = single digestion of the plasmid with EcoRI D = single digestionwith XhoI E = double digestion - both EcoRI and XhoI. http://www.answers.com/topic/agarosegel-jpg

19. 2. Gel Electrophoresis: Visualizing DNA Fragments • Ethidium bromide is a fluorescent dye that makes DNA fragments visible by staining the gel • DNA fragments can then be isolated and purified http://www.answers.com/topic/agarosegel-jpg

21. Paternity Testing

22. 4.4.5: Analyse DNA profiles to draw conclusions about paternity or forensic investigations. [Obj. 3]

23. Animations • http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html • http://learn.genetics.utah.edu/content/labs/gel/ • http://www.dnalc.org/resources/animations/gelelectrophoresis.html

24. 2. Gel Electrophoresis: Proteins too! • Gel electrophoresis can also be used to separate proteins, usually using polyacrylamide gels http://www.biotechlearn.org.nz/var/biotech/storage/images/multimedia/images/protein_electrophoresis/48251-4-eng-GB/protein_electrophoresis_medium.jpg http://www.bio-link.org/vlab/Graphics/Tools/ProteinGel2.jpg

25. 3. Plasmids • Small, circular double-stranded DNA that can enter and exit bacterial cells • Lack a protein coat • Independent of bacterial chromosome • 1000-200,000 base pairs

26. 3. Plasmids: Endosymbiosis • Use host bacterial enzymes and ribosomes to replicate and express plasmid DNA • Carry genes that express proteins to protect bacteria against antibiotics and heavy metals

27. 3. Plasmids • Foreign genes (ie. insulin) can be inserted into plasmids, so bacteria can express gene and make its respective protein • Higher copy number of plasmids (number of individual plasmids) in bacteria • results in larger number of gene copies, thus more of its respective protein is synthesized

28. 3. Plasmids • Restriction endonucleases splice foreign genes into plasmids • DNA ligase reforms phosphodiester bond between the fragments, resulting in recombinant DNA http://www.learner.org/courses/biology/archive/animations/hires/a_gmo1_h.html http://www.accessexcellence.org/RC/VL/GG/inserting.html

29. 4. Transformation • Introduction of foreign DNA (usually a plasmid) into a bacterium • Plasmids can be used as a vector (vehicle that DNA can be introduced to host cells) to carry a specific gene into a host cell http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Siegenthaler/fig2.gif

30. 4. Transformation: Competence • Competent cell - Bacterium that readily takes up foreign DNA (ie. able to undergo transformation) • Most cells are not naturally competent, but can be chemically induced to become competent • Calcium ion in calcium chloride stabilizes negatively charged phosphates on bacterial membrane

31. 4. Transformation: Competence

32. 4.4.9: State two examples of the current uses of genetically modified crops or animals. [Obj. 1] • The transfer of a gene for factor IX which is a blood clotting factor, from humans to sheep so that this factor is produced in the sheep’s milk. • The transfer of a gene that gives resistance to the herbicide glyphosate from bacterium to crops so that the crop plants can be sprayed with the herbicide and not be affected by it. 

33. Issues Surrounding Genetically Modified (GM) Products by Subhuti Dharmananda, Ph.D., Director, Institute for Traditional Medicine, Portland, Oregon

34. Meet the Super Cow • http://www.youtube.com/watch?v=Nmkj5gq1cQU

35. 4.4.10: Discuss the potential benefits and possible harmful effects of one example of genetic modification. [Obj. 3] Benefits Crops • Enhanced taste and quality, Reduced maturation time, Increased nutrients, yields, and stress tolerance, Improved resistance to disease, pests, and herbicides, New products and growing techniques Animals • Better yields of meat, eggs, and milk • Improved animal health and diagnostic methods • Increased resistance, productivity, hardiness, and feed efficiency Environment • "Friendly" bioherbicides and bioinsecticides • Conservation of soil, water, and energy • Bioprocessing for forestry products • Better natural waste management • More efficient processing Society • Increased food security for growing populations

36. Controversies Safety • Potential human health impact: allergens, transfer of antibiotic resistance, unknown effects. • Potential environmental impact: unintended transfer of transgenes through cross-pollination, unknown effects on other organisms (e.g., soil microbes), and loss of flora and fauna biodiversity Access and Intellectual Property • Domination of world food production by a few companies • Increasing dependence on industrialized nations by developing countries • Biopiracy-foreign exploitation of natural resources Ethics • Violation of natural organisms' intrinsic values • Tampering with nature by mixing genes among species • Objections to consuming animal genes in plants and vice versa • Stress for animals Labeling • Not mandatory in some countries (e.g., United States) • Mixing GM crops with non-GM confounds labeling attempts Society • New advances may be skewed to interests of rich countries Dharmananda, S. Issues Surrounding Genetically Modified (GM) Products. Online: http://www.google.ca/imgres?imgurl=http://www.itmonline.org/image/gmo1b.jpg&imgrefurl=http://www.itmonline.org/arts/gmo.htm&usg=__y5yFCjq561X8N0vAy5g8nid85NM=&h=453&w=400&sz=32&hl=en&start=5&zoom=1&tbnid=Kqfgq2YL9RdYEM:&tbnh=127&tbnw=112&ei=k9TfTpasGsfL0QG8hdCuBw&prev=/search%3Fq%3Dgmo%26um%3D1%26hl%3Den%26safe%3Dactive%26sa%3DN%26gbv%3D2%26tbm%3Disch&um=1&itbs=1