Ch. 20 Biotechnology

1. Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly used technologies.

2. Understanding and Manipulating Genomes • Sequenced the human genome in 2003 through: • Biotechnology: manipulation of organisms • Genetic engineering: manipulation of genes • Recombinant DNA: 2 DNAs combined

3. Using Bacteria as Tools • Bacteria • Circular DNA • Plasmid • Extra genetic material • Small, circular DNA • Not necessary, but usually beneficial http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RecombinantPlasmid.gif

4. Using Bacteria as Tools • Bacterial Transformation • Uptake of DNA from the fluid surrounding the cell • Causes genetic recombination • Allow insertion of gene of interest http://biology200.gsu.edu/houghton/4564%20'04/figures/lecture%203/transformation.jpg

5. 20.1: DNA (Gene) Cloning • Uses: make many copies (amplify) quickly and produces proteins • Basic Method: • Use bacterial plasmids (cloning vector). • Insert desired gene (recombinant DNA). • Return plasmid to bacteria. • Bacteria reproduce. • Various applications.

6. Making Recombinant DNA • Restriction enzymes (nucleases) cut DNA in specific places (restriction site) to form restrictionfragments. • Must use same enzyme on plasmid and desired gene • Forms sticky ends:unbonded nucleotides • Add DNA ligase to rebond recombinant DNA.

7. Cloning a Eukaryotic Gene in a Bacterial Plasmid • In gene cloning, the original plasmid is called a cloning vector • A cloning vector is a DNA molecule that can carry foreign DNA into a cell and replicate there

8. Cloning a Eukaryotic Gene in a Bacterial Plasmid • Only a cell that took up a plasmid, which has theampR gene, will reproduce and form a colony. • Colonies with nonrecombinant plasmids will be blue, because they can hydrolyze X-gal. • Colonies with recombinant plasmids, in which lacZ is disrupted, will be white, because they cannot hydrolyze X-gal. • By screening the white colonies with a nucleic acid probe (see Figure 20.5), researchers can identify clones of bacterial cells carrying the gene of interest.

9. Storing Cloned Genes • Genomic Library: complete set of plasmid clones saved. • Phages are also used so they are saved as phage library. • A bacterial artificial chromosome (BAC) is a large plasmid that has been trimmed down and can carry a large DNA insert • Complementary DNA (cDNA) can be made by reverse transcription of mRNA to make a cDNA library.

10. ID Clone Carrying Gene of Interest • Nucleic acid probe (RNA or DNA) radioactively labeled which hybridizes to gene.

11. Eukaryotic Genes in Bacterial Expression Systems • To overcome differences in promoters and other DNA control sequences, scientists usually employ an expression vector, a cloning vector that contains a highly active prokaryotic promoter • To overcome inability to remove introns, use cDNA form of the gene

12. Eukaryotic Cloning and Expression Systems • The use of cultured eukaryotic cells as host cells and yeast artificial chromosomes (YACs) as vectors helps avoid gene expression problems • YACs behave normally in mitosis and can carry more DNA than a plasmid • Eukaryotic hosts can provide the posttranslational modifications that many proteins require http://www.accessexcellence.org/RC/VL/GG/images/YAC.gif

13. Amplifying DNA: Polymerase Chain Reaction 1 DNA strand → billions in hours. • Denature: Heat DNA to break H-bonds • Annealing: Add primers and cool • Extension: Add heat resistant DNA polymerase and nucleotides • Repeat using thermocycler

14. 20.2 Restriction Fragment Analysis Gel Electrophoresis • DNA is – charge; attracted to + • Gel that separates DNA by length; smaller pieces can travel faster/further. • Make fragments by restriction enzymes and separate them. • Alleles have different sequences of DNA so are cut differently.

15. Southern Blotting • A technique called Southern blotting combines gel electrophoresis with nucleic acid hybridization • Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel

16. Restriction Fragment Length Polymorphisms (RFLPs) • Restriction fragments made using the same enzyme on homologues. • Used as a marker (fingerprint) for individuals. Paternity Test

17. DNA Sequencing • Relatively short DNA fragments can be sequenced by the dideoxy chain-termination method • Inclusion of special dideoxyribonucleotides in the reaction mix ensures that fragments of various lengths will be synthesized

18. DNA Sequencing

19. DNA Sequencing http://files.myweb.med.ucalgary.ca/files/64/images/DNA%20Sequencing%20Images/Sample_sequencing_result_2005-10-25_copy.jpg

20. How to ID Unknown Genes • Compare to known genes of other organisms. • Disable the gene and observe the consequence. • In vitro interference: use copies DNA gene, introduce mutagen, reinsert into cell, observe consequence.

21. Studying Expression of Interacting Groups of Genes • DNA Microarray Assays • Take mRNA • Make cDNA (single strand) • Fluorescently label • Apply to array chip (contains known DNA fragments the cDNA will bond to) • Look for fluorescence.

22. Determining Gene Function • One way to determine function is to disable the gene and observe the consequences (knock-outs) • Using in vitro mutagenesis, mutations are introduced into a cloned gene, altering or destroying its function • When the mutated gene is returned to the cell, the normal gene’s function might be determined by examining the mutant’s phenotype A transgenic mouse with an active rat growth hormone gene (left). This transgenic mouse is twice the size of a normal mouse (right). http://web.virginia.edu/Heidi/chapter29/Images/8883n29_30.jpg

23. Comparing Genomes of Different Species • Allows us to look for evolutionary relationships. • Comparative data on simple organisms helps us understand more complex ones. • Closely related species: figure out one and use as a template for the others.

24. Future Directions • Proteomics: study proteins encoded by genomes. • Single Nucleotide Polymorphisms (SNPs): single base-pair differences from one human to another. • People are 99.99% identical on genetic level.

25. 20.3 Cloning • In Plants: • Totipotent: cells can dedifferentiate. • Tranplanting a clipping or root causing a clone to be made.

26. Cloning • In Animals • Remove nucleus from egg • Add nucleus from somatic cell of donor • Grow in culture • Implant in uterus • Clone is born!

27. CC, the first cloned cat Although CC is a clone of her mother, they are not identical due to the X-inactivation mechanism and different environmental influences • Figure 20.20

28. Stem Cells of Animals • Goal of cloning human embryos → stem cell production • Stem cell = undifferentiated cell • Embryonic stem cells have the potential to become anything (pluripotent). • Adult stem cells can’t.

29. Regenerative Medicine? Human ear grown in a lab from stem cells. • Human pluripotent stem cells crucial for the development of regenerative medicine • Can allow for growing a whole new heart or liver, since they can be converted into any cell type in the body http://www.zmescience.com/research/studies/lab-grown-stem-cells-may-mutate-in-time/

30. 20.4 Applications of Genetic Engineering • Medical Applications: • Identifying genes that cause disease/disorders • Gene therapy: changing disease causing genes in humans.

31. 20.4 Applications of Genetic Engineering • Pharmaceutical Products • Insulin • Human growth hormone • Tissue plasminogen activator to dissolve blood clots • HIV blockers • Vaccines http://www.udel.edu/physics/scen103/CGZ/14b.gif

32. Forensic Evidence • DNA fingerprinting using gel electrophoresis • Environmental Cleanup • Mining bacteria (copper, lead, nickel, etc) • Cleaning toxic waster • Clean oil spills

33. Agricultural Applications • Animal Husbandry and “Pharm” animals • Transgenic animals (has recombinant DNA) to make better wool, leaner meat, shorter maturation time, pharmaceutical factories for blood clotting factors. • Genetic Engineering in Plants • Delayed ripening, resistance to spoilage/disease, increase nutritional value. • Uses Ti plasmid recombined with desired genes.

34. Transgenic Animals • Human gene for antithrombin inserted into a goat’s genome and the protein is produced in the milk http://www.livinghistoryfarm.org/farminginthe70s/crops_12.html

35. Genetic Engineering in Plants • Agricultural scientists have endowed a number of crop plants with genes for desirable traits • The Ti plasmid is the most commonly used vector for introducing new genes into plant cells

36. Transgenic Plants Bt transgenic corn is normal corn that contains a gene from the soil bacterium Bacillus thuringiensis. Gene allows production of a toxic protein that can kill many types of caterpillars (http://www.ces.ncsu.edu/plymouth/pubs/btcorn99.html) 1994. Flavr Savr Tomato. 1st engineered food in stores. Engineered to remain firm even as it turns red and ripe.

37. Safety and Ethics • Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures • Most public concern about possible hazards centers on genetically modified (GM) organisms used as food