Biotechnology

1. Chapter 14: Genetic Engineering-Modification of the DNA of an organism to produce new genes with new characteristics

2. Biotechnology • Use of organisms to benefit humanity

3. Recombinant DNA technology • DNA from different organisms is spliced together • Allows scientists to make many copies of any DNA segment (clone) • Can introduce foreign DNA into cells of microorganisms

4. Recombinant DNA technology • Restriction enzymes – cut DNA • Bacteria produce for defense against viruses • Vector – transports DNA into a cell • Ex: bacteriophage • Plasmid – separate, smaller circular DNA that maybe be present and able to replicate inside bacteria • Transformation – uptake of foreign DNA by cells • How plasmids can get into bacteria

5. Bacterial Conjugation and Recombination

6. Recombinant DNA technology • Palindromic sequences – reads the same as complement, in opposite direction • AAGCTT • TTCGAA • Many restriction enzymes cut these sequences • Restriction enzymes cut on a stagger  sticky ends (can pair with complementary single-stranded end of other DNA cut with same enzyme) • DNA Ligase – links 2 fragments  recombinant DNA

7. Fig. 20-3-3 Restriction site 5 3 3 5 DNA Restriction enzymecuts sugar-phosphatebackbones. 1 Sticky end DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs. 2 One possible combination DNA ligaseseals strands. 3 Recombinant DNA molecule

8. Restriction Enzymes

9. Steps of Creating a Recombinant DNA Plasmid (Basic) • 1. Plasmids and desired DNA cut by same restriction enzyme • 2. Mix 2 types of DNA so sticky ends pair • 3. DNA ligase forms bonds between fragments

11. Fig. 20-2 Cell containing geneof interest Bacterium 1 Gene inserted intoplasmid Bacterialchromosome Plasmid Gene ofinterest RecombinantDNA (plasmid) DNA of chromosome 2 Plasmid put intobacterial cell Recombinantbacterium 3 Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest Gene ofInterest Protein expressedby gene of interest Copies of gene Protein harvested Basic research andvarious applications 4 Basicresearchon protein Basicresearchon gene Gene used to alter bacteria for cleaning up toxic waste Gene for pest resistance inserted into plants Protein dissolvesblood clots in heartattack therapy Human growth hor-mone treats stuntedgrowth

12. Cloning a Gene

13. Fig. 20-4-4 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carryingplasmids RESULTS Colony carrying recombinant plasmid with disrupted lacZ gene Colony carrying non-recombinant plasmidwith intact lacZ gene One of manybacterial clones

14. Cloning DNA • Genome – total DNA per cell • Genomic library – collection of DNA fragments more or less representative of all DNA in genome • Genetic Probe – single stranded DNA or RNA that is radioactively labeled and can attach to target sequence by base pairing rules

15. A probe can be synthesized that is complementary to the gene of interest • For example, if the desired gene is – Then we would synthesize this probe … … 5 G G C T A A C T T A G C 3 C C G A T T G A A T C G 5 3

16. DNA Probe

17. Using a DNA probe

18. Polymerase Chain Reaction (PCR) • Can amplify a small sample of DNA quickly • DNA replication in vitro • 2 strands separated by heating so special heat-resistant DNA polymerase called Taq polymerase used (thermophile) • MAJOR BONUS: Only specific sequences can be replicated • Study: crime scenes, archaeological remains

19. PCR

20. Gel Electrophoresis • Separates fragments like DNA, RNA or polypeptides (they carry charge and can migrate in an electrical field • RNA and DNA (-) --- so they move to (+) pole • Smaller fragments go further • Compare sample to standard • Usually “blot” - transfer DNA from gel to nitrocellulose filter for further analysis • DNA Fingerprinting

21. Gel Electrophoresis

22. Fig. 20-9a TECHNIQUE Powersource Mixture ofDNA mol-ecules ofdifferentsizes Anode Cathode – + Gel 1 Powersource – + Longermolecules 2 Shortermolecules

23. Fig. 20-9b RESULTS

27. DNA Fingerprint

28. Transgenic Organisms • Plants and animals in which foreign genes have been incorporated • Animals • Inject DNA into nucleus of egg or stem cell • Eggs implanted in uterus; stem cells injected into blastocysts + then implanted into foster mother • Plants • Disease resistance • Pesticide resistance

29. Transgenics

30. Fig. 20-20 Embryonic stem cells Adult stem cells From bone marrowin this example Early human embryoat blastocyst stage(mammalian equiva-lent of blastula) Cells generatingall embryoniccell types Cells generatingsome cell types Culturedstem cells Differentcultureconditions Differenttypes ofdifferentiatedcells Blood cells Nerve cells Liver cells

31. Fig. 20-18 TECHNIQUE Mammarycell donor Egg celldonor 2 1 Egg cellfrom ovary Nucleusremoved Cells fused 3 Culturedmammary cells 3 Nucleus frommammary cell Grown inculture 4 Early embryo Implantedin uterusof a thirdsheep 5 Surrogatemother Embryonicdevelopment 6 Lamb (“Dolly”)genetically identical tomammary cell donor RESULTS

32. Fig. 20-19

33. Cloning Video

34. GE Plants

35. Application of GE • Human proteins • Insulin • Hormones - HGH • Human treatments for disease • Multiple sclerosis, certain cancers, heart attacks, forms of anemia • Vaccines

36. Fig. 20-23

37. Fig. 20-24 (a) This photo shows EarlWashington just before his release in 2001,after 17 years in prison. Source of sample STRmarker 1 STRmarker 2 STRmarker 3 Semen on victim 17, 19 13, 16 12, 12 Earl Washington 16, 18 14, 15 11, 12 17, 19 13, 16 12, 12 Kenneth Tinsley (b) These and other STR data exonerated Washington andled Tinsley to plead guilty to the murder.

38. Forensics