1. Copyright © 2011 Pearson Education Inc. Lecture prepared by Mindy Miller-Kittrell, University of Tennessee, Knoxville M I C R O B I O L O G Y WITH DISEASES BY TAXONOMY, THIRD EDITION Chapter 8 Recombinant DNA Technology

2. Copyright © 2011 Pearson Education Inc. The Role of Recombinant DNA technology in Biotechnology Recombinant DNA technology – Intentionally modifying genomes of organisms for practical purposes – Three goals – Eliminate undesirable phenotypic traits – Combine beneficial traits of two or more organisms – Create organisms that synthesize products humans need

3. Copyright © 2011 Pearson Education Inc. Overview of recombinant DNA technology Figure 8.1

4. Copyright © 2011 Pearson Education Inc. The Tools of Recombinant DNA Technology Mutagens – Physical and chemical agents that produce mutations – Scientists utilize mutagens to – Create changes in microbes’ genomes to change phenotypes – Select for and culture cells with beneficial characteristics – Mutated genes alone can be isolated

5. Copyright © 2011 Pearson Education Inc. The Tools of Recombinant DNA Technology The Use of Reverse Transcriptase to Synthesize cDNA – Isolated from retroviruses – Uses RNA template to transcribe molecule of cDNA – Easier to isolate mRNA molecule for desired protein first – mRNA of eukaryotes has introns removed – Allows cloning in prokaryotic cells

6. Copyright © 2011 Pearson Education Inc. The Tools of Recombinant DNA Technology Synthetic Nucleic Acids – Molecules of DNA and RNA produced in cell-free solutions – Uses of synthetic nucleic acids – Elucidating the genetic code – Creating genes for specific proteins – Synthesizing DNA and RNA probes to locate specific sequences of nucleotides – Synthesizing antisense nucleic acid molecules

7. Copyright © 2011 Pearson Education Inc. The Tools of Recombinant DNA Technology Restriction Enzymes – Bacterial enzymes that cut DNA molecules only at restriction sites – Categorized into two groups based on type of cut – Cuts with sticky ends – Cuts with blunt ends

8. Copyright © 2011 Pearson Education Inc. Actions of restriction enzymes Figure 8.2

9. Copyright © 2011 Pearson Education Inc. The Tools of Recombinant DNA Technology Animation: Recombinant DNA Technology

10. Copyright © 2011 Pearson Education Inc. The Tools of Recombinant DNA Technology Vectors – Nucleic acid molecules that deliver a gene into a cell – Useful properties – Small enough to manipulate in a lab – Survive inside cells – Contain recognizable genetic marker – Ensure genetic expression of gene – Include viral genomes, transposons, and plasmids

11. Copyright © 2011 Pearson Education Inc. Producing a recombinant vector Figure 8.3

12. Copyright © 2011 Pearson Education Inc. The Tools of Recombinant DNA Technology Gene Libraries – A collection of bacterial or phage clones – Each clone in library often contains one gene of an organism’s genome – Library may contain all genes of a single chromosome – Library may contain set of cDNA complementary to mRNA

13. Copyright © 2011 Pearson Education Inc. Production of a gene library Figure 8.4

14. Copyright © 2011 Pearson Education Inc. Techniques of Recombinant DNA Technology Multiplying DNA in vitro : The Polymerase Chain Reaction (PCR) – Large number of identical molecules of DNA produced in vitro – Critical to amplify DNA in variety of situations – Epidemiologists use to amplify genome of unknown pathogen – Amplified DNA from Bacillus anthracis spores in 2001 to identify source of spores

15. Copyright © 2011 Pearson Education Inc. Techniques of Recombinant DNA Technology Multiplying DNA in vitro : The Polymerase Chain Reaction (PCR ) – Repetitive process consisting of three steps – Denaturation – Priming – Extension – Can be automated using a thermocycler

16. Copyright © 2011 Pearson Education Inc. Techniques of Recombinant DNA Technology Animation: Polymerase Chain Reaction: Overview

17. Copyright © 2011 Pearson Education Inc. Techniques of Recombinant DNA Technology Animation: Polymerase Chain Reaction: Components

18. Copyright © 2011 Pearson Education Inc. Polymerase chain reaction (PCR) Figure 8.5a

19. Copyright © 2011 Pearson Education Inc. Polymerase chain reaction (PCR) Figure 8.5b

20. Copyright © 2011 Pearson Education Inc. Techniques of Recombinant DNA Technology Animation: PCR: The Process

21. Copyright © 2011 Pearson Education Inc. Techniques of Recombinant DNA Technology Selecting a Clone of Recombinant Cells – Must find clone containing DNA of interest – Probes are used

22. Copyright © 2011 Pearson Education Inc. Techniques of Recombinant DNA Technology Separating DNA Molecules: Gel Electrophoresis and the Southern Blot – Gel electrophoresis – Separates molecules based on electrical charge, size, and shape – Allows scientists to isolate DNA of interest – Negatively charged DNA drawn toward positive electrode – Agarose makes up gel; acts as molecular sieve – Smaller fragments migrate faster and farther than larger ones – Determine size by comparing distance migrated to standards

23. Copyright © 2011 Pearson Education Inc. Gel electrophoresis Figure 8.6

24. Copyright © 2011 Pearson Education Inc. Techniques of Recombinant DNA Technology Separating DNA Molecules: Gel Electrophoresis and the Southern Blot – Southern blot – DNA transferred from gel to nitrocellulose membrane – Probes used to localize DNA sequence of interest – Northern blot – used to detect RNA – Uses of Southern blots – Genetic “fingerprinting” – Diagnosis of infectious disease – Demonstrate incidence and prevalence of organisms that cannot be cultured

25. Copyright © 2011 Pearson Education Inc. The Southern blot technique Figure 8.7

26. Copyright © 2011 Pearson Education Inc. Techniques of Recombinant DNA Technology DNA Microarrays – Consist of molecules of immobilized single-stranded DNA – Fluorescently labeled DNA washed over array will adhere only at locations where there are complementary DNA sequences – Variety of scientific uses of DNA microarrays – Monitoring gene expression – Diagnosis of infection – Identification of organisms in an environmental sample

27. Copyright © 2011 Pearson Education Inc. DNA microarray Figure 8.8

28. Copyright © 2011 Pearson Education Inc. Techniques of Recombinant DNA Technology Inserting DNA into Cells – Goal of DNA technology is insertion of DNA into cell – Natural methods – Transformation – Transduction – Conjugation – Artificial methods – Electroporation – Protoplast fusion – Injection – gene gun and microinjection

29. Copyright © 2011 Pearson Education Inc. Artificial methods of inserting DNA into cells Figure 8.9a/b

30. Copyright © 2011 Pearson Education Inc. Artificial methods of inserting DNA into cells Figure 8.9c/d

31. Copyright © 2011 Pearson Education Inc. Applications of Recombinant DNA Technology Genetic Mapping – Locating genes on a nucleic acid molecule – Provides useful facts concerning metabolism, growth characteristics, and relatedness to others Locating Genes – Until 1970, genes identified by labor-intensive methods – Simpler and universal methods now available – Restriction fragmentation – Fluorescent in situ hybridization (FISH)

32. Copyright © 2011 Pearson Education Inc. Fluorescent in situ hybridization Figure 8.10

33. Copyright © 2011 Pearson Education Inc. Automated DNA sequencing Figure 8.11

34. Copyright © 2011 Pearson Education Inc. Applications of Recombinant DNA Technology Environmental Studies – Most microorganisms have never been grown in a laboratory – Scientists know them only by their DNA fingerprints – Allowed identification of over 500 species of bacteria from human mouths – Determined that methane-producing archaea are a problem in rice agriculture

35. Copyright © 2011 Pearson Education Inc. Applications of Recombinant DNA Technology Pharmaceutical and Therapeutic Applications – Protein synthesis – Creation of synthetic peptides for cloning – Vaccines – Production of safer vaccines – Subunit vaccines – Introduce genes of pathogens into common fruits and vegetables – Injecting humans with plasmid carrying gene from pathogen – Humans synthesize pathogen’s proteins

36. Copyright © 2011 Pearson Education Inc. Applications of Recombinant DNA Technology Pharmaceutical and Therapeutic Applications – Genetic screening – DNA microarrays used to screen individuals for inherited disease caused by mutations – Can also identify pathogen’s DNA in blood or tissues – DNA fingerprinting – Identifying individuals or organisms by their unique DNA sequence

37. Copyright © 2011 Pearson Education Inc. DNA fingerprinting Figure 8.12

38. Copyright © 2011 Pearson Education Inc. Applications of Recombinant DNA Technology Pharmaceutical and Therapeutic Applications – Gene therapy – Missing or defective genes replaced with normal copies – Some patients’ immune systems react negatively – Medical diagnosis – Patient specimens can be examined for presence of gene sequences unique to certain pathogens – Xenotransplants – Animal cells, tissues, or organs introduced into human body

39. Copyright © 2011 Pearson Education Inc. Applications of Recombinant DNA Technology Agricultural Applications – Production of transgenic organisms – Recombinant plants and animals altered by addition of genes from other organisms

40. Copyright © 2011 Pearson Education Inc. Applications of Recombinant DNA Technology Agricultural Applications – Herbicide resistance – Gene from Salmonella conveys resistance to glyphosate (Roundup) – Farmers can kill weeds without killing crops – Salt tolerance – Scientists have removed gene for salt tolerance and inserted into tomato and canola plants – Transgenic plants survive, produce fruit, and remove salt from soil

41. Copyright © 2011 Pearson Education Inc. Applications of Recombinant DNA Technology Agricultural Applications – Freeze resistance – Crops sprayed with genetically modified bacteria can tolerate mild freezes – Pest resistance – Bt toxin – Naturally occurring toxin only harmful to insects – Organic farmers used to reduce insect damage to crops – Gene for Bt toxin inserted into various crop plants – Genes for Phytophthora resistance inserted into potato crops

42. Copyright © 2011 Pearson Education Inc. Applications of Recombinant DNA Technology Agricultural Applications – Improvements in nutritional value and yield – Tomatoes allowed to ripen on vine and shelf life increased – Gene for enzyme that breaks down pectin suppressed – BGH allows cattle to gain weight more rapidly, – Have meat with lower fat content and produce 10% more milk – Gene for β -carotene (vitamin A precursor) inserted into rice – Scientists considering transplanting genes coding for entire metabolic pathways

43. Copyright © 2011 Pearson Education Inc. The Ethics and Safety of Recombinant DNA Technology Supremacist view – humans are of greater value than animals Long-term effects of transgenic manipulations are unknown Unforeseen problems arise from every new technology and procedure Natural genetic transfer could deliver genes from transgenic plants and animals into other organisms Transgenic organisms could trigger allergies or cause harmless organisms to become pathogenic

44. Copyright © 2011 Pearson Education Inc. The Ethics and Safety of Recombinant DNA Technology Studies have not shown any risks to human health or environment Standards imposed on labs involved in recombinant DNA technology Can create biological weapons using same technology

45. Copyright © 2011 Pearson Education Inc. The Ethics and Safety of Recombinant DNA Technology Ethical Issues – Routine screenings? – Who should pay? – Genetic privacy rights? – Profits from genetically altered organisms? – Required genetic screening? – Forced correction of “genetic abnormalities”?