1. PowerPoint ® Lecture Presentations prepared by Bradley W. Christian, McLennan Community College C H A P T E R © 2016 Pearson Education, Inc. Biotechnology and DNA Technology 9

2. © 2016 Pearson Education, Inc.

3. Introduction to Biotechnology Learning Objectives 9-1Compare and contrast biotechnology, genetic modification, and recombinant DNA technology. 9-2Identify the roles of a clone and a vector in making recombinant DNA.

4. © 2016 Pearson Education, Inc. Introduction to Biotechnology Biotechnology: the use of microorganisms, cells, or cell components to make a product Foods, antibiotics, vitamins, enzymes Recombinant DNA (rDNA) technology: the insertion or modification of genes to produce desired proteins

5. © 2016 Pearson Education, Inc. An Overview of Recombinant DNA Procedures Vector: self-replicating DNA molecule used to transport foreign DNA into a cell Clone: population of genetically identical cells arising from one cell; each carries the vector

6. © 2016 Pearson Education, Inc. Figure 9.1 A Typical Genetic Modification Procedure.

7. © 2016 Pearson Education, Inc. Check Your Understanding Differentiate biotechnology and rDNA technology. 9-1 In one sentence, describe how a vector and clone are used. 9-2

8. © 2016 Pearson Education, Inc. Tools of Biotechnology Learning Objectives 9-3Compare selection and mutation. 9-4Define restriction enzymes, and outline how they are used to make rDNA. 9-5List the four properties of vectors. 9-6Describe the use of plasmid and viral vectors. 9-7Outline the steps in PCR, and provide an example of its use.

9. © 2016 Pearson Education, Inc. Tools of Biotechnology Selection: selecting for a naturally occurring microbe that produces a desired product Mutation: Mutagens cause mutations that might result in a microbe with a desirable trait Site-directed mutagenesis: a targeted and specific change in a gene

10. © 2016 Pearson Education, Inc. Restriction Enzymes Cut specific sequences of DNA Destroy bacteriophage DNA in bacterial cells Methylated cytosines in bacteria protect their own DNA from digestion Create blunt ends or staggered cuts known as sticky ends

11. © 2016 Pearson Education, Inc. Recombinant DNA Technology PLAY Animation: Recombinant DNA Technology

12. © 2016 Pearson Education, Inc. Table 9.1 Selected Restriction Enzymes Used in rDNA Technology

13. © 2016 Pearson Education, Inc. Figure 9.2 A restriction enzyme's role in making rDNA. A restriction enzyme cuts (red arrows) double-stranded DNA at its particular recognition sites, shown in blue. These cuts produce a DNA fragment with two sticky ends. DNA from another source, perhaps a plasmid, cut with the same restriction enzyme When two such fragments of DNA cut by the same restriction enzyme come together, they can join by base pairing. The joined fragments will usually form either a linear molecule or a circular one, as shown here for a plasmid. Other combinations of fragments can also occur. The enzyme DNA ligase is used to unite the backbones of the two DNA fragments, producing a molecule of rDNA. Recognition sites DNACut Sticky end rDNA

14. © 2016 Pearson Education, Inc. Vectors Carry new DNA to desired cells Must be able to self-replicate Plasmids and viruses can be used as vectors Shuttle vectors exist in several different species and can move cloned sequences among various organisms

15. © 2016 Pearson Education, Inc. Figure 9.3 A plasmid used for cloning. pUC19 amp R lacZ HindIII BamHI EcoRI ori

16. © 2016 Pearson Education, Inc. Check Your Understanding How are selection and mutation used in biotechnology? 9-3 What is the value of restriction enzymes in rDNA technology? 9-4 What criteria must a vector meet? 9-5 Why is a vector used in rDNA technology? 9-6

17. © 2016 Pearson Education, Inc. Polymerase Chain Reaction Process of increasing small quantities (amplifying) of DNA for analysis Used for diagnostic tests for genetic diseases and detecting pathogens Reverse-transcription PCR uses mRNA as template

18. © 2016 Pearson Education, Inc. PCR: Overview PLAY Animation: PCR: Overview

19. © 2016 Pearson Education, Inc. PCR: Components PLAY Animation: PCR: Components

20. © 2016 Pearson Education, Inc. Figure 9.4 The polymerase chain reaction.

21. © 2016 Pearson Education, Inc. Check Your Understanding For what is each of the following used in PCR: primer, DNA polymerase, 94  C? 9-7

22. © 2016 Pearson Education, Inc. Techniques of Genetic Modification Learning Objectives 9-8Describe five ways of getting DNA into a cell. 9-9Describe how a genomic library is made. 9-10Differentiate cDNA from synthetic DNA. 9-11Explain how each of the following is used to locate a clone: antibiotic-resistance genes, DNA probes, gene products. 9-12List one advantage of modifying each of the following: Escherichia. coli, Saccharomyces cerevisiae, mammalian cells, plant cells.

23. © 2016 Pearson Education, Inc. Inserting Foreign DNA into Cells DNA can be inserted into a cell by: Transformation: Cells take up DNA from the surrounding environment Electroporation: Electrical current forms pores in cell membranes Protoplast fusion: Removing cell walls from two bacteria allows them to fuse

24. © 2016 Pearson Education, Inc. Chromosome Plasma membrane Cell wall Bacterial cell walls are enzymatically digested, producing protoplasts. Bacterial cells Protoplasts In solution, protoplasts are treated with polyethylene glycol. Protoplasts fuse. Segments of the two chromosomes recombine. Recombinant cell grows new cell wall. Recombinant cell Figure 9.5 Protoplast fusion.

25. © 2016 Pearson Education, Inc. Inserting Foreign DNA into Cells DNA can be inserted into a cell by: Gene gun Microinjection

26. © 2016 Pearson Education, Inc. Figure 9.6 A gene gun, which can be used to insert DNA-coated "bullets" into a cell.

27. © 2016 Pearson Education, Inc. Figure 9.7 The microinjection of foreign DNA into an egg.

28. © 2016 Pearson Education, Inc. Genomic Libraries Collections of clones containing different DNA fragments An organism's DNA is digested and spliced into plasmid or phage vectors and introduced into bacteria At least one clone exists for every gene in the organism

29. © 2016 Pearson Education, Inc. Figure 9.8 Genomic libraries. Genome to be stored in library is cut up with restriction enzyme Host cell Recombinant plasmid OR Recombinant phage DNA Phage cloning vector Plasmid LibraryPhage Library

30. © 2016 Pearson Education, Inc. Genomic Libraries Complementary DNA (cDNA) is made from mRNA by reverse transcriptase Used for obtaining eukaryotic genes because eukaryotic DNA has introns that do not code for protein mRNA has the introns removed, coding only for the protein product

31. © 2016 Pearson Education, Inc. Figure 9.9 Making complementary DNA (cDNA) for a eukaryotic gene. Exon Intron Exon DNA Nucleus A gene composed of exons and introns is transcribed to RNA by RNA polymerase. RNA transcript Processing enzymes in the nucleus remove the intron-derived RNA and splice together the exon-derived RNA into mRNA. mRNA Cytoplasm mRNA is isolated from the cell, and reverse transcriptase is added. First strand of DNA is synthesized. The mRNA is digested by reverse transcriptase. DNA strand being synthesized DNA polymerase is added to synthesize second strand of DNA. cDNA of gene without introns Test tube

32. © 2016 Pearson Education, Inc. Synthetic DNA Builds genes using a DNA synthesis machine

33. © 2016 Pearson Education, Inc. Figure 9.10 A DNA synthesis machine.

34. © 2016 Pearson Education, Inc. Check Your Understanding Contrast the five ways of putting DNA into a cell 9-8 What is the purpose of a genomic library? 9-9 Why isn't cDNA synthetic? 9-10

35. © 2016 Pearson Education, Inc. Selecting a Clone Blue-white screening Uses plasmid vector containing ampicillin resistance gene (amp R ) and β-galactosidase gene (lacZ) Bacteria is grown in media containing ampicillin and X-gal, a substrate for β-galactosidase

36. © 2016 Pearson Education, Inc. Figure 9.11 Blue-white screening, one method of selecting recombinant bacteria. Ampicillin-resistance gene (amp R ) Plasmid DNA and foreign DNA are both cut with the same restriction enzyme. The plasmid has the genes for lactose hydrolysis (the lacZ gene encodes the enzyme β-galactosidase) and ampicillin resistance. Plasmid Foreign DNA Recombinant plasmid Foreign DNA will insert into the lacZ gene. The bacterium receiving the plasmid vector will not produce the enzyme β-galactosidase if foreign DNA has been inserted into the plasmid. The recombinant plasmid is introduced into a bacterium, which becomes ampicillin resistant. All treated bacteria are spread on a nutrient agar plate containing ampicillin and a β-galactosidase substrate and incubated. The β-galactosidase substrate is called X-gal. Only bacteria that picked up the plasmid will grow in the presence of ampicillin. Bacteria that hydrolyze X-gal produce galactose and an indigo compound. The indigo turns the colonies blue. Bacteria that cannot hydrolyze X-gal produce white colonies. Bacterium Restriction site β-galactosidase gene (lacZ) Restriction sites Colonies with foreign DNA

37. © 2016 Pearson Education, Inc. Selecting a Clone Colony hybridization Use DNA probes: short segments of single-stranded DNA complementary to the desired gene

38. © 2016 Pearson Education, Inc. Figure 9.12 Colony hybridization: using a DNA probe to identify a cloned gene of interest. Make replica of master plate on nitrocellulose filter. Treat filter with detergent (SDS) to lyse bacteria. Treat filter with sodium hydroxide (NaOH) to separate DNA into single strands. Bound DNA probe Replica plate Compare filter with replica of master plate to identify colonies containing gene of interest. Wash filter to remove unbound probe. Probe will hybridize with desired gene from bacterial cells. Fluorescence labeled probes Add labeled probes. Nitrocellulose filter Strands of bacterial DNA Master plate with colonies of bacteria containing cloned segments of foreign genes Gene of interest Single- stranded DNA Colonies containing genes of interest

39. © 2016 Pearson Education, Inc. Making a Gene Product E. coli Advantages: easily grown and its genomics are known Disadvantages: produces endotoxins and does not secrete its protein products

40. © 2016 Pearson Education, Inc. Figure 9.13 E. coli genetically modified to produce gamma interferon, a human protein that promotes an immune response.

41. © 2016 Pearson Education, Inc. Making a Gene Product Saccharomyces cerevisiae Easily grown and has a larger genome than bacteria Expresses eukaryotic genes easily Plant cells and whole plants Express eukaryotic genes easily Plants are easily grown, large-scale, and low-cost Mammalian cells Express eukaryotic genes easily Can make products for medical use Harder to grow

42. © 2016 Pearson Education, Inc. Check Your Understanding How are recombinant clones identified? 9-11 What types of cells are used for cloning rDNA? 9-12

43. © 2016 Pearson Education, Inc. Applications of DNA Technology Learning Objectives 9-13List at least five applications of DN technology. 9-14Define RNAi. 9-15Discuss the value of genome projects. 9-16Define the following terms: random shotgun sequencing, bioinformatics, proteomics.

44. © 2016 Pearson Education, Inc. Applications of DNA Technology Learning Objectives 9-17Diagram the Southern blotting procedure, and provide an example of its use. 9-18Diagram DNA fingerprinting, and provide an example of its use. 9-19Outline genetic engineering with Agrobacterium.

45. © 2016 Pearson Education, Inc. Therapeutic Applications Human enzymes and other proteins such as insulin Subunit vaccines: made from pathogen proteins in genetically modified yeasts Nonpathogenic viruses carrying genes for pathogen's antigens as DNA vaccines Gene therapy to replace defective or missing genes

46. © 2016 Pearson Education, Inc. Table 9.2 Some Pharmaceutical Products of rDNA (1 of 2)

47. © 2016 Pearson Education, Inc. Table 9.2 Some Pharmaceutical Products of rDNA (2 of 2)

48. © 2016 Pearson Education, Inc. Therapeutic Applications Gene silencing Small interfering RNAs (siRNAs) bind to mRNA, which is then destroyed by RNA-induced silencing complex (RISC) RNA interference (RNAi) inserts DNA encoding siRNA into a plasmid and transferred into a cell

49. © 2016 Pearson Education, Inc. Figure 9.14 Gene silencing could provide treatments for a wide range of diseases.

50. © 2016 Pearson Education, Inc. Check Your Understanding Explain how DNA technology can be used to treat disease and to prevent disease. 9-13 What is gene silencing? 9-14

51. © 2016 Pearson Education, Inc. Genome Projects Shotgun sequencing sequences small pieces of genomes which are assembled by a computer Metagenomics is the study of genetic material directly from environmental samples The Human Genome Project sequenced the entire human genome The Human Proteome Project will map proteins expressed in human cells

52. © 2016 Pearson Education, Inc. Figure 9.15 Shotgun sequencing. Isolate DNA. Fragment DNA with restriction enzymes. Clone DNA in a bacterial artificial chromosome (BAC). Construct a gene library Random sequencingClosure phase Sequence DNA fragments. Edit sequences; fill in gaps. Assemble sequences.

53. © 2016 Pearson Education, Inc. Scientific Applications Bioinformatics: understanding gene function via computer-assisted analysis Proteomics: determining proteins expressed in a cell Reverse genetics: discovering gene function from a genetic sequence

54. © 2016 Pearson Education, Inc. Scientific Applications Southern blotting: DNA probes detect specific DNA in fragments (RFLPs) separated by gel electrophoresis

55. © 2016 Pearson Education, Inc. Figure 9.16 Southern blotting. Restriction enzyme DNA containing the gene of interest is extracted from human cells and cut into fragments by restriction enzymes. Fragments are called restriction fragment length polymorphisms, or RFLPs (pronounced "rif-lips"). The DNA bands are transferred to a nitrocellulose filter by blotting. The solution passes through the gel and filter to the paper towels by capillary action. Labeled probes This produces a nitrocellulose filter with DNA fragments positioned exactly as on the gel. The fragments are separated according to size by gel electrophoresis. Each band contains many copies of a particular DNA fragment. The bands are invisible but can be made visible by staining. Smaller Larger Gene of interest The filter is exposed to a labeled probe for a specific gene. The probe will base-pair (hybridize) with a short sequence present on the gene. The fragment containing the gene of interest is identified by a band on the filter. Gel Human DNA fragments Nitrocellulose filter DNA transferred to filter Nitrocellulose filter Gel Sponge Paper towels Salt solution Gel Sealable plastic bag

56. © 2016 Pearson Education, Inc. Forensic Microbiology DNA fingerprinting is used to identify pathogens PCR microarrays and DNA chips can screen samples for multiple pathogens Differs from medicine because it requires: Proper collection of evidence Establishing a chain of custody

57. © 2016 Pearson Education, Inc. Figure 9.17 DNA fingerprints used to track an infectious disease. E. coli isolates from patients whose infections were not juice related Apple juice isolates E. coli isolates from patients who drank contaminated juice

58. © 2016 Pearson Education, Inc. Nanotechnology Bacteria can make molecule-sized particles Nanospheres used in drug targeting and delivery

59. © 2016 Pearson Education, Inc. Figure 9.18 Bacillus cells growing on selenium form chains of elemental selenium.

60. © 2016 Pearson Education, Inc. Check Your Understanding How are shotgun sequencing, bioinformatics, and proteomics related to genome projects? 9-15, 9-16 What is Southern blotting? 9-17 Why do RFLPs result in a DNA fingerprint? 9-18

61. © 2016 Pearson Education, Inc. Agricultural Applications Ti plasmid: occurs in Agrobacterium tumefaciens Integrates into the plant genome and causes a tumorlike growth Can be used to introduce rDNA into a plant

62. © 2016 Pearson Education, Inc. Figure 9.19 Crown gall disease on a rose plant. Crown gall

63. © 2016 Pearson Education, Inc. Figure 9.20 Using the Ti plasmid as a vector for genetic modification in plants. Agrobacterium tumefaciens bacterium Ti plasmid The plasmid is removed from the bacterium, and the T-DNA is cut by a restriction enzyme. The plasmid is reinserted into a bacterium. Recombinant Ti plasmid The foreign DNA is inserted into the T-DNA of the plasmid. Foreign DNA is cut by the same enzyme. A plant is generated from a cell clone. All of its cells carry the foreign gene and may express it as a new trait. The plant cells are grown in culture. The bacterium is used to insert the T-DNA carrying the foreign gene into the chromosome of a plant cell. Inserted T-DNA carrying foreign gene Restriction cleavage site T-DNA

64. © 2016 Pearson Education, Inc. Agricultural Applications Bt toxin Herbicide resistance Suppression of genes Antisense DNA Nutrition Human proteins

65. © 2016 Pearson Education, Inc. Table 9.3 Some Agriculturally Important Products of rDNA Technology

66. © 2016 Pearson Education, Inc. Check Your Understanding Of what value is the plant pathogen Agrobacterium? 9-19

67. © 2016 Pearson Education, Inc. Safety Issues and Ethics of Using DNA Technology Learning Objective 9-20List the advantages of, and problems associated with the use of genetic modification techniques.

68. © 2016 Pearson Education, Inc. Safety Issues and Ethics of Using DNA Technology Need to avoid accidental release into the environment Genetically modified crops must be safe for consumption and for the environment Who will have access to an individual's genetic information?

69. © 2016 Pearson Education, Inc. Check Your Understanding Identify two advantages and two problems associated with genetically modified organisms. 9-20

70. © 2016 Pearson Education, Inc. Clinical Focus: Norovirus—Who Is Responsible for the Outbreak? Are the outbreaks related? What is the source?

71. © 2016 Pearson Education, Inc. Clinical Focus 9.1a

72. © 2016 Pearson Education, Inc. Clinical Focus: Norovirus—Who Is Responsible for the Outbreak? RT-PCR with a norovirus primer

73. © 2016 Pearson Education, Inc. Clinical Focus 9.1b