1. © 2012 Pearson Education, Inc. Lecture by Edward J. Zalisko PowerPoint Lectures for Campbell Biology: Concepts & Connections, Seventh Edition Reece, Taylor, Simon, and Dickey Chapter 12 DNA Technology and Genomics

2.  DNA technology –has rapidly revolutionized the field of forensics, –permits the use of gene cloning to produce medical and industrial products, –allows for the development of genetically modified organisms for agriculture, –permits the investigation of historical questions about human family and evolutionary relationships, and –is invaluable in many areas of biological research. Introduction © 2012 Pearson Education, Inc.

3. Figure 12.0_1 Chapter 12: Big Ideas Gene Cloning DNA Profiling Genetically Modified Organisms Genomics

4. GENE CLONING © 2012 Pearson Education, Inc.

5. 12.1 Genes can be cloned in recombinant plasmids  Biotechnology is the manipulation of organisms or their components to make useful products.  For thousands of years, humans have –used microbes to make wine and cheese and –selectively bred stock, dogs, and other animals.  DNA technology is the set of modern techniques used to study and manipulate genetic material. © 2012 Pearson Education, Inc.

6. Figure 12.1A

7. 12.1 Genes can be cloned in recombinant plasmids  Genetic engineering involves manipulating genes for practical purposes. –Gene cloning leads to the production of multiple, identical copies of a gene-carrying piece of DNA. –Recombinant DNA is formed by joining nucleotide sequences from two different sources. –One source contains the gene that will be cloned. –Another source is a gene carrier, called a vector. –Plasmids (small, circular DNA molecules independent of the bacterial chromosome) are often used as vectors. © 2012 Pearson Education, Inc.

8.  Steps in cloning a gene 1.Plasmid DNA is isolated. 2.DNA containing the gene of interest is isolated. 3.Plasmid DNA is treated with a restriction enzyme that cuts in one place, opening the circle. 4.DNA with the target gene is treated with the same enzyme and many fragments are produced. 5.Plasmid and target DNA are mixed and associate with each other. 12.1 Genes can be cloned in recombinant plasmids © 2012 Pearson Education, Inc.

9. 6.Recombinant DNA molecules are produced when DNA ligase joins plasmid and target segments together. 7.The recombinant plasmid containing the target gene is taken up by a bacterial cell. 8.The bacterial cell reproduces to form a clone, a group of genetically identical cells descended from a single ancestral cell. 12.1 Genes can be cloned in recombinant plasmids © 2012 Pearson Education, Inc. Animation: Cloning a Gene

10. Figure 12.1B E. coli bacterium Bacterial chromosome A plasmid is isolated. Gene of interest The plasmid is cut with an enzyme. Plasmid The cell’s DNA is isolated. The cell’s DNA is cut with the same enzyme. DNA Examples of gene use A cell with DNA containing the gene of interest Gene of interest The targeted fragment and plasmid DNA are combined. DNA ligase is added, which joins the two DNA molecules. Gene of interest Genes may be inserted into other organisms. The recombinant plasmid is taken up by a bacterium through transformation. Examples of protein use Harvested proteins may be used directly. The bacterium reproduces. Clone of cells Recombinant bacterium Recombinant DNA plasmid 1 3 5 4 2 6 7 9 8

11. Figure 12.1B_s1 E. coli bacterium Bacterial chromosome A plasmid is isolated. Gene of interest Plasmid The cell’s DNA is isolated. DNA A cell with DNA containing the gene of interest 1 2

12. Figure 12.1B_s2 E. coli bacterium Bacterial chromosome A plasmid is isolated. Gene of interest Plasmid The cell’s DNA is isolated. DNA A cell with DNA containing the gene of interest 1 3 2 4 The plasmid is cut with an enzyme. The cell’s DNA is cut with the same enzyme. Gene of interest

13. Figure 12.1B_s3 E. coli bacterium Bacterial chromosome A plasmid is isolated. Gene of interest Plasmid The cell’s DNA is isolated. DNA A cell with DNA containing the gene of interest 1 3 2 4 5 The plasmid is cut with an enzyme. The cell’s DNA is cut with the same enzyme. Gene of interest The targeted fragment and plasmid DNA are combined.

14. Figure 12.1B_s4 E. coli bacterium Bacterial chromosome A plasmid is isolated. Gene of interest Plasmid The cell’s DNA is isolated. DNA A cell with DNA containing the gene of interest 1 3 2 4 5 6 The plasmid is cut with an enzyme. The cell’s DNA is cut with the same enzyme. Gene of interest The targeted fragment and plasmid DNA are combined. DNA ligase is added, which joins the two DNA molecules. Gene of interest Recombinant DNA plasmid

15. Figure 12.1B_s5 Gene of interest The recombinant plasmid is taken up by a bacterium through transformation. Recombinant bacterium Recombinant DNA plasmid 7

16. Figure 12.1B_s6 Gene of interest The recombinant plasmid is taken up by a bacterium through transformation. The bacterium reproduces. Clone of cells Recombinant bacterium Recombinant DNA plasmid 7 8

17. Figure 12.1B_s7 Gene of interest The recombinant plasmid is taken up by a bacterium through transformation. Harvested proteins may be used directly. The bacterium reproduces. Clone of cells Recombinant bacterium Recombinant DNA plasmid Genes may be inserted into other organisms. 9 7 8

18. 12.2 Enzymes are used to “cut and paste” DNA  Restriction enzymes cut DNA at specific sequences. –Each enzyme binds to DNA at a different restriction site. –Many restriction enzymes make staggered cuts that produce restriction fragments with single-stranded ends called “sticky ends.” –Fragments with complementary sticky ends can associate with each other, forming recombinant DNA.  DNA ligase joins DNA fragments together. © 2012 Pearson Education, Inc. Animation: Restriction Enzymes

19. Figure 12.2_s1 A restriction enzyme cuts the DNA into fragments. Restriction enzyme recognition sequence Restriction enzyme Sticky end Sticky end DNA 1 2

20. Figure 12.2_s2 A restriction enzyme cuts the DNA into fragments. Restriction enzyme recognition sequence Restriction enzyme Gene of interest A DNA fragment from another source is added. Sticky end Sticky end DNA 1 2 3

21. Figure 12.2_s3 A restriction enzyme cuts the DNA into fragments. Restriction enzyme recognition sequence Restriction enzyme Gene of interest A DNA fragment from another source is added. Two (or more) fragments stick together by base pairing. Sticky end Sticky end DNA 1 2 4 3

22. Figure 12.2_s4 A restriction enzyme cuts the DNA into fragments. Restriction enzyme recognition sequence Restriction enzyme Gene of interest A DNA fragment from another source is added. Two (or more) fragments stick together by base pairing. Sticky end Sticky end DNA ligase pastes the strands together. Recombinant DNA molecule DNA 1 2 4 5 3

23. 12.3 Cloned genes can be stored in genomic libraries  A genomic library is a collection of all of the cloned DNA fragments from a target genome.  Genomic libraries can be constructed with different types of vectors: –plasmid library: genomic DNA is carried by plasmids, –bacteriophage (phage) library: genomic DNA is incorporated into bacteriophage DNA, –bacterial artificial chromosome (BAC) library: specialized plasmids that can carry large DNA sequences. © 2012 Pearson Education, Inc.

24. Figure 12.3 A genome is cut up with a restriction enzyme Recombinant phage DNA Recombinant plasmid Bacterial clone Phage clone or Plasmid libraryPhage library

25. 12.4 Reverse transcriptase can help make genes for cloning  Complementary DNA (cDNA) can be used to clone eukaryotic genes. –In this process, mRNA from a specific cell type is the template. –Reverse transcriptase produces a DNA strand from mRNA. –DNA polymerase produces the second DNA strand. © 2012 Pearson Education, Inc.

26. 12.4 Reverse transcriptase can help make genes for cloning  Advantages of cloning with cDNA include the ability to –study genes responsible for specialized characteristics of a particular cell type and –obtain gene sequences –that are smaller in size, –easier to handle, and –do not have introns. © 2012 Pearson Education, Inc.

27. Figure 12.4 C ELL NUCLEUS DNA of a eukaryotic gene RNA transcript mRNA T EST T UBE Reverse transcriptase cDNA strand being synthesized Direction of synthesis Breakdown of RNA Synthesis of second DNA strand Isolation of mRNA from the cell and the addition of reverse transcriptase; synthesis of a DNA strand cDNA of gene (no introns) Exon Intron Transcription RNA splicing (removes introns and joins exons) 1 2 3 4 5

28. Figure 12.4_1 C ELL NUCLEUS DNA of a eukaryotic gene RNA transcript mRNA ExonIntron Transcription RNA splicing (removes introns and joins exons) 1 2 ExonIntronExon

29. Figure 12.4_2 T EST TUBE Reverse transcriptase cDNA strand being synthesized Direction of synthesis Breakdown of RNA Synthesis of second DNA strand Isolation of mRNA from the cell and the addition of reverse transcriptase; synthesis of a DNA strand cDNA of gene (no introns) 3 4 5

30. GENETICALLY MODIFIED ORGANISMS © 2012 Pearson Education, Inc.

31. 12.6 Recombinant cells and organisms can mass-produce gene products  Recombinant cells and organisms constructed by DNA technologies are used to manufacture many useful products, chiefly proteins.  Bacteria are often the best organisms for manufacturing a protein product because bacteria –have plasmids and phages available for use as gene- cloning vectors, –can be grown rapidly and cheaply, –can be engineered to produce large amounts of a particular protein, and –often secrete the proteins directly into their growth medium. © 2012 Pearson Education, Inc.

32. 12.6 Recombinant cells and organisms can mass-produce gene products  Yeast cells –are eukaryotes, –have long been used to make bread and beer, –can take up foreign DNA and integrate it into their genomes, –have plasmids that can be used as gene vectors, and –are often better than bacteria at synthesizing and secreting eukaryotic proteins. © 2012 Pearson Education, Inc.

33. 12.6 Recombinant cells and organisms can mass-produce gene products  Mammalian cells must be used to produce proteins with chains of sugars. Examples include –human erythropoietin (EPO), which stimulates the production of red blood cells, –factor VIII to treat hemophilia, and –tissue plasminogen activator (TPA) used to treat heart attacks and strokes. © 2012 Pearson Education, Inc.

34. Table 12.6

35. 12.6 Recombinant cells and organisms can mass-produce gene products  Pharmaceutical researchers are currently exploring the mass production of gene products by –whole animals or –plants.  Recombinant animals –are difficult and costly to produce and –must be cloned to produce more animals with the same traits. © 2012 Pearson Education, Inc.

36. Figure 12.6A

37. 12.7 CONNECTION: DNA technology has changed the pharmaceutical industry and medicine  Products of DNA technology are already in use. –Therapeutic hormones produced by DNA technology include –insulin to treat diabetes and –human growth hormone to treat dwarfism. –DNA technology is used to –test for inherited diseases, –detect infectious agents such as HIV, and –produce vaccines, harmless variants (mutants) or derivatives of a pathogen that stimulate the immune system. © 2012 Pearson Education, Inc.

38. Figure 12.7A

39. 12.8 CONNECTION: Genetically modified organisms are transforming agriculture  Genetically modified (GM) organisms contain one or more genes introduced by artificial means.  Transgenic organisms contain at least one gene from another species. © 2012 Pearson Education, Inc.

40. 12.8 CONNECTION: Genetically modified organisms are transforming agriculture  The most common vector used to introduce new genes into plant cells is –a plasmid from the soil bacterium Agrobacterium tumefaciens and –called the Ti plasmid. © 2012 Pearson Education, Inc.

41. Figure 12.8A_s1 Restriction site The gene is inserted into the plasmid. Recombinant Ti plasmid DNA containing the gene for a desired trait 1 Ti plasmid Agrobacterium tumefaciens

42. Figure 12.8A_s2 Restriction site The gene is inserted into the plasmid. The recombinant plasmid is introduced into a plant cell. DNA carrying the new gene Recombinant Ti plasmid Plant cell DNA containing the gene for a desired trait 2 1 Ti plasmid Agrobacterium tumefaciens

43. Figure 12.8A_s3 Restriction site The gene is inserted into the plasmid. The recombinant plasmid is introduced into a plant cell. The plant cell grows into a plant. DNA carrying the new gene A plant with the new trait Recombinant Ti plasmid Plant cell DNA containing the gene for a desired trait 3 2 1 Ti plasmid Agrobacterium tumefaciens

44. 12.8 CONNECTION: Genetically modified organisms are transforming agriculture  GM plants are being produced that –are more resistant to herbicides and pests and –provide nutrients that help address malnutrition.  GM animals are being produced with improved nutritional or other qualities. © 2012 Pearson Education, Inc.

45. Figure 12.8B

46. 12.9 Genetically modified organisms raise concerns about human and environmental health  Scientists use safety measures to guard against production and release of new pathogens.  Concerns related to GM organisms include the potential –introduction of allergens into the food supply and –spread of genes to closely related organisms.  Regulatory agencies are trying to address the –safety of GM products, –labeling of GM produced foods, and –safe use of biotechnology. © 2012 Pearson Education, Inc.

47. 12.10 CONNECTION: Gene therapy may someday help treat a variety of diseases  Gene therapy aims to treat a disease by supplying a functional allele.  One possible procedure is the following: 1.Clone the functional allele and insert it in a retroviral vector. 2.Use the virus to deliver the gene to an affected cell type from the patient, such as a bone marrow cell. 3.Viral DNA and the functional allele will insert into the patient’s chromosome. 4.Return the cells to the patient for growth and division. © 2012 Pearson Education, Inc.

48. 12.10 CONNECTION: Gene therapy may someday help treat a variety of diseases  Gene therapy is an –alteration of an afflicted individual’s genes and –attempt to treat disease.  Gene therapy may be best used to treat disorders traceable to a single defective gene. © 2012 Pearson Education, Inc.

49. Figure 12.10 An RNA version of a normal human gene is inserted into a retrovirus. RNA genome of virus Retrovirus Bone marrow cells are infected with the virus. Viral DNA carrying the human gene inserts into the cell’s chromosome. Bone marrow cell from the patient Bone marrow The engineered cells are injected into the patient. Cloned gene (normal allele) 1 2 3 4

50. Figure 12.10_1 An RNA version of a normal human gene is inserted into a retrovirus. RNA genome of virus Retrovirus Cloned gene (normal allele) 1

51. Figure 12.10_2 Bone marrow cells are infected with the virus. Viral DNA carrying the human gene inserts into the cell’s chromosome. Bone marrow cell from the patient Bone marrow The engineered cells are injected into the patient. 2 3 4

52. 12.10 CONNECTION: Gene therapy may someday help treat a variety of diseases  The first successful human gene therapy trial in 2000 –tried to treat ten children with SCID (severe combined immune deficiency), –helped nine of these patients, but –caused leukemia in three of the patients, and –resulted in one death. © 2012 Pearson Education, Inc.

53. 12.10 CONNECTION: Gene therapy may someday help treat a variety of diseases  The use of gene therapy raises many questions. –How can we build in gene control mechanisms that make appropriate amounts of the product at the right time and place? –How can gene insertion be performed without harming other cell functions? –Will gene therapy lead to efforts to control the genetic makeup of human populations? –Should we try to eliminate genetic defects in our children and descendants when genetic variety is a necessary ingredient for the survival of a species? © 2012 Pearson Education, Inc.

54. DNA PROFILING © 2012 Pearson Education, Inc.

55. 12.11 The analysis of genetic markers can produce a DNA profile  DNA profiling is the analysis of DNA fragments to determine whether they come from the same individual. DNA profiling –compares genetic markers from noncoding regions that show variation between individuals and –involves amplifying (copying) of markers for analysis. © 2012 Pearson Education, Inc.

56. Figure 12.11 DNA is isolated. 1 2 3 The DNA of selected markers is amplified. The amplified DNA is compared. Crime sceneSuspect 1Suspect 2

57. 12.12 The PCR method is used to amplify DNA sequences  Polymerase chain reaction (PCR) is a method of amplifying a specific segment of a DNA molecule.  PCR relies upon a pair of primers that are –short, –chemically synthesized, single-stranded DNA molecules, and –complementary to sequences at each end of the target sequence.  PCR –is a three-step cycle that –doubles the amount of DNA in each turn of the cycle. © 2012 Pearson Education, Inc.

58. Figure 12.12 Cycle 1 yields two molecules Cycle 2 yields four molecules DNA polymerase adds nucleotides. Primers bond with ends of target sequences. Heat separates DNA strands. Genomic DNA Target sequence PrimerNew DNA Cycle 3 yields eight molecules 3 5 5 3 355 5 5 5 5 33 33 53 5 3 3 5 5 321

59. Figure 12.12_1 Cycle 1 yields two molecules DNA polymerase adds nucleotides. Primers bond with ends of target sequences. Heat separates DNA strands. Genomic DNA Target sequence Primer New DNA 5 3 3 5 5 335 5 535 5 3 533 5 53 53 321

60. Figure 12.12_2 Cycle 2 yields four molecules Cycle 3 yields eight molecules

61.  The advantages of PCR include –the ability to amplify DNA from a small sample, –obtaining results rapidly, and –a reaction that is highly sensitive, copying only the target sequence. 12.12 The PCR method is used to amplify DNA sequences © 2012 Pearson Education, Inc.

62. 12.13 Gel electrophoresis sorts DNA molecules by size  Gel electrophoresis can be used to separate DNA molecules based on size as follows: 1.A DNA sample is placed at one end of a porous gel. 2.Current is applied and DNA molecules move from the negative electrode toward the positive electrode. 3.Shorter DNA fragments move through the gel matrix more quickly and travel farther through the gel. 4.DNA fragments appear as bands, visualized through staining or detecting radioactivity or fluorescence. 5.Each band is a collection of DNA molecules of the same length. © 2012 Pearson Education, Inc. Video: Biotechnology Lab

63. Figure 12.13 A mixture of DNA fragments of different sizes Power source Gel Completed gel Longer (slower) molecules Shorter (faster) molecules

64. Figure 12.13_1 A mixture of DNA fragments of different sizes Power source Gel Completed gel Longer (slower) molecules Shorter (faster) molecules

65. Figure 12.13_2

66. 12.14 STR analysis is commonly used for DNA profiling  Repetitive DNA consists of nucleotide sequences that are present in multiple copies in the genome.  Short tandem repeats (STRs) are short nucleotide sequences that are repeated in tandem, –composed of different numbers of repeating units in individuals and –used in DNA profiling.  STR analysis –compares the lengths of STR sequences at specific sites in the genome and –typically analyzes 13 different STR sites. © 2012 Pearson Education, Inc.

67. Figure 12.14A Crime scene DNA Suspect’s DNA STR site 1STR site 2 The number of short tandem repeats match The number of short tandem repeats do not match

68. Figure 12.14B Crime scene DNA Suspect’s DNA Longer STR fragments Shorter STR fragments

69. 12.15 CONNECTION: DNA profiling has provided evidence in many forensic investigations  DNA profiling is used to –determine guilt or innocence in a crime, –settle questions of paternity, –identify victims of accidents, and –probe the origin of nonhuman materials. © 2012 Pearson Education, Inc.

70. 12.16 RFLPs can be used to detect differences in DNA sequences  A single nucleotide polymorphism (SNP) is a variation at a single base pair within a genome.  Restriction fragment length polymorphism (RFLP) is a change in the length of restriction fragments due to a SNP that alters a restriction site.  RFLP analysis involves –producing DNA fragments by restriction enzymes and –sorting these fragments by gel electrophoresis. © 2012 Pearson Education, Inc.

71. Figure 12.16 Restriction enzymes added DNA sample 1DNA sample 2 Cut w x yy z Sample 1 Sample 2 z x w yy Longer fragments Shorter fragments

72. Figure 12.16_1 Restriction enzymes added DNA sample 1DNA sample 2 w x y y z Cut

73. Figure 12.16_2 Sample 1 z x w y y Longer fragments Shorter fragments Sample 2

74. GENOMICS © 2012 Pearson Education, Inc.

75. 12.17 Genomics is the scientific study of whole genomes  Genomics is the study of an organism’s complete set of genes and their interactions. –Initial studies focused on prokaryotic genomes. –Many eukaryotic genomes have since been investigated. © 2012 Pearson Education, Inc.

76. Table 12.17

77. 12.17 Genomics is the scientific study of whole genomes  Genomics allows another way to examine evolutionary relationships. –Genomic studies showed a 96% similarity in DNA sequences between chimpanzees and humans. –Functions of human disease-causing genes have been determined by comparing human genes to similar genes in yeast. © 2012 Pearson Education, Inc.

78. 12.18 CONNECTION: The Human Genome Project revealed that most of the human genome does not consist of genes  The goals of the Human Genome Project (HGP) included –determining the nucleotide sequence of all DNA in the human genome and –identifying the location and sequence of every human gene. © 2012 Pearson Education, Inc.

79. 12.18 CONNECTION: The Human Genome Project revealed that most of the human genome does not consist of genes  Results of the Human Genome Project indicate that –humans have about 20,000 genes in 3.2 billion nucleotide pairs, –only 1.5% of the DNA codes for proteins, tRNAs, or rRNAs, and –the remaining 98.5% of the DNA is noncoding DNA including –telomeres, stretches of noncoding DNA at the ends of chromosomes, and –transposable elements, DNA segments that can move or be copied from one location to another within or between chromosomes. © 2012 Pearson Education, Inc.

80. Figure 12.18 Exons (regions of genes coding for protein or giving rise to rRNA or tRNA) (1.5%) Repetitive DNA that includes transposable elements and related sequences (44%) Introns and regulatory sequences (24%) Unique noncoding DNA (15%) Repetitive DNA unrelated to transposable elements (15%)

81. 12.20 Proteomics is the scientific study of the full set of proteins encoded by a genome  Proteomics –is the study of the full protein sets encoded by genomes and –investigates protein functions and interactions.  The human proteome includes about 100,000 proteins.  Genomics and proteomics are helping biologists study life from an increasingly holistic approach. © 2012 Pearson Education, Inc.

82. 1.Explain how plasmids are used in gene cloning. 2.Explain how restriction enzymes are used to “cut and paste” DNA into plasmids. 3.Explain how plasmids, phages, and BACs are used to construct genomic libraries. 4.Explain how a cDNA library is constructed and how it is different from genomic libraries constructed using plasmids or phages. 5.Explain how a nucleic acid probe can be used to identify a specific gene. You should now be able to © 2012 Pearson Education, Inc.

83. 6.Explain how different organisms are used to mass- produce proteins of human interest. 7.Explain how DNA technology has helped to produce insulin, growth hormone, and vaccines. 8.Explain how genetically modified (GM) organisms are transforming agriculture. 9.Describe the risks posed by the creation and culturing of GM organisms and the safeguards that have been developed to minimize these risks. You should now be able to © 2012 Pearson Education, Inc.

84. 10.Describe the benefits and risks of gene therapy in humans. Discuss the ethical issues that these techniques present. 11.Describe the basic steps of DNA profiling. 12.Explain how PCR is used to amplify DNA sequences. 13.Explain how gel electrophoresis is used to sort DNA and proteins. 14.Explain how short tandem repeats are used in DNA profiling. You should now be able to © 2012 Pearson Education, Inc.

85. 15.Describe the diverse applications of DNA profiling. 16.Explain how restriction fragment analysis is used to detect differences in DNA sequences. 17.Explain why it is important to sequence the genomes of humans and other organisms. 18.Describe the structure and possible functions of the noncoding sections of the human genome. 19.Explain how the human genome was mapped. You should now be able to © 2012 Pearson Education, Inc.

86. 21.Compare the fields of genomics and proteomics. 22.Describe the significance of genomics to the study of evolutionary relationships and our understanding of the special characteristics of humans. You should now be able to © 2012 Pearson Education, Inc.

87. Figure 12.UN01 Cut Bacterium Recombinant DNA plasmids Cut DNA fragments Recombinant bacteria Bacterial clone Genomic library Plasmids

88. Figure 12.UN02 A mixture of DNA fragments A “band” is a collection of DNA fragments of one particular length Longer fragments move slower Shorter fragments move faster DNA is attracted to  pole due to PO 4  groups Power source

89. Figure 12.UN03 DNA amplified via DNA sample treated with Bacterial plasmids (a) (b) (c) DNA fragments sorted by size via Recombinant plasmids are inserted into bacteria are copied via (e) (d) Add Particular DNA sequence highlighted

90. Figure 12.UN03_1 DNA amplified via DNA sample treated with Bacterial plasmids (a) (b) treated with

91. Figure 12.UN03_2 (c) DNA fragments sorted by size via Recombinant plasmids are inserted into bacteria are copied via (e) (d) Add Particular DNA sequence highlighted