Chapter 20Overview: Understanding and Manipulating GenomesSlide 3Slide 4Concept 20.1: DNA cloning permits production of multiple copies of a specific gene or other DNA segmentDNA Cloning and Its Applications: A PreviewLE 20-2Using Restriction Enzymes to Make Recombinant DNALE 20-3Cloning a Eukaryotic Gene in a Bacterial PlasmidProducing Clones of CellsLE 20-4_1LE 20-4_2LE 20-4_3Identifying Clones Carrying a Gene of InterestLE 20-5Storing Cloned Genes in DNA LibrariesLE 20-6Slide 19Cloning and Expressing Eukaryotic GenesBacterial Expression SystemsEukaryotic Cloning and Expression SystemsSlide 23Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR)LE 20-7Concept 20.2: Restriction fragment analysis detects DNA differences that affect restriction sitesGel Electrophoresis and Southern BlottingLE 20-8Slide 29LE 20-9Slide 31LE 20-10Restriction Fragment Length Differences as Genetic MarkersConcept 20.3: Entire genomes can be mapped at the DNA levelGenetic (Linkage) Mapping: Relative Ordering of MarkersLE 20-11Physical Mapping: Ordering DNA FragmentsDNA SequencingLE 20-12Slide 40LE 20-13Concept 20.4: Genome sequences provide clues to important biological questionsIdentifying Protein-Coding Genes in DNA SequencesSlide 44Determining Gene FunctionStudying Expression of Interacting Groups of GenesLE 20-14Comparing Genomes of Different SpeciesFuture Directions in GenomicsConcept 20.5: The practical applications of DNA technology affect our lives in many waysMedical ApplicationsDiagnosis of DiseasesLE 20-15Human Gene TherapyLE 20-16Pharmaceutical ProductsForensic EvidenceLE 20-17Environmental CleanupAgricultural ApplicationsAnimal Husbandry and “Pharm” AnimalsSlide 62Genetic Engineering in PlantsLE 20-19Safety and Ethical Questions Raised by DNA TechnologyCopyright © 2005 Pearson Education, Inc. publishing as Benjamin CummingsPowerPoint Lectures for Biology, Seventh EditionNeil Campbell and Jane ReeceLectures by Chris RomeroChapter 20Chapter 20DNA Technology and GenomicsCopyright © 2005 Pearson Education, Inc. publishing as Benjamin CummingsOverview: Understanding and Manipulating Genomes•Sequencing of the human genome was largely completed by 2003•DNA sequencing has depended on advances in technology, starting with making recombinant DNA•In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule•Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposesCopyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings•DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products•An example of DNA technology is the microarray, a measurement of gene expression of thousands of different genesCopyright © 2005 Pearson Education, Inc. publishing as Benjamin CummingsCopyright © 2005 Pearson Education, Inc. publishing as Benjamin CummingsConcept 20.1: DNA cloning permits production of multiple copies of a specific gene or other DNA segment•To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called gene cloningCopyright © 2005 Pearson Education, Inc. publishing as Benjamin CummingsDNA Cloning and Its Applications: A Preview•Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids•Cloned genes are useful for making copies of a particular gene and producing a gene productLE 20-2LE 20-2BacteriumBacterialchromosomePlasmidGene inserted intoplasmidCell containing geneof interestGene ofinterestDNA ofchromosomeRecombinantDNA (plasmid)Plasmid put intobacterial cellRecombinantbacteriumHost cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interestProtein expressedby gene of interestProtein harvestedGene ofinterestCopies of geneBasicresearchon geneBasicresearchon proteinBasic research andvarious applicationsGene for pestresistance insertedinto plantsGene used to alterbacteria for cleaningup toxic wasteProtein dissolvesblood clots in heartattack therapyHuman growth hor-mone treats stuntedgrowthCopyright © 2005 Pearson Education, Inc. publishing as Benjamin CummingsUsing Restriction Enzymes to Make Recombinant DNA•Bacterial restriction enzymes cut DNA molecules at DNA sequences called restriction sites•A restriction enzyme usually makes many cuts, yielding restriction fragments•The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary “sticky ends” of other fragments•DNA ligase is an enzyme that seals the bonds between restriction fragmentsLE 20-3LE 20-3Restriction siteDNA5335Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow.One possible combinationDNA fragment from anothersource is added. Base pairingof sticky ends producesvarious combinations.Fragment from differentDNA molecule cut by thesame restriction enzymeDNA ligaseseals the strands.Recombinant DNA moleculeSticky endCopyright © 2005 Pearson Education, Inc. publishing as Benjamin CummingsCloning 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 thereCopyright © 2005 Pearson Education, Inc. publishing as Benjamin CummingsProducing Clones of Cells•Cloning a human gene in a bacterial plasmid can be divided into six steps:1. Vector and gene-source DNA are isolated2. DNA is inserted into the vector3. Human DNA fragments are mixed with cut plasmids, and base-pairing takes place4. Recombinant plasmids are mixed with bacteria5. The bacteria are plated and incubated6. Cell clones with the right gene are identifiedLE 20-4_1LE 20-4_1Isolate plasmid DNAand human DNA.Cut both DNA samples withthe same restriction enzyme.Mix the DNAs; they join by base pairing.The products are recombinant plasmidsand many nonrecombinant plasmids.Bacterial celllacZ gene(lactosebreakdown)HumancellRestrictionsiteampR gene(ampicillinresistance)BacterialplasmidGene ofinterestStickyendsHuman DNAfragmentsRecombinant DNA plasmidsLE 20-4_2LE 20-4_2Isolate plasmid DNAand human DNA.Cut both DNA samples withthe same restriction enzyme.Mix the DNAs; they join by base pairing.The products are recombinant plasmidsand many