Introduction – Chapter 12

Introduction – Chapter 12 DNA technology– has rapidly revolutionized the field of forensics,– permits the use of gene cloning to produce medicaland industrial products,– allows for the development of genetically modifiedorganisms for agriculture,– permits the investigation of historical questions abouthuman family and evolutionary relationships, and– is invaluable in many areas of biological research. 2012 Pearson Education, Inc.Figure 12.0 1Chapter 12: Big IdeasGene CloningGenetically ModifiedOrganismsDNA ProfilingGenomicsFigure 12.0 21

12.1 Genes can be cloned in recombinant plasmids Biotechnology is the manipulation of organismsor 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 techniquesused to study and manipulate genetic material. 2012 Pearson Education, Inc.12.1 Genes can be cloned in recombinant plasmids Genetic engineering involves manipulatinggenes 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 nucleotidesequences 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 thebacterial chromosome) are often used as vectors. 2012 Pearson Education, Inc.12.1 Genes can be cloned in recombinant plasmids Steps in cloning a gene1. Plasmid DNA is isolated.2. DNA containing the gene of interest is isolated.3. Plasmid DNA is treated with a restriction enzyme thatcuts in one place, opening the circle.4. DNA with the target gene is treated with the sameenzyme and many fragments are produced.5. Plasmid and target DNA are mixed and associate witheach other. 2012 Pearson Education, Inc.2

12.1 Genes can be cloned in recombinant plasmids6. Recombinant DNA molecules are produced when DNAligase joins plasmid and target segments together.7. The recombinant plasmid containing the target gene istaken up by a bacterial cell.8. The bacterial cell reproduces to form a clone, a groupof genetically identical cells descended from a singleancestral cell. 2012 Pearson Education, Inc.Animation: Cloning a GeneRight click on animation / Click play 2012 Pearson Education, Inc.Figure 12.1BE. coli bacteriumA cell with DNAcontaining the geneof interestPlasmidBacterialchromosome12A plasmidis isolated.The cell’s DNAis isolated.Gene ofinterest3DNAThe plasmid is cutwith an enzyme.Examples of gene use4The cell’s DNA is cutwith the same enzyme.Geneof interest56The targeted fragmentand plasmid DNAare combined.DNA ligase is added,which joins the twoDNA molecules.Genes may be insertedinto other organisms.Examples of protein useRecombinantDNAplasmidGeneof interest7The recombinant plasmidis taken up by a bacteriumthrough transformation.Recombinantbacterium8The bacteriumreproduces.9Harvestedproteinsmay beuseddirectly.Cloneof cells3

12.2 Enzymes are used to “cut and paste” DNA Restriction enzymes cut DNA at specificsequences.– Each enzyme binds to DNA at a different restrictionsite.– Many restriction enzymes make staggered cuts thatproduce restriction fragments with single-strandedends called “sticky ends.”– Fragments with complementary sticky ends canassociate with each other, forming recombinant DNA. DNA ligase joins DNA fragments together. 2012 Pearson Education, Inc.Animation: Restriction EnzymesRight click on animation / Click play 2012 Pearson Education, Inc.Figure 12.2 s41DNARestriction enzymerecognition sequenceA restrictionenzyme cutsthe DNA intofragments.2StickyendA DNA fragmentfrom anothersource is added.RestrictionenzymeStickyendGene ofinterest3Two (or more)fragments sticktogether bybase pairing.4DNA ligasepastes thestrands together.5DNA ligaseRecombinantDNA molecule4

12.3 Cloned genes can be stored in genomiclibraries A genomic library is a collection of all of thecloned DNA fragments from a target genome. Genomic libraries can be constructed withdifferent types of vectors:– plasmid library: genomic DNA is carried by plasmids,– bacteriophage (phage) library: genomic DNA isincorporated into bacteriophage DNA,– bacterial artificial chromosome (BAC) library:specialized plasmids that can carry large DNAsequences. 2012 Pearson Education, Inc.Figure 12.3A genome is cut up witha restriction enzymeorRecombinantplasmidRecombinantphage DNABacterialclonePhageclonePlasmid libraryPhage library12.4 Reverse transcriptase can help make genesfor cloning Complementary DNA (cDNA) can be used toclone eukaryotic genes.– In this process, mRNA from a specific cell type is thetemplate.– Reverse transcriptase produces a DNA strand frommRNA.– DNA polymerase produces the second DNA strand. 2012 Pearson Education, Inc.5

12.4 Reverse transcriptase can help make genesfor cloning Advantages of cloning with cDNA include theability to– study genes responsible for specialized characteristicsof 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.Figure 12.4CELL NUCLEUSExon IntronDNA of aeukaryoticgeneExonIntron Exon1 TranscriptionRNAtranscript2 RNA splicing (removesintrons and joins exons)mRNA3 Isolation of mRNA fromTEST TUBEReverse transcriptasecDNA strandbeing synthesizedthe cell and the additionof reverse transcriptase;synthesis of a DNA strand4 Breakdown of RNADirectionof synthesis5 Synthesis of secondDNA strandcDNA of gene(no introns)12.5 Nucleic acid probes identify clones carryingspecific genes Nucleic acid probes bind very selectively tocloned DNA.– Probes can be DNA or RNA sequencescomplementary to a portion of the gene of interest.– A probe binds to a gene of interest by base pairing.– Probes are labeled with a radioactive isotope orfluorescent tag for detection. 2012 Pearson Education, Inc.6

12.5 Nucleic acid probes identify clones carryingspecific genes One way to screen a gene library is as follows:1. Bacterial clones are transferred to filter paper.2. Cells are broken apart and the DNA is separated intosingle strands.3. A probe solution is added and any bacterial coloniescarrying the gene of interest will be tagged on the filterpaper.4. The clone carrying the gene of interest is grown forfurther study. 2012 Pearson Education, Inc.Figure 12.5Radioactivenucleic acid probe(single-stranded DNA)The probe is mixed withsingle-stranded DNAfrom a genomic library.Single-strandedDNABase pairinghighlights thegene of interest.12.6 Recombinant cells and organisms canmass-produce gene products Recombinant cells and organisms constructed byDNA technologies are used to manufacture manyuseful products, chiefly proteins. Bacteria are often the best organisms formanufacturing a protein product because bacteria– have plasmids and phages available for use as genecloning vectors,– can be grown rapidly and cheaply,– can be engineered to produce large amounts of aparticular protein, and– often secrete the proteins directly into their growthmedium. 2012 Pearson Education, Inc.7

12.6 Recombinant cells and organisms canmass-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 theirgenomes,– have plasmids that can be used as gene vectors, and– are often better than bacteria at synthesizing andsecreting eukaryotic proteins. 2012 Pearson Education, Inc.12.6 Recombinant cells and organisms canmass-produce gene products Mammalian cells must be used to produceproteins with chains of sugars. Examples include– human erythropoietin (EPO), which stimulates theproduction of red blood cells,– factor VIII to treat hemophilia, and– tissue plasminogen activator (TPA) used to treat heartattacks and strokes. 2012 Pearson Education, Inc.Table 12.68

12.6 Recombinant cells and organisms canmass-produce gene products Pharmaceutical researchers are currently exploringthe 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 sametraits. 2012 Pearson Education, Inc.Figure 12.6AFigure 12.6B9

12.7 CONNECTION: DNA technology haschanged the pharmaceutical industry andmedicine Products of DNA technology are already in use.– Therapeutic hormones produced by DNA technologyinclude– 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 derivativesof a pathogen that stimulate the immune system. 2012 Pearson Education, Inc.Figure 12.7AFigure 12.7B10

12.8 CONNECTION: Genetically modifiedorganisms are transforming agriculture Genetically modified (GM) organisms containone or more genes introduced by artificial means. Transgenic organisms contain at least one genefrom another species. 2012 Pearson Education, Inc.12.8 CONNECTION: Genetically modifiedorganisms are transforming agriculture The most common vector used to introduce newgenes into plant cells is– a plasmid from the soil bacterium Agrobacteriumtumefaciens and– called the Ti plasmid. 2012 Pearson Education, Inc.Figure 12.8A s3AgrobacteriumtumefaciensDNA containing thegene for a desired traitTiplasmidRestrictionsite1The gene isinserted intothe plasmid.Plant cell2RecombinantTi plasmidThe recombinantplasmid isintroduced intoa plant cell.DNA carryingthe new gene3The plant cellgrows intoa plant.A plantwith thenew trait11

12.8 CONNECTION: Genetically modifiedorganisms 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 improvednutritional or other qualities. 2012 Pearson Education, Inc.Figure 12.8B12.9 Genetically modified organisms raise concernsabout human and environmental health Scientists use safety measures to guard againstproduction and release of new pathogens. Concerns related to GM organisms include thepotential– 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.12

Figure 12.9B12.10 CONNECTION: Gene therapy maysomeday help treat a variety of diseases Gene therapy aims to treat a disease bysupplying a functional allele. One possible procedure is the following:1. Clone the functional allele and insert it in a retroviralvector.2. Use the virus to deliver the gene to an affected celltype from the patient, such as a bone marrow cell.3. Viral DNA and the functional allele will insert into thepatient’s chromosome.4. Return the cells to the patient for growth and division. 2012 Pearson Education, Inc.12.10 CONNECTION: Gene therapy maysomeday 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 disorderstraceable to a single defective gene. 2012 Pearson Education, Inc.13