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Chapter 17 Lecture Outline See separate PowerPoint slides for all figures and tables pre-inserted into PowerPoint without notes and animations. 1 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Biotechnology Chapter 17 2 Recombinant DNA • Restriction endonucleases revolutionized molecular biology • Enzymes that cleave DNA at specific sites – Used by bacteria against viruses • Restriction enzymes significant – Allow a form of physical mapping that was previously impossible – Allow the creation of recombinant DNA molecules (from two different sources) 3 3 Types of Restriction Enzymes •Type I and III cleave with less precision and are not used in manipulating DNA •Type II – Recognize specific DNA sequences – Cleave at specific site within sequence – Can lead to “sticky ends” that can be joined • Blunt ends can also be joined 4 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. EcoRI DNA duplex Restriction sites EcoRI G A A T T C G A A T T C C T T A A G C T T A A G EcoRI Restriction endonuclease cleaves the DNA A A T T EcoRI Restriction endonuclease cleaves the DNA C G G C Sticky ends T A T T A A Sticky ends G C A T A A T T C G DNA from another source cut with the same restriction endonuclease is added. A A T T C G G C A T A T T A T A C G Recombinant DNA molecule DNA ligase joins the strands. 5 DNA ligase – Joins the two fragments forming a stable DNA molecule – Catalyzes formation of a phosphodiester bond between adjacent phosphate and hydroxyl groups of DNA nucleotides – Same enzyme joins Okazaki fragments on lagging strand in replication 6 Gel Electrophoresis • • • • • Separate DNA fragments by size Gel made of agarose or polyacrylamide Submersed in buffer that can carry current Subjected to an electrical field Negatively-charged DNA migrates towards the positive pole • Larger fragments move slower, smaller move faster • DNA is visualized using fluorescent dyes 7 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Restriction Enzyme Digestion Gel Electrophoresis DNA samples are cut with restriction enzymes in three different reactions producing different patterns off ragments. Samples from the restriction enzyme digests are introduced into the gel. Electric current is applied causing fragments to migrate through the gel. Restriction endonuclease 1 cut site Reaction Reaction Reaction 1 2 3 Power source Reaction 1 Short segment Long segment Mixture of DNA fragments of different sizes in solution placed at the top of “lanes” in the gel Lane Restriction endonuclease 2 cut site – Cathode Reaction 2 Gel Medium segment Medium segment Restriction endonuclease 3 + Reaction 3 Anode Buffer Long segment a. Short segment b. 8 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Visualizing Stained Gel Electrophoresis in the Laboratory Gel is stained with a dye to allow the fragments to be visualized. Longer fragments Shorter fragments c. d. d: Courtesy of Biorad Laboratories 9 Transformation • Introduction of foreign DNA from an outside source into a cell • Natural process in many species – E. coli does not • Temperature shifts can induce artificial transformation in E. coli • Transgenic organisms are all or part transformed cells 10 Molecular Cloning • Clone – genetically identical copy • Molecular cloning – isolation of a specific DNA sequence (usually protein-encoding) – Sometimes called gene cloning • The most flexible and common host for cloning is E. coli – Vector – carries DNA in host and can replicate in the host – Each host–vector system has particular uses 11 Vectors • Plasmids – Small, circular chromosomes – Used for cloning small pieces of DNA – 3 components • Origin of replication – allows independent replication • Selectable marker – allows presence of plasmid to be easily identified • Multiple cloning site (MCS) 12 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. A Plasmid Vector Restriction endonuclease Foreign DNA lacZ gene Transform No DNA inserted Medium contains ampicillin and X-gal Ampicillin resistance gene Restriction enzymes cuts within the laxZ gene Foreign DNA and DNA ligase are added DNA inserted Active lacZ gene produces blue colonies Inactive lacZ gene produces white colonies Transform 13 • Artificial chromosomes – Plasmids have limited insert size – Yeast artificial chromosomes (YACs) – Bacterial artificial chromosomes (BACs) – Allow for larger insert for large-scale analysis of genomes 14 Plant genetic engineering Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Gene of interest Plasmid Agrobacterium Plant nucleus 1. Plasmid is removed and cut open with restriction endonuclease. 2. A gene of interest is isolated from the DNA of another organism and inserted into the plasmid. The plasmid is put back into the Agrobacterium. 3. When used to infect plant cells, Agrobacterium duplicates part of the plasmid and transfers the new gene into a chromosome of the plant cell. 4. The plant cell divides, and each daughter cell receives the new gene. These cultured cells can be used to grow a new plant with the introduced gene. 15 Polymerase chain reaction (PCR) • Developed by Kary Mullis – Awarded Nobel Prize • Allows the amplification of a small DNA fragment using primers that flank the region • Each PCR cycle involves three steps: 1. Denaturation (high temperature) 2. Annealing of primers (low temperature) 3. DNA synthesis (intermediate temperature) • Taq polymerase 16 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA segment to be amplified 5´ 3´ 3´ 5´ PCR machine 1. Sample is first heated to denature DNA. DNA is denatured into single strands 5´ 3´ 3´ 5´ 2. DNA is cooled to a lower temperature to allow annealing of primers. 5´ 3´ Primers anneal to DNA 3´ 5´ 17 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. After 20 cycles, a single fragment produces over one million (220) copies! 3. DNA is heated to 72°C, the optimal temperature for Taq DNA polymerase to extend primers 5´ 3´ 3´ 5´ Taq DNA polymerase 3´ 5´ 3´ 5´ 3´ 3´ 5´ 3´ 5´ 5´ 3´ 3´ 5´ 5´ 3´ 3´ 5´ 5´ 3´ 3´ 5´ 3´ 5´ 3´ 5´ 5´ 5´ 3´ Cycle 2: 4 copies Cycle 3: 8 copies 5´ 3´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 3´ 5´ 5´ 3´ 5´ 5´ 3´ 3´ 5´ 3´ 5´ 5´ 3´ 3´ 5´ 18 • Applications of PCR – Allows the investigation of minute samples of DNA – Forensics – drop of blood, cells at base of a hair – Detection of genetic defects in embryos by analyzing a single cell – Analysis of mitochondrial DNA from early human species 19 DNA Libraries • A collection of DNAs in a vector that taken together represent the complex mixture of DNA • Genomic library – representation of the entire genome in a vector – Genome is randomly fragmented – Inserted into a vector – Introduced into host cells – Usually constructed in BACs 20 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Plasmid Library DNA fragments from source DNA DNA inserted into plasmid vector Transformation Each cell contains a single fragment. All cells together are the library. 21 Complementary DNA (cDNA) – DNA copies of mRNA – mRNA isolated • Represents only actively used genes • No introns – Use reverse transcriptase to make cDNA – cDNA used to make library – All genomic libraries from a cell will be the same but cDNA libraries can be different 22 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. exons introns 1 1 2 2 3 3 4 4 Eukaryotic DNA template Transcription 5´ cap 3´ poly- A tail Primary RNA transcript Introns are cut out, and coding regions are spliced together. 3´ poly- A tail 5´ cap Mature RNA transcript Isolation of mRNA Addition of reverse transcriptase 23 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Reverse transcriptase Reverse transcriptase utilizes mRNA to create cDNA. Addition of mRNAdegrading enzymes mRNA–cDNA hybrid Degraded mRNA DNA polymerase Double-stranded cDNA with no introns 24 Analyzing and Creating DNA Differences • Molecular hybridization – Technique used to identify specific DNAs in complex mixtures such as libraries – Also termed annealing – Known single-stranded DNA or RNA is labeled – Used as a probe to identify its complement via specific base-pairing 25 • Molecular hybridization is the most common way of identifying a clone in a DNA library • This process involves three steps: 1. Plating the library • Physically the library is a collection of bacteria or viruses in bacteria 2. Replicating the library 3. Screening the library • Probe is specific sequence of interest 26 • Southern blotting – Sample DNA is digested by restriction enzymes and separated by gel electrophoresis – Double-stranded DNA denatured into singlestrands – Gel “blotted” with filter paper to transfer DNA – Filter is incubated with a labeled probe consisting of purified, single-stranded DNA corresponding to a specific gene 27 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1. DNA in the gel is transferred, or “blotted, ”onto the nitrocellulose. Gel Nitrocellulose paper now contains nucleic acid “print” Radioactive probe (singlestranded DNA) 2. Nitrocellulose with bound DNA is incubated with radio actively labeled nucleic acids and is then rinsed. Sealed container —AATGG— —TTACC— DNA fragments within bands 3. Photographic film is laid over the filter and is exposed only in areas that contain radioactivity (autoradiography). Bands on the film represent DNA in the gel that is complementary to the probe sequence. Film Hybridized nucleic acids Size markers © SSPL/The Image Works 28 • Northern blotting – mRNA is separated by electrophoresis and then blotted onto the filter • Western blotting – Proteins are separated by electrophoresis and then blotted onto the filter – Detection requires an antibody that can bind to one protein 29 DNA Fingerprinting • RFLP analysis – Restriction fragment length polymorphisms – Generated by point mutations or sequence duplications – Restriction enzyme fragments are often not identical in different individuals – Can be detected by Southern blotting 30 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Original Sequence of Restriction Sites (no mutations) Point Mutations Change the Sequence of Restriction Sites Sequence Repetitions Can Occur Between Restriction Sites Larger fragments restriction endonuclease cutting sites + Single base-pair change Smaller fragments – + – + – + Sequence duplication + a. Three different DNA duplexes b. Cut DNA c. Gel electrophoresis of restriction fragments 31 • DNA fingerprinting – Identification technique used to detect differences in the DNA of individuals – Short tandem repeats (STRs) • Typically 2–4 nt long • Not part of coding or regulatory regions – Population is polymorphic for these markers – Using several probes, probability of identity can be calculated or identity can be ruled out – Also used to identify remains 32 STR analysis Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. STRs and DNA fingerprinting 1 Control Ladder 2 3 4 5 6 7 bp DYS19 STR Y chromosome 202 absent 198 194 190 186 178 absent 172 D12S66 STR Chromosome 12 168 160 156 152 absent 148 absent Data provided by Dr. L. Roewer, DNA Laboratory of the Institute for Forensic Medicine of Charité, Berlin 33 “Knockout” mice • Cloned gene interrupted by replacement with a marker gene • Marker gene codes for resistance to the antibiotic neomycin • Interrupted gene is introduced into embryonic stem cells (ES cells) • ES cells injected into embryo early in development 34 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. neo neo Gene to be knocked out neo 1. Using recombinant DN A techniques, the gene encoding resistance to neomycin (neo) is inserted into the gene of interest, disrupting it. The neo gene also confers resistance to the drug G418, which kills mouse cells. This construct is then introduced into ES cells. 2. In some ES cells, the construct will recombine with the chromosomal copy of the gene to be knocked out. This replaces the chromosomal copy with the neo disrupted construct. This is the equivalent to a double crossover event in a genetic cross. 35 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Embryonic stem (ES) cells with knocked out gene ES cells containing neo G418-containing medium Surrogate mouse Blastocyst Dead cells without knocked out gene 3. The ES cells are placed on G418containing medium. The G418 selects cells that have had a replacement event, and now contain a copy of the knocked out gene. 4. The ES cells containing the knocked out gene are injected into a blastocyst stage embryo and then implanted into a female to complete development. 36 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Heterozygous mouse carrying the knockout gene Homozygous mouse for the knockout gene 5. Offspring will contain one chromosome with the gene of interest knocked out. Genetic crosses can then produce mice homozygous for the knocked out gene to assess the phenotype. This can range from lethality to no visible effect depending on the gene. 37 RNA interference • RNAi is another approach to knockdown or knockout expression of a gene – – – – Permanently alters the DNA Targets a specific RNA sequence to degrade Not translated into protein Used to determine gene function in many organisms 38 Medical Applications • Medically important proteins can be produced in bacteria – Human insulin – Vaccines – Problem has been purification of desired proteins from other bacterial proteins 39 Genetically engineering E. coli to make human insulin Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. In Humans Promoter Exon Intron In Bacterial Culture Exon Intron Exon AmpR β-gal Bacterial promoter Bacterial promoter Insulin A chain minus introns and other “extra” sequence Transcription Exon β-gal Insulin B chain minus introns and other “extra” sequence Exon Transform into E.coli Translation 108 amino acids Preproinsulin Culture cells Posttranslational modification Cut Purify β-gal-insulin fusion proteins Disulfide bonds form Cut A chain Purify A and Bchains B chain Cut Achain NH2 Bchain NH2 Disulfide bond Disulfide bond a. b. COOH COOH Active insulin 40 • Vaccines – Subunit vaccines • Genes encoding a part of the protein coat are spliced into a fragment of the vaccinia (cowpox) genome • Injection of harmless recombinant virus leads to immunity – DNA vaccines • Depend on the cellular immune response (not antibodies) 41 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 2. Herpes simplex gene is isolated. 1. DNA is extracted. 3. Vaccinia DNA is extracted and cleaved. Herpes simplex virus Human immune response 6. Antibodies directed against herpes simplex viral coat are made. Gene specifying herpes simplex surface protein Harmless vaccinia (cowpox) virus 4. Fragment containing surface gene combines with cleaved vaccinia DNA. 5. Harmless engineered virus (the vaccine) with surface like herpes simplex is injected into the human body . 42 • Gene therapy – Adding a functional copy of a gene to correct a hereditary disorder – Severe combined immunodeficiency disease (SCID) illustrates both the potential and the problems • On the positive side, 15 children treated successfully are still alive • On the negative side, three other children treated have developed leukemia (due to therapy) 43 44 Agricultural Applications • Ti (tumor-inducing) plasmid – Most used vector for plant genetic engineering – Obtained from Agrobacterium tumefaciens, which normally infects broadleaf plants – Part of the Ti plasmid integrates into the plant DNA and other genes can be attached to it – However, bacterium does not infect cereals such as corn, rice, and wheat 45 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. SCIENTIFIC THINKING Hypothesis: Petunias can acquire tolerance to the herbicide glyphosate by overexpressing EPSP synthase Prediction: Transgenic petunia plants with a chimeric EPSP synthase gene with strong promoter will be glyphosate tolerant Test: 1. Use restriction enzymes and ligase to “paste” the cauliflower mosaic virus promoter (35S) to the EPSP synthase gene and insert the construct in Ti plasmids. 2. Transform Agrobacterium with the recombinant plasmid. 3. Infect petunia cells and regenerate plants. Regenerate uninfected plants as controls. 4. Challenge plants with glyphosate. 35S EPSP synthase Agrobacterium Glyphosate Transformed, regenerated petunia plant Ti plasmid Cultured petunia cells Non-tolerant petunia Tolerant petunia Result: Glyphosate kills control plants, but not transgenic plants. Conclusion: Additional EPSP synthase provides glyphosate tolerance. FurtherExperiments: The transgenic plants are tolerant, but not resistant (note bleaching at shoot tip). How could you determine if additional copies of the gene would increase tolerance? Can you think of any downsides to expressing too much EPSP synthase in petunia? © Rob Horsch, Monsanto Company 46 • Other methods of gene insertion – Gene guns • Uses bombardment with tiny gold particles coated with DNA • Possible for any species • Copy number of inserted genes cannot be controlled – Modification of Agrobacterium system – Use of other bacteria like Agrobacterium 47 • Herbicide resistance – Broadleaf plants have been engineered to be resistant to the herbicide glyphosate – Benefits • Crop resistant to glyphosate would not have to be weeded • Single herbicide instead of many types • Glyphosate breaks down in environment – In the United States, 90% of soy currently grown is GM soy 48 • Bt crops – Insecticidal proteins have been transferred into crop plants to make them pest-resistant – Bt toxin from Bacillus thuringiensis – Use of Bt maize is the second most common GM crop globally • Stacked crops – Both glyphosate-resistant and Bt-producing 49 • Golden rice – Rice that has been genetically modified to produce b-carotene (provitamin A) – Converted in the body to vitamin A – Interesting for 2 reasons • Introduces a new biochemical pathway in tissue of the transgenic plants • Could not have been done by conventional breeding as no rice cultivar known produces these enzymes in endosperm – Available free with no commercial entanglements 50 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Daffodil phytoene synthase gene (psy ) Bacterial carotene desaturase gene (crtI ) Daffodil lycopene β-cyclase gene (lcy ) Genes introduced Into rice genome Rice chromosome psy crtI lcy Expression In endosperm Phytoene synthase GGPP β-Cyclase Carotene desaturase Phytoene Lycopene β-Carotene (Provitamin A) 51 Marker Assisted Breeding (MAB) • • • • • Combines classic plant breeding with molecular biology DNA extracted from leaf tissue of young seedlings Screen for agriculturally important traits Screening uses DNA fingerprinting Seedlings with desired traits raised to maturity 52 • Adoption of genetically modified (GM) crops has been resisted in some areas because of questions – Crop safety for human consumption – Movement of genes into wild relatives • No evidence so far but it is not impossible 53 • Biopharming – Transgenic plants are used to produce pharmaceuticals – 1990 – Human serum albumin produced in genetically engineered tobacco and potato plants – In development • Recombinant subunit vaccines against Norwalk and rabies viruses • Recombinant monoclonal antibodies against tooth decay-causing bacteria 54 • Transgenic animal technology has not been as successful as that in plants • Molecular techniques combined with the ability to clone domestic animals could produce improved animals for economically desirable traits • Main use thus far has been engineering animals to produce pharmaceuticals in milk (also biopharming) 55 Transgenic animals Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Transgenic salmon Wild salmon 6000 Weight (g) 5000 4000 3000 2000 1000 0 0 100 200 300 400 500 600 700 800 900 Days (from first feeding) a. b. b: © Barrett & MacKay Photo 56