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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 DNA ligase joins the strands. Recombinant DNA molecule 1 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 migr ate 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. 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 2 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 lacZ gene Foreign DNA and DNA ligase are added DNA inserted Active lacZ gene produces blue colonies Inactive lacZ gene produces white colonies Transform 3 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Plasmid Library DNA fragments from source DNA DN A inserted into plasmid vector Transformation Each cell contains a single fragment. All cells together are the library. 4 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 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 5 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5. A comparison with the original plate identifies the colony containing the gene. Filter paper Film 1. Colonies of plasmid containing bacteria, each containing a single DNA from the library, are grown on agar. 4. The only sites on the filter that will retain probe DNA will contain DNA complementary to the probe. These represent the sites of colonies containing the gene of interest. 2. A replica of the plate is made by pressing a piece of filter paper against the agar and bacterial colonies. Some cells from each colony adhere to the filter. 3. The filter is washed with a solution to break the cells open and denature the DNA, which sticks to the filter at the site of each colony. The filter is incubated with a radioactively labeled probe that can form hybrids with complementary DNA in the gene of interest. 6 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Test nucleic acids Electrophoresis 1. Electrophoresis is performed, using radioactively labeled markers as a size guide in the first lane. Radioactively labeled markers with specific sizes Electrophoretic gel 2. The gel is covered with a sheet of nitrocellulose and placed in a tray of buffer on top of a sponge. Alkaline chemicals in the buffer denature the DNA into single strands. The buffer wicks its way up through the gel and nitrocellulose into a stack of paper towels placed on top of the 3. DNA in the gel is transferred, or “blotted,” onto the nitrocellulose. Stack of paper towels Nitrocellulose filter Gel Buffer Sponge Nitrocellulose paper now contains nucleic acid “print” Gel Radioactive probe (singlestranded DNA) 4. Nitrocellulose with bound DNA is incubated with radioactively labeled nucleic acids and is then rinsed. Sealed container —AATGG— —TTACC— DNA fragments within bands 5. 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 7 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 8 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Victim Rapist’s semen Suspect’s blood Victim Rapist’s semen Suspect’s blood Courtesy of Lifecodes Corp, Stamford CT 9 10 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. NH2 N O –O P N O N CH2 5´ O O– 4´ 1´ 3´ H 2´ H 11 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Manual Enzymatic DNA Sequencing Automated Enzymatic DNA Sequencing Template Template DNA polymerase DNA polymerase 5´ 3´ T A G C C A T G C 3´ T Primer Reaction for ddG Reaction for ddC Reaction for ddA Reaction for ddT A T C G 5´ A T C G G 5´ A T C G G 5´ A T C 5´ A T C G G 5´ A 5´ A T 5´ A T C G G T T T 5´ A T C G G T 5´ A T C G G T C A A C G A C A A C G T C A T G C A 5´ A 5´ A T 5´ A T C 5´ A T C G 5´ A T C G G 5´ A T C G G T 5´ A T C G G T A 5´ A T C G G T A C 5´ A T C G G T A C G 5´ A T C G G T A C G T T 3´ – Longer segments A G C Primer 5´ G 5´ A 3´ T G C A T G G C T A 5´ T G Laser C Photo detector reads colors A T G G 5´ A T C G G T A C G T 3´ C Shorter segments + T A 5´ a. b. 12 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Adapter DNA fragment Dense primer lawn in flow cell Adapter DNA Adapters Flow cell 1 cm a. b. Bridge amplification with unlabeled dNTPs Free end binds to primer c. Fragments become doublestranded Denature doublestranded molecules Attached Free terminus Attached e. d. Clusters 35 cycles of bridge amplification f. G T C A T N G C O A A –O P N O T O O– 4´ 1´ 3´ G Image capture for each round of synthesis h. b: © 2007, Illumina Inc. All rights reserved N CH2 5´ C First round of synthesis with labeled dNTPs g. NH2 2´ OH A Reversible terminator 13 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´ 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´ 5´ 3´ 5´ 3´ 5´ 3´ 3´ 5´ 3´ 3´ 5´ 3´ 5´ 3´ 5´ 5´ 5´ 3´ 3´ 5´ 14 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Yeast nucleus Transcriptionactivating domain Yeast cell Gal4 protein DNA DNAbinding domain DNAbinding domain Bait vector RNA polymerase Prey vector Inserted DNA Inserted DNA Fusion proteins Prey protein Bait protein Reporter gene 15 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. neo neo Embryonic stem (ES) cells with knocked out gene Gene to be knocked out ES cells containing neo 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. 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. 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. 16 17 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 . 18 19 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 DN A 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. 20 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 Ti plasmid Cultured petunia cells Transformed, regenerated petunia plant 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? (right): © Rob Horsch, Monsanto Company 21 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Daffodil phytoene synthase gene (psy) Bacterial carotene desaturase gene (crtI ) Daffodil lycopene b-cyclase gene (lcy) Genes introduced into rice genome Rice Rice chromosome chromosome psy psy lcy crtI crtI Expression in endosperm Phytoene synthase GGPP -Cyclase Carotene desaturase Phytoene Lycopene in 1897, -Carotene (Provitamin A) 22 23 24