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Lecture 19, Chapter 11 Analysis of transgenic plants part II

Discussion questions. 1. What are the established methods to determine if a plant is transgenic and whether the transgene(s) is expressed?2. In a Southern or northern blot, through what type of chemical bond does the complementary probe bind to nucleic acid?3. Nucleic acids and proteins are separated according to size in agarose and sodium dodecyl sulfate

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Lecture 19, Chapter 11 Analysis of transgenic plants part II

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    1. Lecture 19, Chapter 11 Analysis of transgenic plants part II Neal Stewart

    2. Discussion questions 1. What are the established methods to determine if a plant is transgenic and whether the transgene(s) is expressed? 2. In a Southern or northern blot, through what type of chemical bond does the complementary probe bind to nucleic acid? 3. Nucleic acids and proteins are separated according to size in agarose and sodium dodecyl sulfate–polyacrylamide gel electrophoreisis (SDS-PAGE) gels, respectively. Why do both types of macromolecules migrate toward the anode in an electrical current? 4. What is gene expression, and how can you measure it? 5. Explain why phenotypic data provide evidence of transformation but not proof of a transformation event. 6. What factors are most important when designing a Southern blot experiment to test for transgenic status?

    3. Restriction digest and gel electrophoresis

    4. Southern blot—DNA transfer to nylon

    8. Northern blot analysis Gives relative amount of gene expression-at the transcript level. Isolate mRNA be a lot and of good quality (not degraded) Separate transcripts on a gel Transfer to nylon filter Probe filter with DNA of interest (transgene) http://www.youtube.com/watch?v=KfHZFyADnNg

    9. Northern blot example

    10. Western blot Also to measure gene expression—at the protein level. Extract proteins Separate proteins on a vertical gel Transfer to a membrane using an electrotransfer system Probe with antibodies. Stain for antibodies

    11. Western blots and ELISAs often use amplification of signal via antibodies

    12. Western blot example

    13. For all blots (and all assays for that matter) Use appropriate controls, such as a non-transgenic plant (negative) and a positive control typically plasmid for Southerns and specific for westerns. Use an appropriate standard or a range of standards. Set up the experiment intelligently

    14. ELISA—Enzyme-linked immunosorbant assay

    15. Real-life example Plant gene as a kanamycin resistance selectable marker An ABC (ATP-binding cassette) transporter from Arabidopsis Used to produce transgenic tobacco Compared against nptII gene Both regulated by 35S promoters

    16. How ABC WBC19 might work

    17. Are the plants transgenic for the ABC transporter?

    18. Segregation analysis of event 30 b. Northern blot analysis c. Root growth (trait)

    19. Another example– producing GFP transgenic horseweed Horseweed is a plant that has never been transformed Glyphosate-resistant biotypes are a growing problem for farmers Transformation procedure needed to enable functional genomics GFP used as a reporter gene

    21. GFP plasmid map

    22. GFP plasmid map (simplified)

    23. Southern blot of 1 transgenic event

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