Molecular genetics of gene expression - UT K：基因表达的分子遗传学UT K.pptVIP

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Molecular genetics of gene expression - UT K：基因表达的分子遗传学UT K.ppt

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Molecular genetics of gene expression - UT K：基因表达的分子遗传学UT K

Lecture 19, Chapter 11Analysis of transgenic plantspart II Neal Stewart 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) 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? Restriction digest and gel electrophoresis Southern blot—DNA transfer to nylon 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) /watch?v=KfHZFyADnNg Northern blot example 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 Western blots and ELISAs often use amplification of signal via antibodies Western blot example 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 ELISA—Enzyme-linked immunosorbant assay Real-life example Plant gene as a kanamycin resistance selectable marker An ABC (ATP-binding cassette) transporter from Arabidopsis Use