Lecture12-Functional Genomics Part II

Lecture12-Functional Genomics Part II - Lecture 12 -...

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1 Lecture 12 - Functional Genomics Thursday Oct 27, 2011 Objective :   Know the types of experimental strategies that can be applied  systematically to understand gene function and know when to apply  them. Hartwell, pp. 352-357 (proteome analysis/protein interactions)
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2 Experimental strategies in functional genomics Basic approaches that describe/identify: Gene expression patterns Protein expression patterns  Protein-protein interactions  Mutant phenotypes
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A. mRNA expression pattern (gene-by-gene) Northern blot  qRT-PCR (quantitative Reverse Transcription-PCR) RNA in situ hybridization Transcriptional reporter genes B. Analyses of transcriptome (high throughput)   DNA microarrays Oligonucleotide arrays “RNA-Seq” - Next-generation sequencing Analysis of gene expression C. Protein expression pattern Western blot Immunohistochemistry Translational reporter gene  Mass spectrometry 3
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Detecting proteins: Western blot 4
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Detecting proteins: Western blot http://www.virology.ws/2010/07/07/virology-toolbox-the-western-blot/ 5
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6 Detecting proteins with antibodies Fluorogenic substrate fluorescence Example
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Immunolocalization example 7 Image from F. Ditengou and K. Palme, Freiburg Cells in an Arabidopsis root You can detect more than one protein in the same sample (here, Green and Red)
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Promoter::gene X -GFP chimeric protein Gene fusion: Reporter shows protein expression patterns X GFP GFP X 8 The GFP reporter shows when and where protein  “X” is located *Is this pattern the same or different as the promoter fusion?
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9 Express a fusion of your protein with an epitope tag  so that you can use  commercial antibodies to the tag Promoter::gene X -epitope tag X FLAG FLAG X Anti-FLAG  antibody Commercially  available
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Proteome  analysis by mass-spectrometry (MS)  See Hartwell pp. 352-354 10 Mass spectrometers can analyze the masses of different peptides in a mixture  of peptides, leading to amino acid sequence data.   If the genome sequence is known, then the mass spectrometry data can be  matched to the predicted peptides (each of unique mass) encoded by the  genome. Can be used to compare with the peptide sequence data in the mass  spec. A couple of thousand proteins in a sample can be identified this way.
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Lecture12-Functional Genomics Part II - Lecture 12 -...

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