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Lecture 1 - Approaches - BIOL308.pdf

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University of Waterloo
BIOL 308
Bruce Wolff

Lecture 1September1412Answering Question 2 How do we study gene expression and functionperformance of certain proteinsIn other words what is it that we need to investigate in the cell Measure of gene expression2 Ways to do soBiochemical Approach1Start from protein which is isolated and working back to the information contained in DNAPutting the whole togetherGenetic Approach2Start with genetic information and work towards the productProtein is general product however it can sometimes be RNA as the productTwo links between both types in other words tools that assist with both methodsRecombinant DNA technologyBioinformaticsBuilt up huge amounts of dataData bases of interactionsAlways continues to growNOTE both approaches will give you the exact same information it all depends on what you have to start withBiochemical approaches protein to geneIsolate protein on the basis of its molecular function Ie Enzymatic or hormonal activity1Determine amino acid sequence for that protein2Generate a DNA possible sequence3Because there are multiple codes it can be hardaCreate oligonucleotides which correspond to certain parts of the amino acid sequence4Use cDNA5Using the oligonucleotides as probes to select cDNA or genomic clone encoding the protein from this alibraryUsing cDNA to find the real gene in the organismbSequence isolated gene6Genetic approaches gene to a proteinIsolate genomic clone that responds to a specific trait this trait is one that is altered trait in mutants Ie 1Nutritional auxotrophy inherited disease developmental defectUse the genomic DNA to isolate a cDNA for the mRNA encoded by that gene2By using the cDNA you can sequence it so that it deduces an estimated amino acid sequence of the encoded 3proteinCompar the deduced amino acids against other known proteins4This gains insight on to the function of that proteinaUsing the cDNA you can put it on a plasma to be produced which in return should create a large amount of that 5proteinBiochemical ApproachIsolate and purify protein of interest based on its function or activityAssay under different conditionsThis confirms that it is pure and it is what it actually is In other words conformation of identity of the protein activityThe Ultimate Goal get the DNA sequence for the specific proteinAn amino acid sequence is used to deduce the nucleotide sequence of the genewhich codes for extracted proteinGenetic ApproachDone through the Identification and isolation of mutants Example fruit flies red vs white eyeHow do you Identify these mutantsGenetic screensEssential genesIs it absolutely necessary for survival NOTES FROM LECTURES Page 1
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