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Lecture14-Model Organisms_Yeast

Lecture14-Model Organisms_Yeast - Lecture 14 Model...

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1 Lecture 14 – Model Organisms_Yeast Thursday Nov 3, 2011 Objectives : Know the various reverse genetics methods for knocking out (or knocking down) individual genes as well as genes on a genome- wide scale (-continued from Lecture 13). Understand the advantages and uses of model organisms. Know basic yeast genetics and how yeast is valuable as a model. Homework #3 is posted on Blackboard: due at the beginning of class on TUESDAY, Nov 8 To supplement my lectures, additional reading material will be posted on Blackboard by tomorrow Also check out recommended animations and videos Hartwell, pp. 600-603 (yeast cell-cycle experiments)
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2 Gene Knockout methods   Targeted Gene Replacement  RNA Interference Insertional or chemical mutagenesis Target the gene of interest Based on random mutagenesis
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3 Gene knockouts can be generated by RANDOM insertional or chemical mutagenesis 1) T-DNA insertions or transposon insertions 2) Tilling, based on chemical mutagenesis *Since these are both untargeted approaches, the mutagenized gene in each individual needs to be identified, in order for the mutant to serve as a reverse genetics resource
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4 Gene X NPTII F R LB RB PCR products: Screening for a T-DNA insertion in a specific gene Screening pools (p1-p5) p1 p2 p3 p4 p5 1kb ladder p1 p2 p p4 p5 LB/F RB/R
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5 Identification of the random sites of T-DNA insertions Gene X LB RB Restriction enzyme digestion Ligase PCR, then  sequence database
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6 Gene knockouts can be generated by RANDOM insertional or chemical mutagenesis 1) T-DNA insertions or transposon insertions 2) Tilling, based on chemical mutagenesis
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Genome-wide mutant collections are being created by ch(T argeted I nduced L ocal L esions in G enomes) A chemical mutagen generates random point mutations which are then identified
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8 How are these two mutant collections different and what are the advantages of each? 1) Genome-wide T-DNA insertions or transposon insertions 3) Genome-wide Tilling lines, based on chemical mutagenesis
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9 For example, 1.
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