week_4_lecture_1 - Required readings for week 4 Weaver...

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Required readings for week 4 Weaver , MOLECULAR BIOLOGY. 5th Edition: Chapter 20, p.646-652 (DNA pol); Chapter 21, p.678- 679 (initiation); Chapter 21, p.683-690 (elongation); Chapter 20, p.639-642 (semi-discontinuous rep); Chapter 21, p.694-695 (termination); Chapter 20, p.642- 645 (replicons); Chapter 21, p.679-683 (yeast ARSs); Chapter 21, p.695-699 (telomeres)
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Week 4 lecture 1 1st hour: recap of week 3, quiz and  discussion (20 min), RNAi,  E. coli  DNA  polymerases, DNA pol III, DNA pol I, initiation  of replication, the replisome  Break 2nd hour: accessory proteins, coordinating  leading and lagging strand synthesis
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Recap of week 3, lecture 2 PCR gene fusion, promoter fusion (yeast pGAL), primer design  with RE sites, “in-frame” fusion of affinity tags (epitope, His6,  GST), affinity purification Bioluminescence, GFP (from the jellyfish  Aequorea victoria )   emits  green fluorescence when activated, can be fused in frame to a  protein of interest (often by PCR) to determine its localization in  LIVE cells Real time PCR (RT-PCR) used to generate cDNA from a single type  of mRNA, but the sequence of mRNA must be known so the  primers for the PCR step can be designed. Dye binding for  unknown RT-PCR amplified mRNA compared to dye binding of  RT-PCR mRNA control Site directed mutagenesis (UBC’s Michael Smith): Introduction of precise  gene changes specifically altering amino acids, PCR with primers with  altered codons   single amino acid substitutions in expressed proteins TRANSGENICS: yeast knockouts (homologous recombination to replace  chromosomal genes with selectable markers), fruit fly (P-element  insertions), mouse (positive/negative selection of interrupted gene in ES  cell culture, inject into mouse blastcysts to make transgenic mouse)
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REPLICATION: CONSIDER THIS M & S experiment: DNA replication semi- conservative Replication 5’ to 3’ - but DNA helix
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  • Spring '11
  • DAVIDSON
  • DNA, DNA pol

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