Topic 9, PCR, sequencing, mutagenesis, Q-PCR.ppt.edu

Topic 9, PCR, sequencing, mutagenesis, Q-PCR.ppt.edu -...

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Topic 9—PCR, sequencing, mutagenesis, Q-PCR
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Exam 1 information Runs from Friday Sept. 23 rd to Monday Sept. 26 th in the Testing Center Exams 1/2/3 are all about 75% multiple choice and 25% essay/diagram questions 2 points per multiple choice question Focus on the learning objectives!!!!!
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Learning Objectives Be able to define the following terms: polymerase chain reaction (PCR), primers, DNA polymerase, mutagenesis, DNA sequencing Be able to explain how PCR works—temperatures used, ingredients for the reaction, what happens during each step Be able to accurately diagram how a PCR reaction takes place Be able to describe the methods and goals of site- directed mutagenesis and Sanger sequencing Be able to explain/diagram the mechanism of quantitative PCR
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What are purposes of PCR? There are several: Detect specific DNA sequences Amplify specific DNA fragments in order to have enough copies for further analysis or manipulation Modify sequence or ends of DNA fragments Essential part of Sanger DNA sequencing Quantify how many copies of a DNA fragment are present in a sample PCR revolutionized molecular biology and it is used commonly in many areas of biology
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PCR requires these reagents: Thermostable DNA polymerase Primers Nucleotides Appropriate pH Divalent cations (e.g., Mg +2 ) DNA template to
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Temperature cycles 94-95° C, denaturation 55-60° C, primer annealing 68-72° C, primer extension Annealing step is shown, initial denaturation step not shown Primer extension step
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PCR Animation http://www.youtube.com/watch?v=eEcy9k_KsDI
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Taq DNA Polymerase From bacteria that lives in very high temperature locations such as thermal vents on the ocean floor or hot springs ( Thermus aquaticus or Taq ) Because these organisms are adapted to life at high temperatures, their enzymes do not denature at high temperatures Any of our polymerases would be completely denatured by the first 94°C incubation step in PCR (denatured = inactive)
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Topic 9, PCR, sequencing, mutagenesis, Q-PCR.ppt.edu -...

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