5 What does PCR do? PCR makes millions of copies of DNA Uses PCR machine A DNA photocopier!
6 Cell divisionDNA polymerase duplicates DNA during cell division
7 DNA polymerase in action! Stars show DNA polymerase bound to DNA
8 Who invented PCR?“EUREKA!!! I stopped the car. Somehow, I thought, it had to be an illusion.Otherwise it would change DNA chemistry forever. Otherwise it would make me famous. It was too easy. Someone else would have done it and surely I would have heard of it.We would be doing it all the time.”Kary Mullis - inventor of PCR, Nobel Prize 1993
9 Mullis’s Nobel prize speech is well worth reading
10 What is in a PCR reaction Use 5μl from tube labelled “Template DNA”Use 20μl from tube labelled “primers”The PCR beadTemplate DNAThe starting material in a PCR reaction.Primers are two short pieces of DNA (0-15 bases long) that determine the region of DNA to be copied.NucleotidesA’s, T’s, G’s and C’s to make up the new DNA strandsTaq DNA polymeraseThe enzyme that makes new DNA strandsMgCl2Required for Taq DNA polymerase to function
11 How does PCR work? Mix the following: Template DNA Nucleotides Primers Taq DNA polymeraseMgCl2
12 Cycle through 3 temperatures Denature: unzips DNAAnneal: primers bind to complementary areas of target DNAExtend: Taq DNA polymerase fills in the blanks!
13 Lots of DNA producedSuccessive cycles double amount of DNA
14 Taq DNA polymeraseThermus aquaticus bacteria that lives at high temperatureDNA polymerase crucial to automate PCR
23 Quick PCR cycles Initial denaturation 94°C for 180 seconds Then 20 cycles of:94°C for 30 seconds71°C for 15 seconds (annealing)71°C for 15 seconds (extension)Annealing & extension are same temperature
24 EdvoCyclerPCR machineEasy to useSelect cat noProgrammable
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