What does PCR do? PCR makes millions of copies of DNA Uses PCR machine A DNA photocopier!
Cell division DNA polymerase duplicates DNA during cell division
DNA polymerase in action! Stars show DNA polymerase bound to DNA
Who invented PCR? Kary Mullis - inventor of PCR, Nobel Prize 1993 “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.”
Mullis’s Nobel prize speech is well worth reading http://www.nobelprize.org/nobel_prizes/c hemistry/laureates/1993/mullis- lecture.html
What is in a PCR reaction Use 5μl from tube labelled “Template DNA” Use 20μl from tube labelled “primers” The PCR bead Template DNA The 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. Nucleotides A’s, T’s, G’s and C’s to make up the new DNA strands Taq DNA polymerase The enzyme that makes new DNA strands MgCl 2 Required for Taq DNA polymerase to function
Mix the following: Template DNA Nucleotides Primers Taq DNA polymerase MgCl 2 How does PCR work? ++ ++ ++
Cycle through 3 temperatures Denature: unzips DNA Anneal: primers bind to complementary areas of target DNA Extend: Taq DNA polymerase fills in the blanks!
Lots of DNA produced Successive cycles double amount of DNA
Taq DNA polymerase Thermus aquaticus bacteria that lives at high temperature DNA polymerase crucial to automate PCR
Quick PCR cycles Initial denaturation 94°C for 180 seconds Then 20 cycles of: 94°C for 30 seconds 71°C for 15 seconds (annealing) 71°C for 15 seconds (extension) Annealing & extension are same temperature
EdvoCycler PCR machine Easy to use Select cat no Programmable