PCR The polymerase chain reaction. Crick and Watson – structure of DNA.

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Presentation transcript:

PCR The polymerase chain reaction

Crick and Watson – structure of DNA

A double helix with two antiparallel strands

“It has not escaped our notice that the specific pairing we have postulated immediately suggests a copying mechanism for the genetic material.”

Ends 5’ end 3’end 5’ end

My gene: 5’TCACGACGTTGTAAAACGACGTCTATACCGAGCTCCTCTCTCTCTCATAGCTGTTTCCTGTGTGAA3’ I design custom oligonucleotides to the “sense” strand at the beginning and the “antisense” at the end Sense 5’TCACGACGTTGTAAAACGACGTCTATACCGAGCTCCTCTCTCTCTCATAGCTGTTTCCTGTGTGAA3’ Anti 3’AGTGCTGCAACATTTTGCTGCAGATATGGCTCGAGGAGAGAGAGAGTATCGACAAAGGACACACTT5’ “forward primer” 5’TCACGACGTTGTAAAACGACG3’ “reverse primer” 3’GTATCGACAAAGGACACACTT5’

DNA hybridization rate is altered by changing Temperature: template dependent Salt Concentration: can be standardized DNA concentration: can be standardized Time: can be standardized

Thermal cycler Peltier effect heating / cooling block Heated lid (prevents evaporation problems) 96 well plate format Microprocessor controlled thermal program

Real-time PCR Quantitative Fast High sample processing speed Very sensitive

Parasitic Worm Inoculation Levels Violet= 200 worms per root Red = 50 worms per root Blue= 20 worms per root