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PCR

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.

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PCR

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  1. PCR The polymerase chain reaction

  2. Crick and Watson – structure of DNA

  3. A double helix with two antiparallel strands

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

  5. Ends 5’ end 3’ end 3’end 5’ end

  6. 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’

  7. http://www.people.virginia.edu/~rjh9u/pcranim.html

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

  9. Thermal cycler Peltier effectheating / cooling block Heated lid (prevents evaporationproblems) 96 well plate format Microprocessor controlledthermal program

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

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

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