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PCR
The polymerase chain reaction
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Crick and Watson – structure of DNA
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A double helix with two antiparallel strands
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“It has not escaped our notice that the specific pairing we have postulated immediately suggests a copying mechanism for the genetic material.”
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Ends5’ end
3’end
3’ end
5’ end
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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’
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http://www.people.virginia.edu/~rjh9u/pcranim.html
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DNA hybridization rate is altered by changing
Temperature: template dependent
Salt Concentration: can be standardized
DNA concentration: can be standardized
Time: can be standardized
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Thermal cyclerPeltier effectheating / coolingblock
Heated lid(preventsevaporationproblems)
96 well plateformat Microprocessor controlled
thermal program
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Real-time PCR
Quantitative
Fast
High sample processing speed
Very sensitive
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Parasitic Worm Inoculation Levels
Violet = 200 worms per root
Red = 50 worms per root
Blue = 20 worms per root