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(PCR)
Sample collection (blood ,tissue ,swabs..etc)
Pre-PCR techniques
DNA extraction
DNA measurement
PCR
Electrophoresis
ost-PCR techniques
Raise temperature to
94oC to separate the
duplex form of DNA into
single strands
(2)Annealing
Annealing is the process of allowing two
sequences of DNA to form hydrogen bonds.
The temperature is lowered (cooling the DNA to
55C)
which allows the primers to bind to the specific
and complementary DNA sequence to be
amplified.
5 TTAACGGCCTTAA . . . TTTAAACCGGTT 3 DNA
strand
AATTGCCGGAATT . . . . . . . . . .> forward primer
reverse primer <. . . . . . . . . . AAATTTGGCCAA
DNA t d 3 TTAACGGCCTTAA strand 3 . . .
TTTAAACCGGTT 5
Annealing temperature
Tm = 4 (G + C) + 2 (A + T)
(3)Extension
The DNA polymerase is able to extend the
primers by adding nucleotides to the developing
DNA strand.
Extend primers raise temp to 72oC, allowing Taq
pol to attach at each priming site and extend a
new DNA strand
Repeat the Denature, Anneal, Extension steps at
their respective temperatures.
Template DNA
Denature (heat to
95oC)
Denaturing
Lower temperature to
56oC Anneal with
primers 1
cycle
Template DNA
Increase temperature to
72oC DNA polymerase +
dNTPs
Elongation
DNA copies vs Cycle number
2500000
2000000
DNA copies
1500000
1000000
500000
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Cycle number