1. Principle of PCR
Principle of PCR
Prof. Dr. Baron

2. PCR - polymerase chain reaction3‘ 5‘ 3‘ 5‘ 3‘ 5‘ 3‘ 5‘ 5‘ 3‘ 5‘ 3‘ 3‘ 5‘ 3‘ 5‘ 5‘ 3‘ 5‘ 3‘ 5‘ 3‘ 5‘ 3‘
Principle of PCR
Prof. Dr. Baron

3. Progression of PCR
Principle of PCR
Prof. Dr. Baron

4. Primer for PCR
Primers are starting molecules for the DNA polymerase and are composed of single stranded DNA (ssDNA)
Primers are 20 to 40 nucleotides in length
Primers have to be synthesized (many companies live on that!)
One primer is complementary to one strand of the DNA to be amplified (template DNA)
Primer software available in the internet facilitates the primer design
Two primers are needed, forward primer and reverse primer
Primers anneale antiparallel to the 3‘ end of template DNA
Both primers should have the same annealing temperatures (Ta)
Ta depends on primer length and GC content
The stronger the primer binds to template DNA the higher is the Ta
The higher the Ta the more specific binds the primer to the template DNA
Ta ranges from 52°C to 65°C
Principle of PCR
Prof. Dr. Baron

5. PCR of 20 Cycles
Principle of PCR
Prof. Dr. Baron

6. PCR of 20 Cycles
Principle of PCR
Prof. Dr. Baron

7. Amplification by PCR of DNA
Amplification from 20 to 50 cycles
20 cycles  220 copies of DNA = 106-fold amplification
30 cycles  230 copies of DNA = 109-fold amplification
40 cycles  240 copies of DNA = 1012-fold amplification
Formula: 2n DNA copies, n = number of PCR cycles
Elaborated by K. Mullis in 1985, nobel price in 1993, use of DNA polymerase from E. coli, no automation possible
Automation was possible after introducing heat stabile DNA polymerases (e.g. Taq-Polymerase)
Automation of PCR by programmable thermal cycler
1st cycle yields DNA of undefined length, two length-defined DNA copies after the 3rd cycle
Principle of PCR
Prof. Dr. Baron

8. Taq-Polymerase origin/organism Thermus aquaticus
habitat geyser, Yellowstone NP ('76)
molecular weight 94 kDa (rec. from E. coli)
pH optimum 7 – 8
temperature optimum in vitro °C
rate of synthesis °C
22 55°C
1.5 37°C °C
error rate in vitro 1 in 5000 nucleotides
stability/biological half life °C
6 97°C
additional enzyme activity 5´ 3´ exonuclease
limitation of numbers of PCR cycles
biological half life of Taq 95°C
shortage of primers and dNTP's
amplified dsDNA binds primer
Principle of PCR
Prof. Dr. Baron

9. End
Principle of PCR
Prof. Dr. Baron