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

Principle of PCRProf. Dr. Baron

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Principle of PCR

Principle of PCRProf. Dr. Baron

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PCR - polymerase chain reaction

primer 1 (forward primer)

primer 2 (reverse primer)

DNA polymerasePo l

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5‘5‘ 3‘

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Principle of PCRProf. Dr. Baron

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Progression of PCR

Principle of PCRProf. Dr. Baron

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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 PCRProf. Dr. Baron

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Cycle Step Temperature Duration

1. DNA denaturation 95 °C 1 min primer annealing 50 - 65 °C 30 sec primer elongation 72 °C 45 sec - 3 min

2. - 19. DNA denaturation 92 - 95 °C 30 sec primer annealing 50 - 65 °C 30 sec primer elongation 72 °C 45 sec - 3 min

20 DNA denaturation 92 - 95 °C 30 sec primer annealing 50 - 65 °C 30 sec final elongation 72 °C 7 min hold 7 °C hours

PCR of 20 Cycles

Principle of PCRProf. Dr. Baron

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Cycle Step Temperature Duration

1. DNA denaturation 95 °C 1 min primer annealing 50 - 65 °C 30 sec primer elongation 72 °C 45 sec - 3 min

2. - 19. DNA denaturation 92 - 95 °C 30 sec primer annealing 50 - 65 °C 30 sec primer elongation 72 °C 45 sec - 3 min

20 DNA denaturation 92 - 95 °C 30 sec primer annealing 50 - 65 °C 30 sec final elongation 72 °C 7 min hold 7 °C hours

PCR of 20 Cycles

Principle of PCRProf. Dr. Baron

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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 PCRProf. Dr. Baron

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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 70 - 80°C• rate of synthesis 150 nucleotides/s @ 75-80°C

22 nucleotides/s @ 55°C 1.5 nucleotides/s @ 37°C 0.25 nucleotides/s @ 22°C

• error rate in vitro 1 in 5000 nucleotides• stability/biological half life 12 min @ 94°C

6 min @ 97°C• additional enzyme activity 5´ 3´ exonuclease

limitation of numbers of PCR cycles• biological half life of Taq polymerase @ 95°C • shortage of primers and dNTP's• amplified dsDNA binds primer

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