PCR OPTIMIZATION

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Last updated 6:26 AM on 7/13/26
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30 Terms

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260 nm

Nucleic acid absorb strongly at ____

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Polymerase Chain Reaction

What does PCR stands for?

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260 and 280

the ratio that gives an estimate of the purity of nucleic acid

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260/280 ratio of 1.8-2.0

Pure preparations of DNA

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1.8-2.0

ratio of pure preparation of DNA

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260/280 ratio 1.8-2.0

260/230 ratio 2.0-2.2

DNA Template

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260/280 ratio 2.0

RNA Template

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1-5mM

Mg2+ concentration

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Increased sensitivity, Decrease specificity

Increase Mg id equivalent to:

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0.1-0.6 uM

Primer concentration

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5 C - 10 C below melting temp of primers

Annealing temperature

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50-500 uM

dnTP concentration

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0.5-3.5 units / 50 uL

DNA polymerase

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Gradient PCR

  • Generate a temperature gradient for the annealing step

  • focused on the annealing temperature

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Thermus aquaticus (Taq)

  • Isolated from hot spring dwelling bacterium

  • Not destroyed at 94 C

  • Optimal temperature is 72 C

  • Error rate 3.0 × 10-4

  • Widely used for endpoint PCR

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72 C

Optimal temperature for heat table DNA polymerase

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3.0 × 10-4

Error rate for Heated Stable DNA Polymerase

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Taq

Widely used for endpoint PCR

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Thermus aquaticus (Taq)

  • Stabilizes the entire DNA polymerase process

  • Thermostable DNA polymerase that catalyzes the primer-dependent incorporation of nucleotides into duplex DNA in the 5’ → 3’ direction in the presence of Magnesium

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Does not proof read

Error rate 1 per 3,300 bases

Newer polymerase have high fidelity

Problems with Taq

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Pyrococcus furiosus (Pfu)

  • Hyperthermophilic archaeon

  • 3’-to-5’ exonuclease activity

  • Excellent for GC-rich templates

  • Error rate 1 per 1,000,000 bases

  • Applications: cloning, DNA sequencing

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Pyrococcus furiosus

What does Pfu stands for?

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230

At what absorbance indicates the presence of phenol

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280

At what absorbance indicates the presence of protein

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Gradient PCR

Optimize the reaction in one PCR run

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94 C

Taq is not destroyed at what temperature?

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3’-5’

Exonuclease activity of Pfu

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High Fidelity DNA Polymerase / Pfu

Excellent for GC-rich templates

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1 per 1,000,000 bases

error rate of High Fidelity DNA Polymerase

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Cloning

DNA Sequencing

Applications of High Fidelity DNA Polymerase / Pfu