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Catalog 2007

Short introduction in  Polymerase-Chain-Reaction (PCR)

Enzymatic amplifikation of DNA with PCR (Polymerase Chain Reaktion)  
---> more literature about PCR

Introduction:
The idea of making unlimited DNA copies from a single copy of DNA which was later called: "Polymerase Chain Reaction" (PCR) was born in 1983 by Kary B. Mullis. Ten years later, he was awarded the Nobel Prize in Stockholm for his brilliant method.

PCR was first published in 1985 (Saiki et al., 1985). For Elongation a Klenow polymerase was used. Because Klenow polymerase is heat instable, new enzyme had to be added for every new cycle. The maximum product length was only 400 bp. In 1988 the first report using thermostable DNA polymerase from Thermophilus aquaticus (Taq-polymerase) was published (Saiki et al., 1988). PCR technique is today a powerful standard tool for molecular biology.

PCR is composed of cycles of heating and cooling. A cycle begins with denaturing the DNA by heating (breaking apart the double strands of the DNA molecule into single-stranded DNAs). Then, primers (short pieces of DNA complementary to a specific sequence) bind to their specific part of every single-stranded DNA during cooling. This step is called annealing. In the final step, extension, the temperature is raised again and a specific enzyme called Taq polymerase is used to add complementary bases to the DNA from the end of the primer along the rest of the single-stranded DNA templates, thereby making multiple copies of the DNA segment between the two primers. The entire process is repeated a large number of times (cycles) to yield a large amount of the desired DNA region. After 25 PCR Cycles you can achieve 3,2 x 10⁷ copies from one target molecule.

Template: The template DNA used for PCR has to be pure (OD 260/280 1,8 - 2,0). The amount of Template depends on the size of DNA. The amount can vary from pg (for plasmid- and page-DNA) up tol µg (for genomic DNA). The DNA used for PCR should be free of substances which can inhibit PCR reaction like EDTA/EGTA.

Primer: The content of Primers in the PCR Mix should be abundantly (0,2 - 0,8 µM).  The ratio of the bases  G/C to A/T should be approximately the same. The length of the primer should be between 12-50 bases. The annealing temperature should be about 5°C below the melting Temperature (Tm) of the designed primer. Calculation formular (valid for primers with a max. of 15 bases): Tm = 4 x (G + C) + 2 x (A + T)

Internal complementary sequences are able to build secundary structures called Hairpin that should be avioded. If additional nucleotides should be adjusted to the primer sequence for example to create a restriction site they should be located at the 5´- end of the primers. In order to create a functional restriction site at least 4 additional nucleotides should be attached to the primer sequences. These additional nucleotides were not considered in the calculation of the Tm value.

MgCl₂ und MgSO₄ - concentration influences strongly the yield and specifity of the PCR reaction. As a rule, concentrations of 1 to 10 mM final concentration were used. The concentrations also depends on the polymerase: MgCl₂ (1 mM -6 mM for Taq Polymerase) and  MgSO₄ (1 mM -10 mM for Pfu and Psp Polymerase). The optimal concentration can vary for different PCRs. If the complete reaction buffer (which includes MgCl₂ or MgSO₄) doesn' t work at all it will be necessary to find the optimal concentration by titration. However too low concentrations of Mg²⁺ can cause poor yield of PCR product. Indeed an excess of free Mg²⁺ ions can cause synthesis of unspecific PCR products. The Mg²⁺ ions are building a complex with the free dNTPs hereby the assembly of the dNTPs by the activity of the polymerase can be enhanced.

dNTPs (Desoxy­nukleoside - triphosphate) The concentration of each dNTP (dATP, dCTP, dGTP and dTTP) should be  200 µM for the PCR set up. To high concentrations can inhibit the PCR reaction.

The 5´- 3´Polymerases vary in different parameters. Even if new genes should be analysed for their sequence or the gene of interest should be cloned for gene expression, it would be necessary to use high fidelity polymerases for accurate amplification. These polymerases have a 3'-5' exonuclease (proofreading) activity. But this activity is often associated with low processivity of the polymerase:  

Psp/Pfu- Polymerases  shows 3´- 5´ Exonukleaseaktivität  ("proofreading" activity) but also shows low processivity compared to the normal Taq Polymerase. An exception is the GC-rich DNA Polymerase. This enzyme (a KOD Polymerase) shows  an elongation rate and processivity which is 5 times and 10 to 15 times higher, respectively, than for Pfu DNA Polymerase, resulting in highly accurate and robust yields in short reaction times.

All proofreading polymerases create PCR fragments which have blunt-ends. The normal Taq Polymerase whereas with its transferase activity adjusts additional A nucleotides at the 3'-end of the PCR products. Because of these adjusted A nucleotides the PCR products can be used for T/A-Cloning in special T/A-Vectors.

Short Profile:

Taq Polymerase: Thermus aquaticus                                    optimum: 72°C

• For efficent amplification of fragments < 10 kb, no 3´- 5´ exonukleaseactivity.
• The polymerase shows transferase activity and attaches additonal A nukleotides at the 3´- end. The PCR product can be used for T/A - cloning.
• Ion: MgCl2
• 1000 bp/min
• For 50µl reaction volume you should use 0,5 - 2 units of Taq DNA Polymerase

Pfu DNA Polymerase: Pyrococcus furiosus (Archaebakterium)     optimum: 75°C

• "Proofreading" DNA Polymerase (3´- 5´ exonukleaseactivity)
• Pfu und Psp Polymerases generates DNA-fragments with blut-ends up to 10 kb.
• Ion: MgSO4
• 500 bp/min
• For 50µl reaction volume you should use 1,25 - 2, 5 units of Pfu Polymerase

Abkürzung: Psp     Name: Pyrococcus species                                      Optimum: 75°C

• The optimun depends strongly on the  Pyrococcus spec. and can vary between  68 - 78 ° C.
• Ion: MgSO4
• 500 bp/min
• For 50µl reaction volume you should use 1,25 - 2, 5 units of Psp.

Unit - definition :

One unit of activity is the amount of enzyme required to incorporate 10 mmoles of dNTPs into acid-insoluble DNA fraction in 30 minutes at 68° - 75°C.

Procedure:

The PCR reaction volume can vary between 20 to 100 µl depending on the issue of application.

The choice of the right polymerase depends on the issue of application (normal Taq or proofreading polymerase). The reaction buffer can be supplied as complete buffer (including MgCl₂/MgSO₄) or as incomplete buffer (without MgCl₂/MgSO₄). For the last version the right MgCl₂/MgSO₄ concentrations should be adjusted to the reaction mix.  The amount of template can also strongly vary. (see also the table below). It is often recommended to set up an negativ control without template (you can use H₂O instead of DNA). It is also recommended to set up a positve control. Important for the set up of all PCR mixes is to add the polymerase at the last step.

Example for 50 µl PCR reaction mix:

For a large number of samples it is recommended to set up a master mix. 

Formular for the calculation for the amount of components:

Q = X + N + P + 1  (Q= Faktor for the volume of the different components; X= number of samples; N= number of negativ-controls; P= number of positiv-controls)

PCR -Cycler program (standard, not optimized)

1.   step : 5 min. at 94°C denaturation.

2.   step : 30 sec.  at 94°C denaturieren.

3.   step : annealing (primers bind to their specific part of every single-stranded
      DNA  at   55°C für 30 - 120 sec., The temperature can vary and depends on the 
      designed primers.

4.   step : elongation (synthesis of the  DNA) at 68° - 78°C. The temperature depends
      on which polymerase is used for the PCR reaction (elongation rate: 500 -
      1000bp/min).

5.   step : elongation at 68° - 78°C for 5-15 min.

6.   step : After the PCR is finished the reaction mix should be cooled down in the cycler 
      to 4°C -  8°C. As a rule 25 to 35 Cycles are enough for standard PCR. That  means
      step 2 to step 4 should be repeated 25 to 35 times. If genomic DNA or a   proof-      reading polymerase is use 30-35 repeats of step 2-4 should be used.

 Troubleshouting:

• Titration of MgCl₂ or MgSO₄

If the PCR doesn't work  with the complete buffer provided with the polymerase (low yield or unspecific products) it is necessary to titrate the amount of Mg²⁺ ion in order to find the optimum for PCR reaction.

 Taq:  1 - 4 mM  MgCl2

Pfu bzw. Psp:  1- 10 mM MgSO4  

For the titration you use the 10x incomplete reaction buffer (without Mg²⁺ ions) and add different amounts of the suppllied MgCl2  (25-100mM solution) for Taq DNA Polymerase or MgSO4 (25 mM solution) for Psp/Pfu Polymerase

 The volume left in the PCR mixture should be adjusted to the final volume with water.

More literature around the PCR:

For more questions around PCR reaction and optimization please contact our technical support.

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