Water for PCR

Impact of Water

Impact of Water Quality on PCR and Related Techniques

• Ions
  Polymerase is sensitive to heavy metals, such as Cd and Zn as well as a number of divalent metals, in particular transition metals (e.g. Fe, Co, Cu, Ni), that may bind to the site of the enzyme and impact the catalytic activity of the polymerase.
  
  In addition, very specific magnesium concentration is required for optimum activity of the polymerase.
  
  In order to ensure that no transition metals and magnesium are present in the high purity water selected to prepare buffer, water with a resistivity of 18.2 MΩ.cm is highly recommended. 
   
• Organics
  Organic molecules that are negatively charged, such as organic, fulvic and humic acids, mimic the DNA charge and may interfere with the catalytic process. They behave as non-competitive inhibitors onto the polymerase. Although they do not react with the enzyme, they come in and out the active site, reducing the turn over of the polymerase. Low TOC value (< 10 ppb or µg/L) is recommended to ensure good water quality in terms of organic content. 

• Bacteria
  Microorganisms are particularly detrimental to PCR based reactions:
• Bacteria liberate nucleases that destroy the nucleic acids being worked on
• Bacteria contain DNA that can end-up being amplified together with the target DNA
• Bacteria release ions and organics that can interference with the polymerase activity.

Data

• Nuclease-free
• Resistivity 18.2 MΩ.cm
• TOC < 10 ppb
• Bacteria < 1 cfu/mL

In RT-PCR experiments, a known concentration of an external piece of plasmid mRNA was added. This plasmid RNA is used as an external control. Since the amount of RNA molecules added is known, it is possible to predict the amount of DNA that should result from the amplification of the cDNA, assuming “ideal” conditions.

Figure 1. RT-PCR on plasmid RNA (pAW109). Various concentrations of RNA are added. The ideal points are obtained by the theoretical calculation of doubling the amount of DNA at each cycle.

Figure 2: Amplification of plasmid cDNA. The same amount of cDNA was added in all cuvettes.

Conclusion

Reference

• Sensitive, real-time PCR detects low-levels of contamination by Legionella pneumophila in commercial reagents. Shen H, Rogelj S, Kieft TL. Mol Cell Probes. 2006 Jun-Aug;20(3-4):147-53.
  http://www.ncbi.nlm.nih.gov/pubmed/16632318

More Information

• http://biology.about.com/gi/dynamic/offsite.htm?site=http://www.ornl.gov/hgmis
• http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#
• http://cmgm.stanford.edu/biochem118/Stem%20Cell.html 

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