Is it important to have a dNTP preparation free from inorganic pyrophosphate (PPi)?
Yes. An excess of PPi can inhibit PCR reactions. Typically, DNA replication proceeds in low PPi concentrations because of the hydrolytic action of cellular pyrophosphatases (PPiases). Due to PPi being very effective in chelating Mg, an imbalance of Mg may occur, which could have a negative effect on the PCR. When dNTPs are synthesised chemically,excess PPi is normally added to the reaction. During the nucleotide purification process, free unincorporated PPi cannot be detected by conventional HPLC methods; NMR-detection is required. The dNTP enzymatic synthesis method bypasses this risk and the absence of these PCR inhibitors is guaranteed.
