1. Why are dNTPs important?
2. How can dNTPs be presented?
3. Is it better to use a dNTP mix rather than a set?
4. Which PCR inhibitors can be present in a non-ultrapure dNTP preparation?
5. Does dNTP quality affect fidelity?
6. Does dNTP quality affect processivity?
7. Is the pH of the dNTP solution important for stability?
8. Is dNTP concentration important?
9. Are nucleotides in solution more stable than its lyophilised form?
10. Is it important to have a dNTP preparation free from heavy and transition metals?
11. Which methods for manufacturing dNTPs exist?
12. What are the advantages of dNTP enzymatic synthesis over chemical synthesis?
13. What are the advantages of dNTPs being presented in Lithium salts as opposed to Sodium salts?
14. Is it important to have a dNTP preparation free from inorganic pyrophosphate (PPi)?
15. Is it important to have a dNTP preparation free from tetraphosphates?
    
      

[Translate to English:] dNTPs or deoxynucleotide triphosphates are the "building blocks" for DNA. Purity and stability of dNTPs are two of the essential factors to achieve a successful PCR. The use of a highly purified dNTP preparation is particularly recommended for sensitive techniques such as long-range PCR, RT-PCR,multiplex, mutagenesis experiments and Real-Time applications. The purity of dNTPs is also important when the starting amount of template is minimal.

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[Translate to English:] dNTPs can be supplied as either a set or a mix. The mix is presented in a single tube containing a premixed solution of dATP, dCTP, dGTP and dTTP. This solution is ready for use and is optimised for PCR and other applications. The set contains four separate tubes,one for each deoxynucleotide. dNTPs can be supplied in lyophilised form or dissolved in either Lithium or Sodium salts.

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[Translate to English:] Yes. This preparation offers added convenience, minimises pipetting steps and can be added directly to amplification reactions. Using a dNTP mix ensures a better reproducibility in your experiments.

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[Translate to English:] A dNTP preparation containing PCR inhibitors resulting from an inadequate manufacturing process can dramatically affect your experiment. Several parameters must be taken into account when purity is defined. Each dNTP should preferably be free of NTPs, other dNTPs, modified nucleotides (methylated, deaminated, etc.), deoxynucleoside diphosphates (dNDPs) and monophosphates (dNMPs), heavy/transition metals, inorganic pyrophosphates (PPi) and tetraphosphates. HPLC is a suitable method of testing dNTP purity, but some contaminants (i.e. PPi) cannot be detected using this technique.

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[Translate to English:] Yes. Taq polymerase does not discriminate between correct and modified nucleotides, so point mutations may occur. When proof-reading DNA polymerases are used, this problem is only partially eliminated since the presence of methylated/deaminated nucleotides often blocks DNA synthesis.

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[Translate to English:] Yes. The quality of dNTPs is especially important for sophisticated reactions such as amplification of long products and Real-Time PCR. Methylated and deaminated nucleotides exhibit inferior results with proofreading DNA Polymerases.

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[Translate to English:] Yes. The pH for Li salts solutions is not as temperature-dependant as with Na salts so no dramatic shifts in pH occur when dNTPs are repeatedly freeze/thawed. This results in the dNTP preparation being more stable and, consequently, having a much longer shelf-life than with Na salts.

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[Translate to English:] Yes. Significant hydrolysis occurs when dNTPs are stored at low concentrations. Always store nucleotide stock solutions at a concentration of >/- 10mM. 100mM is the ideal concentration for storage.

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Yes. Preparations of dNTPs decompose into nucleoside di- and mono- phosphates via a disproportionation reaction. At temperatures above 4°C, lyophilised preparations of deoxynucleotides undergo disproportionation faster than nucleotides in solution. In contrast, at -20°C, the rate of degradation for both forms is less than 1% per year.

Nucleotides in solution are also generally purer. Some lyophilised preparations approach 98% purity or more but rarely match the >99% achieved by extremely pure solutions. Generally, solution nucleotides are purer than the lyophilised version.

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