FAQ dNTPs PCR

Text nur auf Englisch verfügbar.  

  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?

      

1. Why are dNTPs important?

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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2. How can dNTPs be presented?

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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3. Is it better to use a dNTP mix rather than a set?

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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4. Which PCR inhibitors can be present in a non-ultrapure dNTP preparation?

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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5. Does dNTP quality affect fidelity?

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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6. Does dNTP quality affect processivity?

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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7. Is the pH of the dNTP solution important for stability?

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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8. Is dNTP concentration important?

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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9. Are nucleotides in solution more stable than its lyophilised form?

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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10. Is it important to have a dNTP preparation free from heavy and transition metals?

Yes. The presence of these metals increases dNTP degradation into dNDPs and dNMPs. Thus, metal-free preparations are more stable.

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11. Which methods for manufacturing dNTPs exist?

dNTPs are normally manufactured from deoxynucleosides or deoxynucleoside monophosphates (dNMPs) by either chemical phosphorylation or enzymatic synthesis. Chemical synthesis involves the addition of phosphate groups or inorganic pyrophosphates (PPi) to deoxynucleosides or dNMPs, whereas the enzymatic method employs a phosphorylation process carried out by highly specific enzymes.

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12. What are the advantages of dNTP enzymatic synthesis over chemical synthesis?

The enzymatic synthesis of dNTPs uses highly specific enzymatic systems which eliminate impurities and PCR inhibitors, such as modified nucleotides, PPi and deoxynucleoside tetraphosphates. The presence of contaminants resulting from the chemical manufacturing process, such as traces of dNDPs, pyrophosphates or other ionic species (e.g. acetate) interfere with the PCR reaction and may lead to poor yields or no PCR product at all. Chemically synthesised dNTPs, unless thoroughly purified, often contain deoxynucleoside tetraphosphates, which are powerful PCR inhibitors. Chemical synthesis can also lead to deamination and other nucleotide modifications. This is not the case with enzymatic synthesis.

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13. What are the advantages of dNTPs being presented in Lithium salts as opposed to Sodium salts?

As mentioned in question 7, dNTPs presented in Lithium salts are more resistant to repeated freeze/thawing than those in Sodium salts, and remain sterile during the entire storage period (the lithium ion has been shown to have significant bacteriostatic activity towards various microorganisms).

dNTPs are more soluble in Lithium salts than in Sodium salts. This is particularly important for dGTP, which has a tendency to precipitate during freezing, therefore causing an imbalance in the final dNTP concentration.

Lithium salts are also more soluble in ethanol than sodium salts. Thus, removal by ethanol precipitation of lithium salts is more efficient than removal of sodium salts. Using lithiumsalt nucleotide preparations reduces saltinduced artifacts and increases the legibility of sequencing gels.

Lithium salts are highly suited to PCR, sequencing, and labelling applications.

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14. 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.

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15. Is it important to have a dNTP preparation free from tetraphosphates?

Yes. Chemical dNTP synthesis is normally carried out by unspecific addition of PPi to dNMPs. If PPi is accidentally added to dNDPs this will result in the formation of tetraphosphates, which may cause strand termination (smearing) and other PCR problems. This is especially important in long-distance PCR applications and Reverse Transcription of long fragments.

Such problems are not encountered with enzymatically synthesised dNTPs, since phosphorylation is carried out using very specific enzymes that only incorporate one phosphate group at a time.

* PCR is a registered Trademark Roche Molecular Systems.

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Letzte Aktualisierung: 05.09.2012