Deoxynucleotide Triphosphates and Buffer Components

Deoxynucleotide triphosphates (dNTPs) are the essential building blocks of nucleic acid molecules, and as such are necessary components of PCR mixes as no new (amplified) DNA could be generated without them. The four individual deoxynucleotides which make up a DNA sequence (i.e. deoxyadenosine triphosphate, dATP; deoxythymidine triphosphate, dTTP; deoxycytosine triphosphate, dCTP; and deoxyguanosine triphosphate, dGTP) are usually added to PCR and RT-PCR mixes in equimolar amounts, though if the target DNA sequence to be PCR amplified comprises mainly dATP/dTTP (AT ratio) or mainly dGTP/dCTP (GC ratio), then the molar ratio of dNTPs added to the PCR mix may be altered in order to take into account this imbalance. dNTPs are usually purchased either individually or as an (equimolar) mix from commercial suppliers (e.g. Roche, Promega, etc.), and are chemically stable when stored in slightly alkaline aqueous solutions at –20°C. However, it should be noted that dNTPs are naturally acidic in solution; hence working and stock solutions may have to be neutralized with alkaline compounds prior to long-term storage. Neutralized dNTP solutions are normally adjusted to 10 mM stock solutions by spectrophotometry, or by adding the correct volume of sterile water to the lyophilized product directly after its chemical synthesis. Some PCRs may profit from the use of highly purified “PCR-grade” dNTPs (Roche) which contain less than 0.9% deoxynucleotide diphosphates (dNDP), are >99% pure, and free from contaminants such as modified nucleotides and tetrapyrophosphate. These (more expensive) high-purity dNTPs may be helpful in successfully amplifying PCR DNA from preparations containing very low copy numbers of target DNA.

In some PCR applications and protocols, one of the four dNTPs may be replaced by an analogous, dNTP, e.g. inosine,7-deaza-2′ -deoxyguanosine, or a modified dNTP, e.g. a biotin-, fluorophore- or radioactively-labeled dNTP. Such modifications allow further downstream post-PCR processing applications to be performed.


Deoxynucleotide Triphosphate Buffer Component Nucleic Acid Molecule Tetramethyl Ammonium Chloride dNTP Concentration 
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  1. Al-Soud W, Radstrom P. 2000. Effects of amplification facilitators on diagnostic PCR in the presence of blood, feces and meat. J Clin Microbiol 38:4463–4470.PubMedGoogle Scholar
  2. Chakrabarti R, Schutt CE. 2001. The enhancement of PCR amplification by low molecular-weight sulfones, Gene 274:293–298.PubMedCrossRefGoogle Scholar
  3. Chien A, Edgar DB, Trela JM. 1976. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J Bacteriol 127:1550–1557.PubMedGoogle Scholar
  4. Ely JJ, Reeves-Daniel A, Campbell ML, Kohler S, Stone WH. 1998. Influence of magnesium ion concentration and PCR amplification conditions on cross-species PCR. Biotechniques 25(1):38–40, 42.PubMedGoogle Scholar
  5. Hengen PN. 1997. Optimizing multiplex and LA PCR with betaine. Trends Biochem Sci 22:225–226.PubMedCrossRefGoogle Scholar
  6. Henke W, Loening SA. 1998. Recently, betaine has been introduced as an additive in different PCR strategies. Nucleic Acids Res 26:687.PubMedGoogle Scholar
  7. Honore B, Madsen P. 1997. The tetramethylammonium chloride (TMAC) method for screening cDNA libraries with highly degenerate oligonucleotide probes obtained by reverse translation of amino acid sequences. Methods Mol Biol 69:139–146.PubMedGoogle Scholar
  8. Jung A, Ruckert S, Frank P, Brabletz T, Kirchner T. 2002. 7-Deaza-2´-deoxyguanosine allows PCR and sequencing reactions from CpG islands. Molecular Patholog 55:55–57.CrossRefGoogle Scholar
  9. Kreader CA. 1996. Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein Appl Environ Microbiol.62:1102–1106.PubMedGoogle Scholar
  10. Lantz PG, Knutsson R, Blixt Y, Al Soud WA, Borch E, Radstrom P. 1998. Detection of pathogenic Yersinis enterocoloitica in enrichment media and pork by a multiplex PCR: a study of sample preparation and PCR inhibitory components. Int J Food Microbiol 45:93–105.PubMedCrossRefGoogle Scholar
  11. Lin CH, Patel DJ. 1997. Structural basis of DNA folding and recognition in an AMP-DNA aptamer complex: distinct architectures but common recognition motifs for DNA and RNA aptamers complexed to AMP. Chem Biol 4:817–832.PubMedCrossRefGoogle Scholar
  12. Lin Y, Jayasana SD. 1997. Inhibition of multiple thermostable DNA polymerases by a heterodimeric aptamer. J Mol Biol 271:100–111.PubMedCrossRefGoogle Scholar
  13. McEvoy CR, Seshadri R, Firgaira FA. 1998. Large DNA fragment sizing using native acrylamide gels on an automated DNA sequencer and GENESCAN software. Biotechniques 25:464–470.PubMedGoogle Scholar
  14. Moretti S, Pinzi C, Spallanzani A, Berti E, Chiarugi A, Mazzoli S, Fabiani M, Vallecchi C, Herlyn M. 1999. Immunohistochemical evidence for cytokine networks during progression of human melanocytic lesions. Int J Cancer 84:160–168.PubMedCrossRefGoogle Scholar
  15. Nichols R, Andrews PC, Zhang P, Bergstrom DE. 1994. A universal nucleoside for use at ambiguous sites in DNA primers. Nature 369:492–493.PubMedCrossRefGoogle Scholar
  16. Park YH, Kohel RJ. 1994. Effect of concentration of MgCl2 on random-amplified DNA polymorphism. Biotechniques 16(4):652–656.PubMedGoogle Scholar
  17. Sturzenbaum SR. 1999. Transfer RNA reduces the formation of primer artifacts during quantitative PCR. Biotechniques 27:50–52.PubMedGoogle Scholar
  18. Taylor TB, Winn-Deen ES, Picozza E, Woudenberg TM, Albin M. 1997. Optimization of the performance of the polymerase chain reaction in silicon-based microstructures. Nucleic Acids Res 25:3164–3168.PubMedCrossRefGoogle Scholar

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