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.

Keywords

Magnesium DMSO Adenosine Gelatin Deoxythymidine 

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