dGTP

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 PCR, mutagenesis experiments and real-time applications. The purity of dNTPs is also important when the starting amount of template is minimal.

dNTPs can be supplied as either a mix or a set. 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 optimized for PCR and other applications. The set contains four separate tubes, one for each deoxynucleotide. dNTPs can be supplied in lyophilized form or in solution as lithium salts.

The standard concentration of each dNTP in a PCR reaction is 0.2 mM. If the starting stock is a 100mM solution of each dNTP, you need to add 0.1 µl of each nucleotide to a 50 µL standard PCR reaction. Since this is not convenient, it is recommended to prepare mixes: If the 100 mM dNTP stock solutions are mixed in equimolar amounts, the concentration of the mix will be 100mM total or 25 mM of each nucleotide. From a 100x stock, you need to add 0.5 µL to a 50 µL reaction. Meridian also offers more diluted mixes of 40mM total (10mM each), which is a 50x stock solution and 10 mM total (2.5 mM each) 10x working stock. Several different dNTP Mix formats are available from Meridian as ready-to-go solutions.

Yes. A factory pre-dispensed and certified dNTP Mix preparation offers added convenience by minimizing pipetting steps and errors, which could lead to concentration imbalances. And it can be added directly to amplification reactions. Using a dNTP Mix ensures reproducibility in your experiments.

Your PCR assay can be dramatically affected by a dNTP preparation containing inhibitors, which have resulted from an inadequate manufacturing process. Several parameters must be taken into account when purity is sought and each dNTP should preferably be free of ribonucleoside triphosphates, other dNTPs, modified nucleotides (methylated, deaminated etc.), deoxynucleoside di- and monophosphates (dNDPs and dNMPs), heavy transition metals, inorganic pyrophosphates (PPi) and nucleoside tetraphosphates.

It depends. You probably can increase the DNA yield but you will have to optimize the complete PCR reaction, adjust the buffer, the Mg2+ and so on. It is not a matter pertaining only to nucleotides.

Yes. Taq polymerase does not discriminate between correct and modified nucleotides, so point mutations may occur. When proofreading DNA polymerases are used, this problem is only partially eliminated since the presence of methylated/deaminated nucleotides often blocks DNA synthesis.

Yes. The quality of dNTPs is especially important for sophisticated reactions such as amplification of long templates and real-time PCR. Methylated and deaminated nucleotides exhibit inferior results with proofreading DNA polymerases.

There is consensus that the most stringent tests to qualify the purity of dNTP preparations are long distance PCR or the synthesis of long cDNAs in a reverse transcription reaction. dNTP quality is also a very important factor for real-time assays.

Yes. The optimal pH for storage of nucleotides is from pH 7.5-8.2 (pH at 20°C). An acidic pH will cause hydrolysis of dNTPs to dNDPs and dNMPs, rendering them less suitable for PCR applications. During freezing/thawing cycles, the pH of the dNTP solutions can differ from the pH at 20°C. The pH for lithium salt solutions is not as temperature-dependent as with sodium salts, hence where lithium salts are used, no dramatic shifts in pH occur when dNTPs are repeatedly frozen and thawed. This results in the dNTP preparation being more stable and, consequently, having a much longer shelf life than with sodium salts. 

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

The enzymatic synthesis of dNTPs uses highly specific enzymatic systems which eliminate impurities and PCR inhibitors, such as modified nucleotides, PPi and deoxynucleoside tetraphosphates. PCR reactions are impeded by the presence of contaminants resulting from chemical manufacturing processes, such as traces of dNDPs, pyrophosphates or other ionic species (e.g. acetate). Such contamination may lead to poor yields or to no PCR product at all. Unless thoroughly purified, chemically synthesized dNTPs often contain deoxynucleoside tetraphosphates which are powerful PCR inhibitors. Chemical synthesis can also lead to deamination and other nucleotide modifications whereas enzymatic synthesis of dNTPs bypasses these risks.

Yes. Preparations of dNTPs decompose into nucleoside di- and mono-phosphates via a disproportionation reaction. At temperatures above 4°C, lyophilized preparations of deoxynucleotides undergo disproportionation faster than nucleotides in solution. By contrast, at -20°C, the rate of degradation for both forms is less than 1% per year. Nucleotides in solution are also generally purer than the lyophilized form. Some lyophilized preparations approach 98% purity or more but rarely match the >99% achieved with extremely pure solutions.

Reference was made earlier to the greater solubility of dNTPs in lithium salts than in sodium salts. Also, dNTPs presented in lithium salts are more resistant to repeated freezing and thawing than those presented in sodium salts. Furthermore, they remain sterile during the entire storage period (the lithium ion has been shown to have significant bacteriostatic activity towards various microorganisms). Finally, using lithium-salt nucleotide preparations reduces salt-induced artifacts and increases the legibility of sequencing gels. Lithium salts are highly suited to PCR sequencing and labeling applications.

Yes, an excess of inorganic pyrophosphate can inhibit PCR reactions since DNA replication is favored by a low concentration of pyrophosphates on account of the hydrolytic action of cellular pyrophosphates. Inorganic pyrophosphate is often present in chemically synthesized dNTPs and this contamination can be detected by NMR detection and not by conventional HPLC methods. The enzymatic synthesis employed by Meridian produces dNTPs which are entirely free of inorganic pyrophosphate, thus allowing accurate purity determination by HPLC and the identity of the letter and the concentration can be confirmed by photometric measurements.

Yes, chemical dNTP synthesis is normally carried out by addition of PPi to dNMPs. If PPi is added to dNDP, it will result in the formation of nucleoside tetraphosphates which may cause strand termination as manifested by gel “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 synthesized dNTPs, since phosphorylation is carried out using highly specific enzymes that only incorporate one phosphate group at a time.

All the concentrations of our dNTP Mixes are totalled, e.g. our 100 mM dNTP Mix is made up of 25 mM of each dNTP (dATP, dCTP, dGTP and dTTP).

When dUTP is used in place of or in conjunction with dTTP, the resulting PCR product is a suitable substrate for Uracil DNA Glycosylase (UDGase), which allows the user to completely destroy any contaminating DNA from a previous PCR reaction prior to commencing the current amplification. At Meridian we provide dUTP both as a stand-alone product (BIO-39035) and as part of a dUTP Mix (BIO-39041).

We recommend that you dilute your dNTPs using Molecular Biology or PCR Grade Water.