Deoxynucleoside triphosphates dNTPs

Nucleotides 2 -deoxynucleoside triphosphates (dNTPs) are purchased from Sigma (St. Louis, MO), and the 2, 3 -dideoxynucleoside triphosphates (ddNTPs) from Boehringer-Mannheim Biochemicals (Mannheim, Germany)... 

To minimize the risk of contamination, we have eliminated all unnecessary components from the PCR buffer, such as ammonium sulfate and 2-mercaptoethanol. A typical 25 pi double-stranded amplification mixture contains 67 mAf Tris (pH 8.8), 2 mAf MgCl2, bovine serum albumin (20 /ig/ml), 1 mAf of each deoxynucleoside triphosphate (dNTP), 1 pM of each primer, template DNA (10-1000 ng), and Taq polymerase (2 units, Perkin-Elmer Cetus, Norwalk, CT). Amplifications always include two important controls an extract control, which is a blank extraction to control for contamination of extraction components as explained above, and several negative PCR controls, which should control for contamination of PCR components during preparation of reagents or setup of the amplification reactions. The controls should be made identical to the... 

Although the number and location of mismatched bases will affect PCR amplification, mismatches at the 3 terminus are expected to have the greatest effect on the PCR. Kwok et al.9 reported on the effects of 3 -termi-nal mismatches on amplification of HIV sequence. In their system, A-G, G-A, C-C and A-A mismatches were detrimental while all other mismatches had minimal effect. Most notably, oligonucleotides with a 3 -ter-minal T served efficiently as primers even when mismatched with T, C, or G. The concentration of deoxynucleoside triphosphates (dNTPs) in the reaction mix also affects Taq polymerase extension. For example, whereas most 3 -terminal mismatches with the exception of A-G, G-A, C-C, and A-A amplified efficiently in the presence of 800 fiM dNTPs, only T-G and G-T mismatches and perfectly matched sequences amplified in the presence of 6 fiM dNTPs. Thus, 3 -terminal mismatches can be more efficiently extended if the primers terminate in a T and if amplifications are carried out at higher dNTP concentrations. 

Deoxynucleoside triphosphate (dNTP) mix Mix equal volumes of 0.5 mMdATP, 0.5 mMdGTP, and 0.5 mMdTTP... 

Deoxynucleoside triphosphate (dNTP) kit (high-performance liquid chromatography [HPLC] purified, USB, cat. no. US 77100). 

This reaction is the initial step in production of deoxynucleoside triphosphates (dNTPs) for DNA synthesis. Four dNTPs are needed for DNA synthesisdATP, dCTP, dGTP, and TTP (TTP comes from dUDP). The proportions of these dNTPs need to be balanced for efficient synthesis. The feedback molecules, or effectors, are the final products of the pathway, the dNTPs, and they act on ribonucleotide reductase to modify its substrate specificity in order to balance the production of dNTPs. In addition to the specificity control site, an additional allosteric control site determines the overall rate of the reaction. At this site, ATP acts as a positive regulator and dATP as a negative regulator. 

To understand the mechanism of potentiation of ACV/GCV by SDC, the effect of SDC on intracellular deoxynucleoside triphosphate (dNTP) pools was determined. As shown in Table 6, SDC had no effect on the levels of dCTP, dATP and dGTP, but significantly reduced the level of dTTP (p<0.05). Combination treatment of infected cells with SDC and ACV resulted in a consistent increase in dNTP pools which were statistically significant. Furthermore, SDC caused a more than 4-fold increase in the estimated pool of the ACV-TP (p<0.005), as compared with that in infected cells treated with ACV alone. Combination of SDC... 

The extension phase during which, in the presence of the four deoxynucleoside triphosphates (dNTPs) and a thermostable DNA polymerase, the template strands are replicated from the 3 ends of the primers at 68-72 °C. 

Deoxynucleoside triphosphates dNTPs are typically used at a concentration of 200 pmol 1 each dNTP in a PCR reaction. Excessively high concentrations promote nonspecific product formation. Modified dNTPs are sometimes used to label PCR products with radioactive or fluorescent markers or with haptens such as biotin, fluorescein, or digoxygenin. The modified dNTP is typically used at a concentration (0.1-1 pmoll" ) much lower than the unmodified dNTP ( 200pmoll ). Probes can be easily generated using PCR amplification with a labeled nucleotide, followed by removal of the unincorporated label. 

In the chain termination method, a single-stranded DNA of unknown sequence is mixed with primer and divided into four separate reaction mixtures. To each reaction mixture are added all four deoxynucleoside triphosphates (dNTPs), one of which is labeled in the 5 phosphoryl group with phosphorus-32 ( P) so that the newly synthesized fragments can be visualized by autoradiography. 

Deoxynucleoside Triphosphate (dNTP) Nucleotide used to synthesize strands of DNA during sequencing. 

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