Deoxynucleotide triphosphates

DNA polymerases normally use 3 -deoxynucleotide triphosphates as substrates for polymerization. Given an adequate concentration of substrate, DNA polymerase synthesizes a long strand of new DNA complementary to the substrate. The use of this reaction for sequencing DNA depends on the inclusion of a single 2/3 -dideoxynucleoside triphosphate (ddNTP) in each of four polymerization reactions. The dideoxynucleotides ate incorporated normally in the chain in response to a complementary residue in the template. Because no 3 -OH is available for further extension, polymerization is... 

The double-stranded DNA to be amplified is heated in the presence of Taq polymerase, Mg2+ ion. the four deoxynucleotide triphosphate monomers (dNTPs), and a large excess of two short oligonucleotide primers of about 20 bases each. Each primer is complementary to the sequence at the end of one of the target DNA segments. At a temperature of 95 °C, double-stranded DNA denatures, spontaneously breaking apart into two single strands. 

Polymerase chain reaction (PCR) The process by which a specific sequence of DNA can be amplified (copied many times) in vitro. It requires a pair of primers and template DNA, thermostable DNA polymerase (e.g. Taq polymerase), deoxynucleotide triphosphates and a thermocycler. The process can amplify large... 

The components listed in Table 20-1 are typical for PCR. As can be seen, it is a fairly simple reaction to set up. The template and primer concentrations must be determined beforehand the buffer, enzyme, and deoxynucleotide triphosphate (dNTP) mixture are commercially available. 

The concentrations of the deoxynucleotide triphosphates have been estimated from information in the scientific literature and are presented below. The concentrations are presented on the assumption that they are distributed equally between the nucleus and cytosol. 

Regulation of the balance of the concentrations of the four deoxyribonucleotides depends on the properties of only two enzymes, the ribonucleotide reductase complex and deoxy-CMP deaminase. The balance between pyrimidine deoxynucleotides is brought about by the properties of the deoxy-CMP deaminase, which is inhibited by deoxy-TTP and stimulated by deoxy-CTP. The ribonucleotide reductase also possesses allosteric sites which bind all four deoxynucleotide triphosphates, the effect of which is to maintain approximately similar concentrations of all the triphosphates. 

Estimates of the concentrations of all four deoxynucleotide triphosphates have been collated from the small number of studies so far published (on proliferating lymphocytes). As expected from the properties of the reductase, the concentrations of three nucleotides are similar but that for dCTP is considerably higher dATP, 0.02mM/L, dGTP 0.02mM/L, dTTP 0.04mM/L and... 

Figure 4.40 Assessment of spot quality using dye-labeled deoxynucleotide triphosphates (dNTPs). (From Shearstone, J.R. et al.. Biotechniques, 32, 1051-1057, 2002. With permission.)...

The two new daughter strands are formed along the template by base pairing with deoxynucleotide triphosphates (dNTPs) serving as the building blocks. 

D. Bhattacharyya, D. B. Nama and S. Reynolds, Deoxynucleotide triphosphates and oligonucleotides by P DNP NMR. 2008. Oxford Instruments Application Note, HypAppNote/31P/0108. 

Foscarnet (phosphonoformic acid) is an inorganic pyrophosphate analog (Figure 49-2) that inhibits viral DNA polymerase, RNA polymerase, and HIVreverse transcriptase directly without requiring activation by phosphorylation. Foscarnet blocks the pyrophosphate binding site of these enzymes and inhibits cleavage of pyrophosphate from deoxynucleotide triphosphates. It has in vitro activity against HSV, VZV, CMV, EBV, HHV-6, HHV-8, and HIV-1. 

The first step of a PCR involves DNA denaturation at 90-95 °C, in a buffered, neutral, aqueous solution containing DNA polymerase, the four deoxynucleotide triphosphates and Mg++, in the presence of a large excess of the two primers (Fig. 27). In the second step, the temperature of the reaction is lowered to about 10 °C below the melting temperature of the primers and the primers (which are considerably smaller than the DNA) are allowed to hybridize to their complementary sequence on the DNA template molecule. This temperature is still too high for the DNA to fully renature. The temperature is then raised to 72 °C, the optimal temperature for extension of the primers by the DNA polymerase, which catalyses the addition of nucleotide triphosphates to extend the sequence in each direction from the... 

Figure 6.6. Amplification of DNA by PCR. Target DNA sequence from a complex genome can be amplified by heat denaturation, providing appropriate conditions for the enzyme (Taq DNA polymerase) that allow it to cause exponential amplification of a particular DNA segment. Among components besides the enzyme that are essential for amplification process are oligonucleotide primers in opposite orientation to each other, shown by dotted arrows, deoxynucleotide triphosphates (dNTPs), Mg2+, and buffer. A 30-cycle amplification leads to a many-million-fold amplification of the discrete DNA segment, flanked by oligonucleotide primer sequences. (Reproduced from Short Protocols in Molecular Biology, 4th ed., F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, eds., Wiley, New York, 1999, p. 15-1.)...

Gold (ABI-Perkin Elmer, Foster City, CA). Too much DNA can lead to PCR artifacts and hence should be avoided. The four deoxynucleotide triphosphates (dNTP) include dATP, dCTP, dGTP, and dTTP. The mixture can be prepared and stored in aliquots at — 80°C. The commercial vendors that sell the Taq DNA polymerase enzyme also provide PCR reaction buffer either with or without Mg2+. The basic constituents of PCR buffer include 100 mM Tris-Cl, pH 8.3 (at room temperature), 500 mM KC1, and other additives in some brands. 

Four separate reactions are set up in the tips of drawn out capillary tubes. Each reaction mixture contains the four deoxynucleotide triphosphates one of which in each case is present in limiting amounts corresponding to the dideoxynucleotide added. In the protocol described a-[32P]-dATP is used as the label but, with the appropriate changes in the compositions of the different dNTP mixes, any of the a-pP] labelled nucleotides could be used. The extension reaction is catalysed using DNA polymerase (Klenow subfragment)... 

Hi) Deoxynucleotide triphosphate (dNTP°) mixes Stocks of 20 mM dNTP s (in 5mM Tris-HCl, pH 7.4, 0.1 mM EDTA) are stored frozen. The 0.5 mM working solutions are made freshly by dilution into water. 

Add 1 /xl CTAG mix (equal vols. of 20 mM solutions of the four deoxynucleotide triphosphates). 

For PCR labeling of Ml3, follow the directions in Step 3 except modify the deoxynucleotide triphosphate mix to include three unlabeled nucleotides and one labeled nucleotide (yielding a final concentration for each at 10 pM). Ten cycles of amplification should suffice. 

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