Deoxynucleotide triphosphates dNTPs

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. 

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. 

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. 

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. 

HIV-1 RT bound to double-stranded ohgonucleotide template-primers both in the presence and in the absence of a deoxynucleotide triphosphate (dNTP) substrate... 

Prepare a 100 pL 20 mM stock of deoxynucleotide triphosphate (dNTP) mix by combining 20 pL aliquots of each of the four dNTPs, with 20 pL of depH20. This dNTP mixture is usually freshly prepared, although it can be stored at -20°C for a short period of time. 

The DHBV pol gene product is produced by in vitro transcription of a cDNA copy of the DHBV genome and subsequent translation of the RNA using an in vitro rabbit reticulocyte translation system. Aliquots of the translated polymerase are monitored, without further purification, for RNA-dependent DNA polymerase activity. The activity is monitored by measuring the incorporation of [35S]-labeled deoxynucleotide triphosphate (dNTP) into nascent DNA. 

Primer— A short oligonucleotide designed to anneal to single-stranded DNA and from which DNA polymerase can add deoxynucleotide triphosphate (dNTPs) in a complementary fashion to the template DNA. A primer pair flanks the target DNA to be amplified in PCR and creates the specificity of the reaction. 

Next, one of the four deoxynucleotide triphosphates (dNTPs), dATP, dGTP, dCTP or dlTP, is added to the reaction mixture. If the nucleotide is complementary to the next base in the strand, then DNA polymerase catalyses its incorporation. This reaction is accompanied by the release of a pyrophosphate (PPi) molecule (Fig. 5.27). The amount of PPi released is equimolar to the amount of nucleotide incorporated. For example, if three nucleotides are incorporated, then three molecules of PPi are released. 

DNA polymerase catalyzes the synthesis of the complementary strand of DNA in vitro, using single-stranded DNA as a template, provided that both the four deoxynucleotide triphosphate (dNTP) monomers and a primer are present.The primer provides a short stretch of double-stranded DNA by base pairing with its complement on the single-stranded DNA. 

Note that other Taq DNA polymerases, Taq buffers, and Deoxynucleotide triphosphate (dNTP) mixes can be used instead of the PCR beads. The beads are suggested only for simplicity. o Primer cocktail (mixture of the forward and reverse primers). 

PCRcycles are performed in 50 pi of 10 mM Tris-HCl, 2.5 mM MgClj, 50 mM KCl, pH 8.3, containing 2.5 U Taq and Pwo DNApolymerases mixmre (Expand High Fidelity PCRSystem, Roche), and the four deoxynucleotide triphosphates (dNTP) at a concentration of 200 pM each. 

---