DdNTP

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... 

I Very small amounts of the four 2, 3 -dicleoxyrtbonucleoside triphosphates (ddNTPs), each of which is labeled with a fluorescent dye of a different color (A 2 ,3 -d/ fco.Yyribonucleoside triphosphate is one in which both 2 and 3 -OH groups are missing from ribose.)... 

While the ddNs and ANPs must be converted intracellularly to their 5 -triphosphates (ddNTPs) or diphosphate derivatives before they can interact as competitive inhibitors/alternate substrates with regard to the natural substrates (dNTPs), the NNRTIs do not need any metabolic conversion to interact, noncompetitively with respect to the dNTPs, at an allosteric, non-substrate binding site of the HIV-1 RT. Through the analysis of NNRTI-resistant mutants, combined with site-directed mutagenesis studies, it has become increasingly clear which amino acid residues are involved in the interaction of the NNRTIs with HIV-1 RT, and, since the conformation of the HIV-1 RT has been resolved at 3.0 A resolution [73], it is now possible to visualize the binding site of the NNRTIs [74],... 

In a more modern procedure, the four ddNTPs are covalently marked with fluorescent dyes, which produce a different color for each ddNTP on laser illumination. This allows the sequence in which the individual fragments appear at the lower end of the gel to be continuously recorded and directly stored in digital form. 

Five microliters of these purified amplicons is then mixed with 1 pi of primer (10 pM) and 4 pi of the Big Dye Terminator reaction mix and the mixture is cycled for the sequencing reaction with the following conditions initial denaturation at 94°C for 45 s, followed by 25 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 90 s. The sequencing reaction is then stored at 4°C until clean up with Autoseq G-50 columns to eliminate the labeled ddNTP and the dNTPs from the reaction mixture. Ten microliters of this purified sequencing reaction is then mixed with 12 pi of the template suppression reagent, heated to 95°C for 7 min, and then cooled on ice. 

A sample of the DNA was reacted with DNA polymerase and each of the nucleotide mixtures (in an appropriate buffer) listed below. Dideoxynucleotides (ddNTPs) were added in relatively small amounts. 

Detailed structures of several DNA polymerases are available, including those with double- or single-stranded DNA bound.31-35 Site-directed mutagenesis (Chapter 14) has been used to identify residues that are in the active sites for polymerization and exonuclease activity.36,37 Mutants that are defective in the exonuclease activity are used for the production of stable complexes with single-stranded DNA or single-stranded 3 ends of duplexes bound in the exonuclease site.31,33 2, 3 -Dideoxynucleotides (ddNTPs) that lack the nucleophilic 3 -OH have been used to make templates that are inert so that productive ternary enzyme DNA ddNTP complexes may be observed.34,35,38... 

Recent kinetic work on RB69 polymerase (family B) and structural comparison between the RB69 polymerase and other polymerase families led to the postulation of a different initial binding event for dNTP (Yang et al, 2002a). The crystal structure of the closed ternary E p/t ddNTP complex of the RB69 polymerase (see Section VII) shows interactions between the fingers subdomain and the ddNTP s triphosphate moiety... 

Li, Z.-P., Tsunoda, H., Okano, K., Nagai, K., Kambara, H., Microchip electrophoresis of tagged probes incorporated with one-colored ddNTP for analyzing single-nucleotide polymorphisms. Anal. Chem. 2003, 75, 3345-3351. 

Figure 2 Minisequencing reaction on with extension primers immobilized on the microarray surface. The multiplex PCR products of the regions containing the SNPs are allowed to hybridize to the oligonucleotides immobilized on the microarray (A). The primers are extended with fluorescently labelled ddNTPs complementary to the nucleotide at the SNP position. Four different fluorophores, one for each nucleotide is used allowing for a simultaneous detection of the four nucleotides in one single reaction (B). After washing the slide with sodium hydroxide and salt buffers, only the extended primers covalently attached to the surface remains and the fluorescence is measured (C) and the genotypes assigned.

An alternative to the ddNTP s (as chain terminators) are the arabinonucleoside triphosphates. Arabinose is a stereoisomer of ribose in which the 3 -hydroxyl group is trans with respect to the 2 -hydroxyl group. Such arabinosyl-(ara) nucleotides act as chainterminating inhibitors of E. coli DNA polymerase I in a manner comparable to ddT. The structures of the chain-terminating inhibitors are shown in Figure 3.1. and the principle of the method in Figure 3.2. 

Nick translation with DNA polymerase I in the presence of dNTP s and one ddNTP (eg ddA). The 5 — 3 exonuclease activity of the enzyme hydrolyses the5 -end of each nick as the 3 -end is simultaneously extended by the 5 — 3 polymerase p activity (for clarity only one nick per strand is shown)... 

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