DdNTPs 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... 

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],... 

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... 

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. 

One p of a different dideoxynucleotide triphosphate solution is then added to each capillary. The concentrations of the stock solutions of each ddNTP which give similar extensions are ... 

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)... 

A number of nucleotide analog compounds function by blocking further chain growth at the replication fork. The 2 3 -dideoxynucleosides can be converted to the triphosphates (ddNTPs). In the case of bacteria, these are incorporated onto the 3 -hydroxyl end of a growing DNA chain, and because the new end now lacks a 3 hydroxyl, no further additions can occur. They are used in conjunction with DNA polymerase I in the dideoxy method of Sanger for determining DNA sequences. 

A 2 deoxyribonucleoside triphosphate (dNTP) A 2, 3 "dideoxyribonucleoside triphosphate (ddNTP) ... 

Each reaction mixture contains the heteroduplex, DNA polymerase, a mixture of the four dNTP species (N = G, A, T, or C), and a small amount of one of the four dideoxynucleotide triphosphates (ddNTP), where no 3 -OH group is present. The ratio of dNTP/ddNTP is high, about 1200 1. DNA polymerase sequentially extends the primer with bases complementary to those present on the opposite strand, but stops when one of the ddNTP species is incorporated. This yields a mixture of fragment lengths, all labeled, where polymerization has stopped at one... 

Synthesis of truncated daughter stands is accomplished by use of 2, 3 -dldeoxyribonucleoside triphosphates (ddNTPs). These molecules, in contrast to normal deoxyiibonucleotides (dNTPs), lack a 3 hydroxyl group (Figure 9-22). Although ddNTPs can be Incorporated into a growing DNA chain by... 

A EXPERIMENTAL FIGURE 9-23 Cloned DNAs can be sequenced by the Sanger method, using fluorescent-tagged dideoxyribonucleoside triphosphates (ddNTPs). (a)... 

DNA polymerase, once incorporated they cannot form a phosphodiester bond with the next incoming nucleotide triphosphate. Thus incorporation of a ddNTP terminates chain synthesis, resulting in a truncated daughter strand. 

For many years, the standard approach was to set up four simultaneous reactions with equivalent, defined concentrations of complementary DNA strand, primer and four de-oxynucleotide triphosphate (dNTP) substrates. Each reaction differed only in having a low mol per cent of a single radioactively labelled dideoxynucleotide (ddNTP) substrate (either ddATP, ddGTP, ddCTP or ddTTP). These ddNTPs possess neither l nor 3 hydroxyl group and therefore prevented further DNA polymerisation whenever they were incorporated into... 

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