Selected article for: "base extension and high fidelity"
Author: Jingyue Ju; Xiaoxu Li; Shiv Kumar; Steffen Jockusch; Minchen Chien; Chuanjuan Tao; Irina Morozova; Sergey Kalachikov; Robert N. Kirchdoerfer; James J. Russo
Title: Nucleotide Analogues as Inhibitors of SARS-CoV Polymerase
  • Document date: 2020_3_14
    • ID: hj675z1b_3
    Snippet: We first carried out DNA polymerase extension reactions with the active form of Sofosbuvir (2'-F,Me-UTP) using Thermo Sequenase as an example of high fidelity, host-like polymerases, and two mutated DNA polymerases which are known to be more promiscuous in their ability to incorporate modified nucleotides, Therminator II and Therminator IX, as examples of viral-like low fidelity enzymes. A DNA template-primer complex, in which the next two availa.....
    Document: We first carried out DNA polymerase extension reactions with the active form of Sofosbuvir (2'-F,Me-UTP) using Thermo Sequenase as an example of high fidelity, host-like polymerases, and two mutated DNA polymerases which are known to be more promiscuous in their ability to incorporate modified nucleotides, Therminator II and Therminator IX, as examples of viral-like low fidelity enzymes. A DNA template-primer complex, in which the next two available bases were A (Fig. 3 ), was incubated with either 2'-F,Me-UTP (structure shown in Fig. 2a) , or dTTP as a positive control, in the appropriate polymerase buffer. If the 2'-F,Me-UTP is incorporated and inhibits further incorporation, a single-base primer extension product will be produced. By contrast, dTTP incorporation will result in primer extension by 2 bases. After performing the reactions, we determined the molecular weight of the extension products using MALDI-TOF-mass spectrometry (MALDI-TOF MS).

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