| Assay Method Information | |
| | Primer Extension Assay |
| Description: | (((Guanine-9-yl) propan-2-oxy) methyl) phosphonic acid diphosphate (PMPGpp) acts as a chain terminator and competes with dGTP.Purified human telomerase was incubated with d(TTAGGG)3 (SEQ ID NO: 1) and deoxy-nucleoside triphosphates for 90 minutes at 37° C. All reactions were done in the presence of 200 dTTP and 10 μM dATP (50,000 cpm/pmol a-33P-dATP). Activity was determined by incubating affinity purified telomerase extract with 1 μM primer [d(TTAGGG)3] (SEQ ID NO: 1), 200 μM dTTP, 50 μM dGTP, 10 μM dATP, 10 μCi [α-33P]dATP (2000-4000 Ci/mmol) in a buffer containing 50 mM (N-2-hydroxyethyl piperazine-N′-3 propanesulfonic acid)-NaOH pH 8.5, 1 mM MgCl2, 1 mM DTT, 5% glycerol, 0.5 mM EGTA and 100 mM KOAc in a final reaction volume of 40 μl. Primer extension products were analyzed on a 15% polyacrylamide gel containing 7 M urea. A characteristic telomerase ladder has multiple bands. If a compound is not a substrate then (TTAGGG)3TTA (21 nt) (SEQ ID NO: 2). If a compound is a chain terminator then (TTAGGG)3TTAG* (22 nt) (SEQ ID NO: 3).FIG. 1 is a photograph of a 15% polyacrylamide gel containing 7 M urea showing the primer extension products in the presence of compounds. Reactions containing 50 or 100 μM dGTP, (lanes 1 and 2) show a characteristic 6 nucleotide ladder. Lanes 8-11 contain increasing concentrations of PMPGpp (ID#142692) in addition to 50 μM dGTP. Lane 12 contains only 50 μM PMPGpp (ID#142692). Lanes 3-7 show a comparison using the known chain terminator, 3′-azido-dGTP.IC 50s for (R)-PMPGpp and (S)-PMPGpp are shown in Table 3. (R)-(((Guanine-9-yl) propan-2-oxy) methyl) phosphonic acid diphosphate is efficiently recognized by human telomerase and added to the 3′ end of a telomeric primer resulting in chain termination. The molecule competes with dGTP and when measured in a cell free assay, using purified telomerase and dGTP concentrations of 50 μM, shows an IC50 of about 1.0 μM. |
| Affinity data for this assay | |
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