An autoradiogram from a typical competition assay

Copyright information: Taken from "Selection of primer-template sequences that bind human immunodeficiency virus reverse transcriptase with high affinity" Nucleic Acids Research 2006;34(1):130-139. Published online 5 Jan 2006 PMCID:PMC1325207. © The Author 2006. Published by Oxford University Press. All rights reserved Competition assay with 12-1 loop-back competed against itself or 1-1 or 12-1 3′ ddG loop-backs. Shown is an autoradiogram of a competition assay using 5′ P-32 end-labeled 12-1 loop-back substrate (10 nM). The substrate was incubated with HIV-RT (2 nM) and increasing amounts of cold competitor substrate (from left to right, 0, 5, 10, 20, 30, 40, 60, 80 and 100 nM) for 1 h. Extension was then initiated by the addition of dNTPs and heparin trap and incubation was continued for 2 min. Lane A, no enzyme added; lane B, trap control with 2 nM HIV-RT. (B) A graph of relative extension versus concentration of competitor is shown for the experiment in (A). Values are relative to the reaction with no competitor added (labeled ‘0’ in each set). The amount of extended substrate in this reaction was quantified using a phosphoimager and set equal to 1. This experiment was repeated with similar results.