Continuous time-resolved resonance energy-transfer assay for polynucleic acid polymerases

A method for identifying a candidate compound as a modulator of polynucleic acid polymerase activity, the method comprising:

• (a) providing a candidate compound, a polynucleic acid primer-template complex labeled with an energy-emitting chemical species and a nucleotide labeled with an energy-emitting chemical species;
• (b) mixing the candidate compound, the polynucleic acid primer-template complex and the nucleotide with a polynucleic acid polymerase;
• (c) prior to, contemporaneously with or after the mixing of step (b), exposing the labeled polynucleic acid primer-template complex and the labeled nucleotide to radiation of excitation wavelength for one of the energy-emitting chemical species to thereby excite that energy-emitting chemical species;
• (d) detecting a signal produced by energy transfer between the excited energy-emitting chemical species and the other energy-emitting chemical species as a result of incorporation of the nucleotide into the polynucleic acid primer-template complex via the activity of the polynucleic acid polymerase, the detected signal indicating an amount of polynucleic acid polymerase activity;
• and
• (e) identifying the candidate compound as a modulator of polynucleic acid polymerase activity based on the amount of signal detected as compared to a control sample.

A method for identifying a candidate compound as a modulator of polynucleic acid polymerase activity, the method comprising:

• (a) providing a candidate compound, a polynucleic acid primer-template complex labeled with a light-emitting chemical species and a nucleotide labeled with a light-emitting chemical species;
• (b) mixing the candidate compound, the polynucleic acid primer-template complex and the nucleotide with a polynucleic acid polymerase;
• (c) prior to, contemporaneously with or after the mixing of step (b), exposing the labeled polynucleic acid primer-template complex and the labeled nucleotide to radiation of excitation wavelength for one of the light-emitting chemical species to thereby excite that light-emitting chemical species;
• (d) detecting a signal at a plurality of time points over a predetermined time period, the signal produced by energy transfer between the excited light-emitting chemical species and the other light-emitting chemical species as a result of incorporation of the nucleotide into the polynucleic acid primer-template complex via the activity of the polynucleic acid potymerase, the detected signal indicating an amount of polynucleic acid polymerase activity;
• and
• (e) identifying the candidate compound as a modulator of polynucleic acid polymerase activity based on the amount of signal detected as compared to a control sample.