Polymerase Assay with a FRET Substrate
AssigneeLife Technologies Corporation
InventorsLei Xi, Keith P. Bjornson, Stephen P. Hendricks
Life of this patent
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Abstract
This specification generally relates to non-radioactive methods of detecting nucleic acid polymerase activity and methods of detecting compounds that modulate nucleic acid polymerase activity. The activity may be measured in real-time using a real-time PCR instrument.
Claims50
A method of detecting nucleic acid polymerase activity, the method comprising:
- a) providing a primer-template duplex comprising a nucleic acid template and a nucleic acid primer, wherein the template comprises a single-stranded tail having a 5′ end having a first FRET label attached, and wherein the template, or the primer comprises a second FRET label, wherein one FRET label is a quencher and the other FRET label is a fluorophore;
- b) contacting the duplex with a nucleic acid polymerase;
- c) contacting the duplex with at least one nucleotide triphosphate or analog thereof;
- and
- d) detecting a signal from the fluorophore, wherein a change in the signal compared to a control is indicative of nucleic acid polymerase activity.
A substrate for detecting or measuring polymerase activity comprising, a primer-template duplex comprising a nucleic acid template and a nucleic acid primer, wherein the template comprises a single stranded tail having a 5′ end having a first FRET label attached, and wherein the template, or the primer comprises a second FRET label, wherein one FRET label is a quencher and the other FRET label is a fluorophore, wherein the primer and template are complementary to each other.
A kit for detecting or measuring polymerase activity comprising:
- a) a primer-template duplex comprising a nucleic acid template and a nucleic acid primer, wherein the template comprises a single stranded tail having a 5′ end having a first FRET label attached, and wherein the template, or the primer comprises a second FRET label, wherein one FRET label is a quencher and the other FRET label is a fluorophore;
- b) at least one nucleotide triphosphate or analog thereof;
- and
- c) at least one polymerase for use as a control.
A method of screening for compounds that modulate nucleic acid polymerase activity, the method comprising:
- a) providing a primer-template duplex comprising a nucleic acid template and a nucleic acid primer, wherein the template comprises a single stranded tail having a 5′ end having a first FRET label attached, and wherein the template, or the primer comprises a second FRET label, wherein one FRET label is a quencher and the other FRET label is a fluorophore;
- b) contacting the duplex with a nucleic acid polymerase;
- c) contacting the duplex with at least one nucleotide triphosphate or analog thereof;
- d) contacting the duplex with a compound;
- and
- e) detecting a signal from the fluorophore, wherein a change in the signal compared to a control is indicative that the compound modulates nucleic acid polymerase activity.
A method of quantitating nucleic acid polymerase activity, the method comprising:
- a) providing a substrate comprising a primer-template duplex comprising a nucleic acid template and a nucleic acid primer, wherein the template comprises a single stranded tail having a 5′ end having a first FRET label attached, and wherein the template, or the primer comprises a second FRET label, wherein one FRET label is a quencher and the other FRET label is a fluorophore;
- b) contacting the duplex with a nucleic acid polymerase;
- c) contacting the duplex with at least one nucleotide triphosphate or analog thereof;
- d) admixing various concentrations of a standard;
- and
- e) detecting a signal from the fluorophore, wherein a change in the signal compared to a control is indicative of nucleic acid polymerase activity, wherein the standard comprises a “filled-in” version of the substrate.
A method of quantitating nucleic acid polymerase activity, the method comprising:
- a) providing a primer-template duplex comprising a nucleic acid template and a nucleic acid primer, wherein the template comprises a single-stranded tail having a 5′ end having a first FRET label attached, and wherein the template, or the primer comprises a second FRET label, wherein one FRET label is a quencher and the other FRET label is a fluorophore;
- b) contacting the duplex with a nucleic acid polymerase;
- c) contacting the duplex with at least one nucleotide triphosphate or analog thereof;
- d) detecting a signal from the fluorophore;
- e) providing a standard, the standard comprising the primer-template duplex having a “filled-in” single stranded tail;
- f) detecting a signal from the fluorophore;
- and
- g) using the signal obtained at different concentrations of the standard and the signal obtained from the primer-template hybrid to quantitate the activity of the enzyme in units.
The method of claim 44, wherein the distance is between about 4 and 20 bases.
Dependencies are inferred from the claim text.