Polymerase Assay with a FRET Substrate

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.

1 - 17 . (canceled) 
     
     
         18 . 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. 
     
     
         19 . The substrate of  claim 18 , wherein the first FRET label is a quencher and the second FRET label is a fluorophore. 
     
     
         20 . The substrate of  claim 18 , wherein the first FRET label is a fluorophore and the second FRET label is a quencher. 
     
     
         21 . The substrate of  claim 18 , wherein the second FRET label is not located at the 3′ end of the primer. 
     
     
         22 . The substrate of  claim 18 , wherein the primer and template are covalently connected. 
     
     
         23 . The substrate of  claim 22 , wherein the primer and template are covalently connected as a stem loop structure. 
     
     
         24 . The substrate of  claim 18 , wherein the second FRET label is attached to the primer. 
     
     
         25 . The substrate of  claim 18 , wherein the quencher and fluorophore are separated from each other at a distance such that when the duplex is not polymerized the fluorophore is quenched by the quencher and when polymerized the fluorophore is not quenched by the quencher. 
     
     
         26 . The substrate of  claim 25 , wherein the distance is between about 4 and 20 bases. 
     
     
         27 . The substrate of  claim 18 , wherein the primer-template duplex is a hybrid primer-template duplex. 
     
     
         28 . 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.   
     
     
         29 . 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.   
     
     
         30 . The method of  claim 29 , wherein the primer-template duplex is a primer-template hybrid duplex. 
     
     
         31 . The method of  claim 29 , wherein the quencher and fluorophore are separated at a distance such that when the duplex is not polymerized the fluorophore is quenched by the quencher and when polymerized the fluorophore is not quenched by the quencher. 
     
     
         32 - 50 . (canceled) 
     
     
         51 . The substrate of  claim 18 , wherein the quencher is selected from the group consisting of a Black Hole Quencher®, an Iowa Black® quencher, an Eclipse® Dark quencher and a DABCYL quencher. 
     
     
         52 . The substrate of  claim 18 , wherein the fluorophore is selected from the group consisting of FAM, TET, HEX, Cy3, TMR, ROX, Texas Red®, LC red 640, Cy5, and LC red 705. 
     
     
         53 . The substrate of  claim 51 , wherein the quencher is a Black Hole Quencher®, an Iowa Black® quencher, or an Eclipse® Dark quencher, and wherein a distance between the quencher and the fluorophore is about 15 bases. 
     
     
         54 . The substrate of  claim 51 , wherein the quencher is a DABCYL quencher, and wherein a distance between the quencher and the fluorophore is about 5 bases. 
     
     
         55 . The substrate of  claim 18 , wherein the primer-template duplex is a DNA/RNA hybrid duplex.