Dna sequencing method

Abstract

A method for determining the sequence of a polynucleotide, the method relying on the detection of a conformational change in an enzyme that interacts with and processes along the polynucleotide. The detection of a conformational change may be carried out by measuring changes in a fluorophore bound to the enzyme.

1 - 6 . (canceled) 
     
     
         7 . A method for determining the sequence of a polynucleotide, comprising the steps of:
 (i) contacting a labeled polynucleotide processive enzyme with an unlabeled target polynucleotide to form a complex, wherein the detectable characteristics of the label alter as the enzyme processes along the target polynucleotide;   (ii) detecting an effect as the enzyme processes along the polynucleotide, thereby determining the sequence of the target polynucleotide, wherein the effect is detected by measuring the change of the detectible characteristics of the label.   
     
     
         8 . The method of  claim 7 , wherein the effect is a conformational change in the enzyme. 
     
     
         9 . The method according to  claim 7 , wherein a second detectable label is bound to a nucleotide brought into contact with the enzyme. 
     
     
         10 . The method according to  claim 9 , wherein the second detectable label interacts with the label on the enzyme, wherein the degree of interaction of the labels is dependent on the proximity of the label on the enzyme and the second detectable label. 
     
     
         11 . The method according to  claim 10 , wherein the label on the enzyme is an energy acceptor and the second detectable label is an energy donor, or wherein the label on the enzyme is an energy donor and the second detectable label is an energy acceptor, and wherein the detection step is carried out by measuring energy transfer between the two types of labels. 
     
     
         12 . The method according to  claim 7 , wherein the enzyme is a polymerase enzyme and the method further comprises contacting the complex with one or more nucleoside triphosphates selected from the group consisting of dATP, dTTP, dGTP, and dCTP, under conditions sufficient to induce polynucleotide processive enzyme activity. 
     
     
         13 . The method according to  claim 12 , wherein the effect is a conformational change in the polymerase that is brought about when the polymerase incorporates a nucleoside triphosphate into a nascent nucleotide strand complementary to the target polynucleotide. 
     
     
         14 . The method according to  claim 12 , wherein the nucleoside triphosphates are labeled. 
     
     
         15 . The method according to  claim 7 , wherein the enzyme is a helicase enzyme or a primase enzyme. 
     
     
         16 . The method according to  claim 7 , wherein the enzyme and/or the complex is immobilized on a solid support. 
     
     
         17 . The method according to  claim 16 , comprising a plurality of enzymes and/or complexes immobilized on the solid support. 
     
     
         18 . The method according to  claim 7 , wherein the labeled polynucleotide processive enzyme comprises a single label or a single type of label. 
     
     
         19 . The method according to  claim 7 , wherein the detection is carried out by measuring a polarisation effect consequent on the altered characteristics of the label. 
     
     
         20 . The method according to  claim 19 , wherein the detection is carried out by fluorescence polarisation anisotrophy. 
     
     
         21 . The method according to  claim 7 , wherein the detecting step is repeated one or more times utilizing the same complex. 
     
     
         22 . The method according to  claim 21 , wherein the contacting and/or detecting step occur under conditions sufficient to induce enzyme activity.