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 . A method for determining the sequence of a polynucleotide, comprising:
 (a) introducing a polymerase enzyme to a target-primer complex to form a primed complex, wherein the target-primer complex comprises a target polynucleotide and a complementary primer sequence;   (b) introducing one or more nucleotides to the primed complex under conditions sufficient for a polymerase reaction; and   (c) detecting a change in local pH as the polymerase enzyme processes along the target polynucleotide, wherein the local pH change results from the extension of the complementary primer sequence by the incorporation of a nucleotide complementary to the target polynucleotide,   wherein steps (b) and (c) are repeated one or more times utilizing the primed complex, thereby determining the sequence of the target polynucleotide.   
     
     
         2 . The method according to  claim 1 , wherein steps (b) and (c) are conducted with each of the different nucleotides in turn, until incorporation is detected, and then repeated. 
     
     
         3 . The method of  claim 2 , wherein each of the different nucleotides is selected from the group comprising dATP, dTTP, dCTP, and dGTP. 
     
     
         4 . The method of  claim 1 , wherein step (c) is repeated one or more times utilizing the primed complex without repeating step (b). 
     
     
         5 . The method of  claim 1 , wherein step (c) is repeated one or more times utilizing the primed complex in the presence of the nucleotides of step (b). 
     
     
         6 . The method according to  claim 1 , wherein the detecting step occurs in a flow cell. 
     
     
         7 . The method according to  claim 1 , wherein the primed complex is immobilized on a solid support. 
     
     
         8 . The method of  claim 1 , comprising a plurality of primed complexes immobilized on the solid support. 
     
     
         9 . The method of  claim 1 , wherein the method is used to identify a mutation in a polynucleotide. 
     
     
         10 . The method according to  claim 2 , wherein the detecting step occurs in a flow cell. 
     
     
         11 . The method according to  claim 2 , wherein the primed complex is immobilized on a solid support. 
     
     
         12 . The method of  claim 2 , comprising a plurality of primed complexes immobilized on the solid support. 
     
     
         13 . The method of  claim 2 , wherein the method is used to identify a mutation in a polynucleotide. 
     
     
         14 . The method according to  claim 4 , wherein the primed complex is immobilized on a solid support. 
     
     
         15 . The method of  claim 4 , comprising a plurality of primed complexes immobilized on the solid support. 
     
     
         16 . The method of  claim 4 , wherein the method is used to identify a mutation in a polynucleotide. 
     
     
         17 . The method according to  claim 5 , wherein the primed complex is immobilized on a solid support. 
     
     
         18 . The method of  claim 5 , comprising a plurality of primed complexes immobilized on the solid support. 
     
     
         19 . The method of  claim 5 , wherein the method is used to identify a mutation in a polynucleotide. 
     
     
         20 . A system for determining the sequence of a polynucleotide according to the method of  claim 1 .