Electrochemical Detection Of Polymerase Reactions By Specific Metal-Phosphate Complex Formation

Abstract

The present invention relates to a nucleic acid sequencing method that determines the sequence of a template nucleic acid by electrochemical means via the depletion of metal ions as a result of its binding to and/or precipitation with pyrophosphate.

1 . An electrochemical nucleic acid sequencing method comprising the steps of:
 a. providing a sample comprising a template nucleic acid to be sequenced;   b. annealing a primer molecule to said template nucleic acid;   c. carrying out nucleic acid elongation steps with a polymerase on said template in the presence of metal ions, wherein each step comprises the addition of a single type of nucleotide, wherein pyrophosphate is released in a stoichiometric ratio to the number of nucleotides incorporated, and wherein the metal ions are capable of specifically binding to pyrophosphate;   d. determining in said sample the potential, current or charge at an electrode at least twice, wherein between the first time and the second time one or more elongation steps take place or, alternatively, determining the potential, current or charge continuously; and   e. correlating the change in potential, current or charge over the number of elongation steps with the type of nucleotides added.   
     
     
         2 . The method according to  claim 1 , wherein the nucleic acid sequencing method is based on pyrosequencing, reversible-terminator-pyrosequencing, or closed complex formation sequencing. 
     
     
         3 . The method according to  claims 1  or  2 , wherein step d. and optionally step e. is done after each elongation step c. 
     
     
         4 . A method for detecting a nucleic acid polymerase reaction comprising the steps of:
 a. providing a sample comprising a nucleic acid;   b. carrying out a nucleic acid polymerase reaction step on said sample in the presence of metal ions, wherein pyrophosphate is released or depleted depending on the type of polymerase reaction, and wherein the metal ions are capable of specifically binding to pyrophosphate;   c. determining in said sample the potential, current or charge at an electrode at least twice, wherein between the first time and the second time one or more nuclei acid polymerase reaction steps take place or, alternatively, determining the potential, current or charge continuously; and   d. correlating the difference in potential, current or charge over the number of polymerase reaction steps and/or time with the progress of the nucleic acid polymerase reaction.   
     
     
         5 . The method according to  claim 4 , wherein the nucleic acid polymerase reaction is a synthesis reaction, an amplification reaction and/or a transcription reaction. 
     
     
         6 . The method according to  claim 4  or  5 , wherein step c. and optionally step d. is done after each polymerase reaction step b. 
     
     
         7 . A method of detecting the formation or depletion of pyrophosphate In a sample comprising the steps of:
 a. providing a sample in which pyrophosphate is formed or depleted;   b. adding to said sample metal ons, wherein the metal ions are capable of specifically binding pyrophosphate;   c. determining in said sample the potential, current or charge at an electrode a first and at least a second time or continuously;   d. correlating the difference in potential, current or charge over time with the formation or depletion of pyrophosphate in the sample.   
     
     
         8 . A method of electrochemically quantifying pyrophosphate in a sample comprising the steps of:
 a. adding to a sample comprising pyrophosphate a predetermined amount of metal ions or metal ions of a known potential, current or charge, wherein the metal ions are capable of specifically binding pyrophosphate;   b. determining in said sample the potential, current or charge at an electrode; and   c. correlating the potential, current or charge with the amount of pyrophosphate present in said sample.   
     
     
         9 . The method according to any of the preceding claims, wherein the metal ions are selected from the group of manganese ions, aluminium ions, tin ions and iron ions. 
     
     
         10 . The method according to any of the preceding claims, wherein the metal ions are manganese ions. 
     
     
         11 . The method according to any of the preceding claims, wherein the electrode is based on manganese oxide. 
     
     
         12 . An electrochemical nucleic ad sequencing apparatus comprising:
 a. means for carrying out the steps of annealing and elongating a primer molecule to a template nucleic ad to be sequenced; and   b. an electrochemical cell comprising metal ions being capable of specifically binding to pyrophosphate and a working electrode responsive to said metal ions.   
     
     
         13 . The electrochemical nucleic acid sequencing apparatus according to  claim 12 , wherein the metal ions are manganese ions and the working electrode is based on manganese oxide.