Method of determining the nucleotide sequence of oligonucleotides and dna molecules

Abstract

The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions.

1 - 32 . (canceled) 
     
     
         33 . A method for sequencing a nucleic acid molecule, the method comprising the steps of:
 (a) providing a nucleic acid duplex comprising a primer nucleic acid hybridized to a template nucleic acid, wherein the duplex is bound to a polymerase that is attached to a substrate;   (b) contacting the duplex with a single type of nucleotide under conditions that allow extension of the primer by incorporation of at least one nucleotide to the 3′ end of the primer to form an extended primer;   (c) detecting whether extension of the primer has occurred; and   (d) sequentially repeating steps (b) and (c) at least once, wherein sequential detection of incorporated nucleotide determines the sequence of the nucleic acid.   
     
     
         34 . The method according to  claim 33 , wherein the nucleotide comprises a detectable label. 
     
     
         35 . The method according to  claim 34 , wherein the detectable label is an optically detectable label. 
     
     
         36 . The method according to  claim 35 , wherein the optically detectable label is a fluorescent label. 
     
     
         37 . The method according to  claim 35 , wherein the optically detectable label is a chemiluminescent label. 
     
     
         38 . The method according to  claim 33 , wherein the polymerase is a DNA polymerase. 
     
     
         39 . The method according to  claim 33 , wherein the polymerase is a reverse transcriptase. 
     
     
         40 . The method according to  claim 33 , wherein the polymerase has reduced exonuclease activity. 
     
     
         41 . The method according to  claim 33 , wherein the polymerase is covalently attached to the substrate. 
     
     
         42 . The method according to  claim 33 , wherein the polymerase is non-covalently attached to the subject. 
     
     
         43 . The method according to claim  1 , wherein detecting is observing pyrophosphate production. 
     
     
         44 . A method for sequencing a nucleic acid molecule, the method comprising the steps of:
 (a) providing a nucleic acid duplex comprising a primer nucleic acid hybridized to a template nucleic acid, wherein the duplex is bound to a polymerase that is attached to a substrate;   (b) contacting the duplex with a single type of detectably labeled nucleotide under conditions that allow extension of the primer by incorporation of at least one labeled nucleotide to the 3′ end of the primer to form an extended primer;   (c) detecting whether extension of the primer has occurred;   (d) detecting the number of labeled nucleotides incorporated into the primer;   (e) removing or silencing the label on the incorporated nucleotide; and   (f) optionally repeating steps (b) through (e) at least once to determine the nucleotide sequence of the nucleic acid molecule.   
     
     
         45 . The method according to  claim 44 , wherein the detectably labeled nucleotide is an optically detectably labeled nucleotide. 
     
     
         46 . The method according to  claim 45 , wherein the optically detectably labeled nucleotide is a fluorescently labeled nucleotide. 
     
     
         47 . The method according to  claim 45 , wherein the optically detectably labeled nucleotide is a chemiluminescently labeled nucleotide. 
     
     
         48 . The method according to  claim 44 , wherein the polymerase is a DNA polymerase. 
     
     
         49 . The method according to  claim 44 , wherein the polymerase is a reverse transcriptase. 
     
     
         50 . The method according to  claim 44 , wherein the polymerase has reduced exonuclease activity. 
     
     
         51 . The method according to  claim 44 , wherein the fluorescent label on the incorporated nucleotide is silenced by reaction with compounds capable of extracting an electron from the excited state of the fluorescent moiety. 
     
     
         52 . The method of  claim 51  wherein the compound is a diphenyliodonium salt. 
     
     
         53 . A method for sequencing a nucleic acid molecule, the method comprising the steps of:
 (a) providing a nucleic acid duplex comprising a primer nucleic acid hybridized to a template nucleic acid;   (b) contacting the duplex with a single type of nucleotide under conditions that allow extension of the primer by incorporation of at least one nucleotide to the 3′ end of the primer to form an extended primer, wherein the nucleotide comprises a non-optically detectable label;   (c) detecting whether extension of the primer has occurred; and   (d) sequentially repeating steps (b) and (c) at least once, wherein sequential detection of incorporated nucleotide determines the sequence of the nucleic acid.   
     
     
         54 . The method according to  claim 53 , wherein the duplex is bound to a polymerase that is attached to a substrate.