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 of synchronizing enzyme-catalyzed extension of DNA primers during reactive sequencing, comprising the steps of:
 (a) providing one or more DNA template systems, each system comprising at least two nucleic acid molecules of identical but unknown sequence hybridized to identical primer oligonucleotides;   (b) contacting the DNA template system with a single type of deoxyribonucleoside triphosphate in the presence of an exonuclease deficient DNA polymerase enzyme under conditions that allow extension of the primers at the 3′ ends by incorporation of at least one deoxyribonucleoside monophosphate, which is complementary to the adjacent DNA template base, to form extended primers;   (c) determining the number of deoxyribonucleoside monophosphates incorporated at the 3′ ends of each of the extended primers by measuring the amplitude of an electrical signal with a voltmeter;   (d) identifying the type of the at least one incorporated deoxyribonucleoside monophosphate on each extended primer;   (e) removing unincorporated deoxyribonucleoside triphosphate;   (f) repeating steps (a) through (e) with each of the remaining three single types of deoxyribonucleoside triphosphates not used in step (b); and   (g) repeating steps (a) through (f) thereby sequencing the DNA through synchronous extension of DNA primers.   
     
     
         34 . The method according to  claim 33 , further comprising the step of removing any contaminating deoxyribonucleoside triphosphates from each single type of deoxyribonucleoside triphosphate. 
     
     
         35 . A method of successive single nucleotide DNA polymerase mediated extension reactions, comprising the steps of:
 (a) providing one or more DNA template systems, each system comprising at least one nucleic acid molecule of unknown sequence hybridized to a primer oligonucleotide;   (b) contacting the DNA template system with a single type of deoxyribonucleoside triphosphate in the presence of an exonuclease deficient DNA polymerase enzyme under conditions that allow extension of the primers at the 3′ ends by incorporation of at least one deoxyribonucleoside monophosphate, which is complementary to the adjacent DNA template base, to form extended primers;   (c) detecting whether extension of the primer has occurred by measuring the amplitude of an electrical signal;   (d) removing unincorporated deoxyribonucleoside triphosphate;   (e) repeating steps (a) through (d) successively with each of the remaining three single types of deoxyribonucleoside triphosphates not used in step (b) and   (f) repeating steps (a) through (e).   
     
     
         36 . The method according to  claim 35 , further comprising the step of removing any contaminating deoxyribonucleoside triphosphates from each single type of deoxyribonucleoside triphosphate. 
     
     
         37 . The method of  claim 35 , wherein said measuring the amplitude of an electrical signal comprises measurement using a voltmeter. 
     
     
         38 . The method of  claim 35 , wherein said deoxyribonucleoside triphosphate is unlabeled. 
     
     
         39 . A method of minimizing out-of-phase extended primer strands during reactive sequencing comprising the steps of:
 (a) providing one or more DNA template systems, each system comprising at least two nucleic acid molecules of identical but unknown sequence hybridized to identical primer oligonucleotides;   (b) contacting, the DNA template system with a single type of deoxyribonucleoside triphosphate in the presence of an exonuclease deficient DNA polymerase enzyme under conditions that allow extension of the primers at the 3′ ends by incorporation of at least one deoxyribonucleoside monophosphate, which is complementary to the adjacent DNA template base, to form extended primers;   (c) determining the number of deoxyribonucleoside monophosphates incorporated at the 3′ ends of each of the extended primer strands by measuring the amplitude of an electrical signal;   (d) identifying the type of the at least one incorporated deoxyribonucleoside monophosphate on each extended primer strand;   (e) removing, unincorporated deoxyribonucleoside triphosphate;   (f) repeating steps (a) through (e) with each of the remaining three single types of deoxyribonucleoside triphosphates not used in step (b); and   (g) repeating steps (a) through (f) thereby minimizing out-of-phase extended primer strands during reactive sequencing.   
     
     
         40 . The method a cording to  claim 39 , further comprising the step of removing any contaminating deoxyribonucleoside triphosphates from each single type of deoxyribonucleoside triphosphate. 
     
     
         41 . The method of  claim 39 , wherein said determining comprises measuring an electrical signal with a voltmeter. 
     
     
         42 . The method of  claim 39 , wherein said determining comprises measuring fluorescence. 
     
     
         43 . The method of  claim 39 , wherein said deoxyribonucleoside triphosphates are in concentration excess relative to said DNA template system. 
     
     
         44 . A method of controlling the phase of enzyme-catalyzed extension of DNA primers during reactive sequencing comprising the steps of:
 (a) providing one or more DNA template systems, each system comprising at least two nucleic acid molecules of identical but unknown sequence hybridized to identical primer oligonucleotides;   (b) contacting the DNA template system with a single type of deoxyribonucleoside triphosphate in the presence of an exonuclease deficient DNA polymerase enzyme under conditions that allow extension of the primers at the 3′ ends by incorporation of at least one deoxyribonucleoside monophosphate, which is complementary to the adjacent DNA template base, to form extended primers;   (c) determining the number of deoxyribonucleoside monophosphates incorporated at the 3′ ends of each of the extended primers by measuring the amplitude of an electrical signal;   (d) identifying the type of the at least one incorporated deoxyribonucleoside monophosphate on each extended primer;   (e) removing unincorporated deoxyribonucleoside triphosphate;   (f) repeating steps (a) through (e) with each of the remaining three single types of deoxyribonucleoside triphosphates not used in step (b); and   (g) repeating steps (a) through (f) thereby controlling the phase of the enzyme-catalyzed extension of DNA primers during reactive sequencing.   
     
     
         45 . The method according to  claim 44 , further comprising the step of removing any contaminating deoxyribonucleoside triphosphates from each single type of deoxyribonucleoside triphosphate. 
     
     
         46 . The method of  claim 44 , wherein said measuring the amplitude of an electrical signal comprises measurement using a voltmeter. 
     
     
         47 . The method of  claim 44 , wherein said deoxyribonucleoside triphosphates is unlabeled. 
     
     
         48 . A method of detecting successive single nucleotide DNA polymerase mediated extension reactions, comprising the steps of:
 (a) providing one or more DNA template systems, each system comprising at least one nucleic acid molecule of unknown sequence hybridized to a primer oligonucleotide;   (b) contacting the DNA template system with a single type of deoxyribonucleoside triphosphate in the presence of an exonuclease deficient DNA polymerase enzyme under conditions that allow extension of the primers at the 3′ ends by incorporation of at least one deoxyribonucleoside monophosphate, which is complementary to the adjacent DNA template base, to form extended primers;   (c) detecting whether extension of the primer has occurred by measuring the amplitude of an electrical signal;   (d) removing unincorporated deoxyribonucleoside triphosphate; and   (e) repeating steps (a) through (d) with each of the remaining three single types of deoxyribonucleoside triphosphates not used in step (b); and   (f) repeating steps (a) through (e).   
     
     
         49 . The method according to  claim 48 , further comprising the step of removing any contaminating deoxyribonucleoside triphosphates from each single type of deoxyribonucleoside triphosphate. 
     
     
         50 . The method of  claim 48 , wherein said measuring the amplitude of an electrical signal comprises measurement using a voltmeter. 
     
     
         51 . The method of  claim 48 , wherein said deoxyribonucleoside triphosphates is unlabeled.