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AssigneeArizona Board of Regents
InventorsPeter Williams, Thomas J. Taylor, Daniel J.B. Williams, Ian Gould, Mark A. Hayes
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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.
-32. (canceled)
A method of synchronizing enzyme-catalyzed extension of DNA primers during reactive sequencing, comprising the steps of:
The method according to claim 1, further comprising the step of removing any contaminating deoxyribonucleoside triphosphates from each single type of deoxyribonucleoside triphosphate.
The method of claim 1, wherein said determining comprises measuring an electrical signal with a voltmeter.
The method of claim 1, wherein said determining comprises measuring fluorescence.
The method of claim 1, wherein said deoxyribonucleoside triphosphates are in concentration excess relative to said DNA template systems.
A method of successive single nucleotide DNA polymerase mediated extension reactions, comprising the steps of:
The method according to claim 6, further comprising the step of removing any contaminating deoxyribonucleoside triphosphates from each single type of deoxyribonucleoside triphosphate.
The method of claim 6, wherein said measuring the amplitude of an electrical signal comprises measurement using a voltmeter.
The method of claim 6, wherein said deoxyribonucleoside triphosphate is unlabeled.
A method of minimizing out-of-phase extended primer strands during reactive sequencing comprising the steps of:
The method according to claim 10, further comprising the step of removing any contaminating deoxyribonucleoside triphosphates from each single type of deoxyribonucleoside triphosphate.
The method of claim 10, wherein said determining comprises measuring an electrical signal with a voltmeter.
The method of claim 10, wherein said determining comprises measuring fluorescence.
The method of claim 10, wherein said deoxyribonucleoside triphosphates are in concentration excess relative to said DNA template system.
A method of controlling the phase of enzyme-catalyzed extension of DNA primers during reactive sequencing comprising the steps of:
The method according to claim 15, further comprising the step of removing any contaminating deoxyribonucleoside triphosphates from each single type of deoxyribonucleoside triphosphate.
The method of claim 15, wherein said measuring the amplitude of an electrical signal comprises measurement using a voltmeter.
The method of claim 15, wherein said deoxyribonucleoside triphosphates is unlabeled.
A method of detecting successive single nucleotide DNA polymerase mediated extension reactions, comprising the steps of:
The method according to claim 19, further comprising the step of removing any contaminating deoxyribonucleoside triphosphates from each single type of deoxyribonucleoside triphosphate.
The method of claim 19, wherein said measuring the amplitude of an electrical signal comprises measurement using a voltmeter.
The method of claim 19, wherein said deoxyribonucleoside triphosphates is unlabeled.
Dependencies are inferred from the claim text.