Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
First Claim
1. A method for sequencing DNA, comprising:
- a) providing a primer/template system comprising a template sequence hybridized to a primer oligonucleotide and a DNA polymerase that lacks 5′
to 3′
exonuclease activity;
b) contacting the primer/template system with a single type of deoxyribonucleotide under conditions that produce a detectable signal when the DNA polymerase incorporates a deoxyribonucleotide onto the 3′
end of the primer oligonucleotide, wherein the single type of deoxyribonucleotide is an unlabeled and unblocked deoxyribonucleotide, and wherein the contacting occurs in a reaction chamber;
c) converting, with a device, the detectable signal into an electrical signal based on an electrical potential generated across the device by the detectable signal, wherein the converting occurs in the reaction chamber, and wherein the amplitude of the electrical signal corresponds to the number of nucleotides incorporated onto the 3′
end of the primer oligonucleotide by the DNA polymerase; and
d) detecting the electrical signal generated by the detectable signal produced in the reaction chamber.
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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.
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Citations
11 Claims
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1. A method for sequencing DNA, comprising:
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a) providing a primer/template system comprising a template sequence hybridized to a primer oligonucleotide and a DNA polymerase that lacks 5′
to 3′
exonuclease activity;b) contacting the primer/template system with a single type of deoxyribonucleotide under conditions that produce a detectable signal when the DNA polymerase incorporates a deoxyribonucleotide onto the 3′
end of the primer oligonucleotide, wherein the single type of deoxyribonucleotide is an unlabeled and unblocked deoxyribonucleotide, and wherein the contacting occurs in a reaction chamber;c) converting, with a device, the detectable signal into an electrical signal based on an electrical potential generated across the device by the detectable signal, wherein the converting occurs in the reaction chamber, and wherein the amplitude of the electrical signal corresponds to the number of nucleotides incorporated onto the 3′
end of the primer oligonucleotide by the DNA polymerase; andd) detecting the electrical signal generated by the detectable signal produced in the reaction chamber. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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Specification