Method of determining the nucleotide sequence of Oligonucleotides and DNA molecules
First Claim
1. An apparatus for DNA sequencing using chemically-modified dNTPs comprising:
- (a) at least one reaction chamber comprising a surface, wherein said reaction chamber comprises;
i) a primer/template system comprising a template sequence hybridized to a universal primer, wherein said primer/template system is tethered to said surface;
ii) an excess of chemically modified dNTPs, wherein said chemically modified dNTPs each comprise;
A) a dNTP, b) a fluorescent label, and c) a chemically-cleavable linker between said dNTP and said fluorescent label; and
iii) a polymerase mutant, wherein said polymerase mutant is capable of more efficiently incorporating said chemically modified dNTPs into said primer-template system than the corresponding wild-type enzyme;
(b) a component for introducing into, and evacuating from, said reaction chamber at least one reagent selected from the group consisting of;
buffers, electrolytes, DNA template, DNA primer, deoxyribonucleotides, and polymerase enzymes;
(c) a component for illuminating said chemically modified dNTPs with optical radiation at a wavelength absorbed by said fluorescent label, and(d) a device capable of sensing fluorescence from said chemically modified dNTPs.
4 Assignments
0 Petitions
Accused Products
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.
-
Citations
6 Claims
-
1. An apparatus for DNA sequencing using chemically-modified dNTPs comprising:
-
(a) at least one reaction chamber comprising a surface, wherein said reaction chamber comprises; i) a primer/template system comprising a template sequence hybridized to a universal primer, wherein said primer/template system is tethered to said surface; ii) an excess of chemically modified dNTPs, wherein said chemically modified dNTPs each comprise;
A) a dNTP, b) a fluorescent label, and c) a chemically-cleavable linker between said dNTP and said fluorescent label; andiii) a polymerase mutant, wherein said polymerase mutant is capable of more efficiently incorporating said chemically modified dNTPs into said primer-template system than the corresponding wild-type enzyme; (b) a component for introducing into, and evacuating from, said reaction chamber at least one reagent selected from the group consisting of;
buffers, electrolytes, DNA template, DNA primer, deoxyribonucleotides, and polymerase enzymes;(c) a component for illuminating said chemically modified dNTPs with optical radiation at a wavelength absorbed by said fluorescent label, and (d) a device capable of sensing fluorescence from said chemically modified dNTPs.
-
-
2. A method of DNA sequencing comprising:
-
(a) providing a primer/template system comprising a template sequence hybridized to a primer oligonucleotide, (b) contacting said primer/template system with a polymerase mutant and an excess of chemically-modified dNTPs under conditions such that a chemically modified dNTP is incorporated onto the 3′
end of said primer oligonucleotide,wherein said chemically modified dNTPs each comprise;
A) a dNTP, b) a fluorescent label, and c) a chemically-cleavable linker between said dNTP and said fluorescent label; andwherein said polymerase mutant is capable of more efficiently incorporating said chemically modified dNTPs into said primer-template system than the corresponding wild-type enzyme; (c) illuminating said chemically modified dNTP that is incorporated into said primer with optical radiation at a wavelength absorbed by said fluorescent label; (d) detecting said chemically modified dNTP; and (e) treating said chemically modified dNTP that is incorporated into said primer oligonucleotide such that said fluorescent label, but not said linker, is cleaved off. - View Dependent Claims (3, 4, 5, 6)
-
Specification