Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
View Patent ↗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. A method of nucleic acid sequencing, the method comprising the steps of:
(a) providing a nucleic acid template/primer system comprising individual template/primer duplexes immobilized on a surface;
(b) exposing said individual template/primer duplexes to a polymerase and one or more type of nucleotide comprising an optically-detectable label, wherein said optically-detectable label is not attached to the 3′ position of the sugar moiety of said nucleotide;
(c) removing nucleotide that is not incorporated into a primer;
(d) detecting incorporated nucleotide by the presence of said label;
(e) removing said label from said incorporated nucleotide;
(f) repeating steps (b) through (e) at least once with a different type of nucleotide comprising an optically-detectable label for incorporation into said primer; and
(g) determining a nucleic acid sequence based upon said incorporated nucleotides.
2. The method of claim 1 , wherein said label is a fluorescent label.
3. The method of claim 2 , wherein said fluorescent label is selected from the group consisting of fluorescein, rhodamine and BODIPY.
4. The method of claim 2 , wherein said detecting step comprises sensing said label.
5. The method of claim 1 , wherein said detecting step comprises resolving an image of said duplex.