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 poly-merase-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 for sequencing nucleic acid, the method comprising the steps of:
hybridizing nucleic acid to a complementary nucleic acid sequence to form one or more duplexes;
adding at least one nucleotide comprising a detectable label to said duplex, wherein said nucleotide is not a dideoxy nucleotide and wherein said nucleotide has a free 3′ hydroxyl when added to said duplex;
identifying said nucleotide comprising said detectable label; and
repeating said adding and identifying steps, thereby to determine the sequence of said nucleic acid.
2. The method of claim 1 , wherein said identifying step comprises detecting pyrophosphate production.
3. The method of claim 1 , further comprising amplifying nucleic acid prior to said hybridizing step.
4. The method of claim 1 , further comprising immobilizing said one or more duplexes on a substrate.
5. The method of claim 4 , wherein said one or more duplexes are immobilized such that said one or more duplexes are individually optically resolvable on said substrate.
6. The method of claim 1 , wherein said detectable label is an optically-detectable label.
7. The method of claim 6 , wherein said optically-detectable label is a fluorescent label.
8. The method of claim 6 , wherein said optically-detectable label is a chemiluminescent label.