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 DNA sequencing comprising: providing a) a DNA template system comprising a nucleic acid molecule hybridized to a universal primer oligonucleotide and a DNA polymerase enzyme, and b) at least one deoxyribonucleotide comprising a detectable fluorescent label that is not attached to the 3′ hydroxyl; extending under conditions wherein said DNA polymerase extends said primer oligonucleotide by at least one nucleotide base to form an extended primer; identifying said at least one nucleotide base on said extended primer by observing the DNA template system; and repeating said extending and identifying steps at least once to determine the nucleotide sequence of the nucleic acid molecule.
2. The method of claim 1 , wherein said at least one deoxyribonucleotide comprises a single type of deoxyribonucleotide.
3. The method of claim 1 , wherein said identifying comprising detecting said detectable fluorescent label on said extended primer.
4. The method of claim 1 , further comprising a step wherein excess deoxyribonucleotide is removed after said extending and prior to said identifying.
5. The method of claim 1 , wherein said polymerase is a mutant polymerase.
6. The method of claim 1 , wherein said label is attached to said nucleotide via a chemically-cleavable linker.
7. The method of claim 1 , wherein said nucleotides comprising the labels are present in excess concentration relative to said template.
8. The method of claim 1 , wherein said identifying step comprises real-time detection of polymerase-catalyzed incorporation of said incorporated nucleotide.
9. The method of claim 8 , wherein said real-time detection comprises detecting said incorporated nucleotide while an incorporation reaction is occurring.
10. The method of claim 1 , wherein said template is an unknown sequence.
11. The method of claim 1 , wherein said template is generated via PCR amplification.
12. The method of claim 1 , wherein said template is directly attached to said solid phase.
13. The method of claim 1 , wherein said template is indirectly attached to said solid phase.
14. The method of claim 1 , wherein said primer/template system is immobilized such that said primer/template system is individually optically resolvable on said solid phase.
15. The method of claim 1 , wherein said solid phase is in a microfluidic system.