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. An apparatus for DNA sequencing comprising:
a) at least one reaction chamber including a DNA primer/template system which produces a detectable reaction event when a DNA polymerase enzyme incorporates an unlabeled and unblocked deoxyribonucleotide monophosphate onto the 3′ end of the primer strand;
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, unlabeled and unblocked deoxyribonucleotides, and polymerase enzymes;
c) a system for converting said detectable reaction event into an electrical signal based on an electrical potential generated from said detectable reaction event in the at least one reaction chamber; and
d) a voltmeter configured to measure said electrical signal in the at least one reaction chamber.
2. The apparatus of claim 1 , wherein said at least one reaction chamber further comprises said deoxyribonucleotide monophosphate, and wherein said deoxyribonucleotide monophosphate is non-chemically modified.
3. The apparatus of claim 1 , wherein said at least one reaction chamber further includes a solid support, wherein said primer/template system comprises a primer and a template, and wherein said template is tethered to said solid support.
4. The apparatus of claim 3 , wherein said template comprises a linker moiety for tethering to said solid support.
5. The apparatus of claim 1 , wherein said DNA polymerase lacks 5′ to 3′ exonuclease activity.
6. The apparatus of claim 1 , wherein the amplitude of said electrical signal corresponds to the number of nucleotides added to said primer strand.
7. The apparatus of claim 1 , wherein said apparatus provides real-time detection of incorporation of said deoxyribonucleotide monophosphate onto said primer strand.
8. A method of DNA sequencing comprising:
a) providing in at least one reaction chamber a primer/template system comprising a template sequence hybridized to a primer oligonucleotide in the presence of a DNA polymerase;
b) contacting said primer/template system with a single type of an unlabeled and unblocked deoxyribonucleotide under conditions which produces a detectable reaction event when said DNA polymerase incorporates a deoxyribonucleotide onto the 3′ end of said primer oligonucleotide;
c) converting said detectable reaction event into an electrical signal based on an electrical potential generated from said detectable reaction event in the at least one reaction chamber; and
d) measuring said electrical signal with a voltmeter in the at least one reaction chamber.
9. The method of 8 , further comprising (e) flushing said reaction chamber with dNTP free buffer.
10. The method of claim 9 , further comprising repeating steps (b) through (e).
11. The method of claim 9 , further comprising repeating steps (b) through (e) until the complete nucleotide sequence of said template sequence is determined.
12. The method of claim 8 , wherein said deoxyribonucleotide is non-chemically modified.
13. The method of claim 8 , wherein said at least one reaction chamber further includes a solid support, and wherein said template sequence is tethered to said solid support.
14. The method of claim 13 , wherein said template sequence comprises a linker moiety for tethering to said solid support.
15. The method of claim 8 , wherein said DNA polymerase lacks 5′ to 3′ exonuclease activity.
16. The method of claim 8 , wherein the amplitude of said electrical signal corresponds to the number of nucleotides added to said primer strand by said DNA polymerase.
17. The method of claim 8 , wherein said electrical signal provides real-time detection of incorporation of said deoxyribonucleotide monophosphate onto said primer strand by said DNA polymerase.
18. The apparatus of claim 1 , wherein the at least one reaction chamber comprises a plurality of reaction chambers formed on an array device.
19. The method of claim 8 , wherein the at least one reaction chamber comprises a plurality of reaction chambers formed on an array device.
20. An apparatus for DNA sequencing, comprising:
a) a reaction chamber including a polymerase and a DNA primer/template system that produces a non-optical signal when a DNA polymerase enzyme incorporates an unlabeled and unblocked deoxyribonucleotide monophosphate onto the 3′ end of the primer strand;
b) a component for introducing into, and evacuating from, said reaction chamber a solution including a single type of unlabeled and unblocked deoxyribonucleotide;
c) a system attached to a surface of the reaction chamber and configured to convert said detectable signal into an electrical signal; and
d) a voltmeter configured to measure said electrical signal.
21. The apparatus of claim 20 , wherein the reaction chamber further comprises the unlabeled and unblocked deoxyribonucleotide monophosphate.
22. The apparatus of claim 20 , wherein the reaction chamber further includes a solid support, wherein said DNA primer/template system comprises a primer and a template, and wherein said template is tethered to said solid support.
23. The apparatus of claim 22 , wherein said template comprises a linker moiety for tethering to said solid support.
24. The apparatus of claim 20 , wherein said DNA polymerase lacks 5′ to 3′ exonuclease activity.
25. The apparatus of claim 20 , wherein the amplitude of said electrical signal corresponds to the number of nucleotides added to said primer strand.
26. The apparatus of claim 20 , wherein said apparatus provides real-time detection of incorporation of said deoxyribonucleotide monophosphate onto said primer strand.
27. The apparatus of claim 20 comprising a plurality of reaction chambers, wherein the plurality of reaction chambers is formed on an array device.
28. A method for DNA sequencing, comprising:
a) providing in a reaction chamber a polymerase and a DNA primer/template system;
b) contacting said DNA primer/template system with a single type of unlabeled and unblocked deoxyribonucleotide monophosphate under conditions which produces a non-optical signal when a DNA polymerase enzyme incorporates said unlabeled and unblocked deoxyribonucleotide monophosphate onto the 3′ end of the primer strand;
c) converting with a system attached to a surface of the reaction chamber said non-optical signal into an electrical signal; and
d) measuring said electrical signal with a voltmeter.
29. The method of claim 28 , further comprising (e) flushing said reaction chamber with dNTP free buffer.
30. The method of claim 29 , further comprising repeating steps (b) through (e).
31. The method of claim 29 , further comprising repeating steps (b) through (e) until the complete nucleotide sequence of said template sequence is determined.
32. The method of claim 28 , wherein said reaction chamber further includes a solid support, and wherein said template sequence is tethered to said solid support.
33. The method of claim 32 , wherein said template sequence comprises a linker moiety for tethering to said solid support.
34. The method of claim 28 , wherein said DNA polymerase lacks 5′ to 3′ exonuclease activity.
35. The method of claim 28 , wherein the amplitude of said electrical signal corresponds to the number of nucleotides added to said primer strand by said DNA polymerase.
36. The method of claim 28 , wherein said electrical signal provides real-time detection of incorporation of said deoxyribonucleotide monophosphate onto said primer strand by said DNA polymerase.
37. The method of claim 28 , wherein said reaction chamber comprises a plurality of reaction chambers formed on an array device.