Single nucleotide detection method
A method for determining the sequence of nucleotide bases in a polynucleotide analyte is provided. It is characterised by the steps of (1) generating a stream of single nucleotide bases from the analyte; (2) producing captured molecules by reacting each single nucleotide base with a capture system; (3) amplifying at least part of the captured molecule to produce a plurality of amplicons characteristic of the single nucleotide base; (4) labelling the amplicons with a corresponding probe having a characteristic detectable element and (5) detecting a property characteristic of the detectable element.
1. A method for determining the sequence of nucleotide bases in a polynucleotide analyte, the method comprising steps of:
(1) generating a stream of single nucleotide bases from the polynucleotide analyte, wherein the stream of single nucleotide bases is obtained by progressive pyrophosphorolysis of the polynucleotide analyte;
(2) producing captured molecules by reacting each single nucleotide base with a capture system, wherein the capture system comprises different sets of capture molecules, each set being selective for a different nucleotide base;
(3) for each captured molecule, amplifying at least part of the captured molecule to produce a plurality of amplicons;
(4) for each captured molecule, labelling the amplicons with a corresponding probe selective for the amplicons and comprising a different detectable element, wherein the probes are present in a mixture of probes each selective for a different nucleotide base;
(5) for each captured molecule, detecting a property characteristic of the detectable element; and
(6) from the totality of the results of step (5), determining the sequence of nucleotide bases in the polynucleotide analyte.
2. The method as claimed in claim 1 , wherein the capture system is comprised of two components for each different nucleotide base: (a) a first oligonucleotide comprising a double-stranded region and a single-stranded region and (b) a second oligonucleotide that is single-stranded and whose nucleotide base sequence is at least partially complementary to that of the single-stranded region of the first oligonucleotide one nucleotide base beyond the end of the double-stranded region.
3. The method as claimed in claim 2 , wherein the first oligonucleotide is j-shaped.
4. The method as claimed in claim 2 , wherein the total length of the first oligonucleotide is from 20 to 100 nucleotide bases.
5. The method as claimed in claim 1 , wherein the capture system comprises a single oligonucleotide for each different nucleotide base, consisting of a single-stranded nucleotide region the ends of which are attached to two different double-stranded oligonucleotide regions.
6. The method as claimed in claim 5 , wherein each double-stranded oligonucleotide region is comprised of from 10 to 30 nucleotide pairs.
7. The method as claimed in claim 5 , wherein two discrete double-stranded oligonucleotide regions are employed, each comprising ends remote from the single-stranded nucleotide region which are closed-looped.
8. The method as claimed in claim 5 , wherein the double-stranded oligonucleotide regions are derivable from a single-stranded oligonucleotide precursor by folding the ends back on themselves to leave a gap comprising the single-stranded nucleotide region.
9. The method as claimed in claim 1 , wherein the polynucleotide analyte is bound to a surface.
10. The method as claimed in claim 1 , wherein step (1) is carried out under non-equilibrium conditions in the presence of a flowing aqueous medium comprising an enzyme, a pyrophosphate anion and magnesium cations, and wherein the single nucleotide bases are continuously removed from a reaction zone where they are generated.
11. The method as claimed in claim 10 , wherein between steps (1) and (2), any residual pyrophosphate anion is destroyed by means of a pyrophosphatase.
12. The method as claimed in claim 1 , wherein the capture system comprises four different first oligonucleotides, each of which has one of four different single-stranded regions, and four different second oligonucleotides, each of which has a sequence complementary to a part of one of the four different single-stranded regions in the four different first oligonucleotides.
13. The method as claimed in claim 1 , wherein steps (3) and (4) are carried out simultaneously.
14. The method as claimed in claim 1 , wherein the amplification in step (3) is carried out using a method selected from the polymerase chain reaction, recombinase polymerase amplification and rolling circle amplification.
15. The method as claimed in claim 1 , wherein step (2) employs a ligase which is deactivated before step (4) occurs.
16. The method as claimed in claim 1 , wherein the probes are molecular beacons.
17. The method as claimed in claim 1 , wherein in step (5) the detectable element is a fluorophore and fluorescence emitted by fluorophores is detected.
18. The method as claimed in claim 1 , wherein at least one of steps (1) to (5) is carried out in microdroplets.
19. A method for determining the sequence of nucleotide bases in a polynucleotide analyte, the method comprising steps of:
(1) generating a stream of single nucleotide bases from the polynucleotide analyte;
(2) producing captured molecules by reacting each single nucleotide base with a capture system, wherein the capture system comprises different sets of capture molecules, each set being selective for a different nucleotide base, and wherein the capture system is comprised of two components for each different nucleotide base: (a) a first oligonucleotide comprising a double-stranded region and a single-stranded region and (b) a second oligonucleotide that is single-stranded and whose nucleotide base sequence is at least partially complementary to that of the single-stranded region of the first oligonucleotide one nucleotide base beyond the end of the double-stranded region;
(3) for each captured molecule, amplifying at least part of the captured molecule to produce a plurality of amplicons;
(4) for each captured molecule, labelling the amplicons with a corresponding probe that is selective for the amplicons and comprising a different detectable element, wherein the probes are present in a mixture of probes each selective for a different nucleotide base;
(5) for each captured molecule, detecting a property characteristic of the detectable element; and
(6) from the totality of the results of step (5), determining the sequence of nucleotide bases in the polynucleotide analyte.
20. The method as claimed in claim 19 , wherein the stream of single nucleotide bases in step (1) is obtained by action of (i) a kinase and (ii) an exonuclease.
21. The method as claimed in claim 19 , wherein the polynucleotide analyte is bound to a surface.
22. The method as claimed in claim 19 , wherein step (1) is carried out under non-equilibrium conditions in the presence of a flowing aqueous medium comprising an enzyme, a pyrophosphate anion and magnesium cations, and wherein the single nucleotide bases are continuously removed from a reaction zone where they are generated.
23. The method as claimed in claim 22 , wherein between steps (1) and (2), any residual pyrophosphate anion is destroyed by means of a pyrophosphatase.
24. The method as claimed in claim 19 , wherein the first oligonucleotide is j-shaped.
25. The method as claimed in claim 19 , wherein the total length of the first oligonucleotide is from 20 to 100 nucleotide bases.
26. The method as claimed in claim 19 , wherein the polynucleotide analyte is DNA or RNA, and the capture system comprises four different sets of capture molecules, each set being selective for a different nucleotide base of the DNA or RNA.
27. The method as claimed in claim 19 , wherein steps (3) and (4) are carried out simultaneously.
28. The method as claimed in claim 19 , wherein the amplification in step (3) is carried out using a method selected from the polymerase chain reaction, recombinase polymerase amplification and rolling circle amplification.
29. The method as claimed in claim 19 , wherein step (2) employs a ligase which is deactivated before step (4) occurs.
30. The method as claimed in claim 19 , wherein the probes are molecular beacons.
31. The method as claimed in claim 19 , wherein in step (5) the detectable element is a fluorophore and fluorescence emitted by fluorophores is detected.
32. The method as claimed in claim 19 , wherein at least one of steps (1) to (5) is carried out in microdroplets.
33. A method for determining the sequence of nucleotide bases in a polynucleotide analyte, the method comprising steps of:
(1) generating a stream of single nucleotide bases from the polynucleotide analyte, wherein step (1) is carried out in the presence of a reaction medium comprising a polymerase that exhibits neither exonuclease or endonuclease behaviour under conditions of a pyrophosphorolysis reaction;
(2) producing captured molecules by reacting each single nucleotide base with a capture system, wherein the capture system comprises different sets of capture molecules, each set being selective for a different nucleotide base;
(3) for each captured molecule, amplifying at least part of the captured molecule to produce a plurality of amplicons;
(4) for each captured molecule, labelling the amplicons with a corresponding probe that is selective for the amplicons and comprising a different detectable element, wherein the probes are present in a mixture of probes each selective for a different nucleotide base;
(5) for each captured molecule, detecting a property characteristic of the detectable element; and
(6) from the totality of the results of step (5), determining the sequence of nucleotide bases in the polynucleotide analyte.
34. The method as claimed in claim 33 , wherein the capture system is comprised of two components for each different nucleotide base: (a) a first oligonucleotide comprising a double-stranded region and a single-stranded region and (b) a second oligonucleotide that is single-stranded and whose nucleotide base sequence is at least partially complementary to that of the single-stranded region of the first oligonucleotide one nucleotide base beyond the end of the double-stranded region.
35. The method as claimed in claim 34 , wherein the first oligonucleotide is j-shaped.
36. The method as claimed in claim 34 , wherein the total length of the first oligonucleotide is from 20 to 100 nucleotide bases.
37. The method as claimed in claim 33 , wherein the capture system comprises a single oligonucleotide for each different nucleotide base, consisting of a single-stranded nucleotide region the ends of which are attached to two different double-stranded oligonucleotide regions.
38. The method as claimed in claim 37 , wherein each double-stranded oligonucleotide region is comprised of from 10 to 30 nucleotide pairs.
39. The method as claimed in claim 37 , wherein two discrete double-stranded oligonucleotide regions are employed, each comprising ends remote from the single-stranded nucleotide region which are closed-looped.
40. The method as claimed in claim 37 , wherein the double-stranded oligonucleotide regions are derivable from a single-stranded oligonucleotide precursor by folding the ends back on themselves to leave a gap comprising the single-stranded nucleotide region.
41. The method as claimed in claim 33 , wherein the polynucleotide analyte is bound to a surface.
42. The method as claimed in claim 33 , wherein step (1) is carried out under non-equilibrium conditions in the presence of a flowing aqueous medium comprising the polymerase, a pyrophosphate anion and magnesium cations, and wherein the single nucleotide bases are continuously removed from a reaction zone where they are generated.
43. The method as claimed in claim 42 , wherein between steps (1) and (2), any residual pyrophosphate anion is destroyed by means of a pyrophosphatase.
44. The method as claimed in claim 33 , wherein the polynucleotide analyte is DNA or RNA, and the capture system comprises four different sets of capture molecules, each set being selective for a different nucleotide base of the DNA or RNA.
45. The method as claimed in claim 33 , wherein steps (3) and (4) are carried out simultaneously.
46. The method as claimed in claim 33 , wherein the amplification in step (3) is carried out using a method selected from the polymerase chain reaction, recombinase polymerase amplification and rolling circle amplification.
47. The method as claimed in claim 33 , wherein step (2) employs a ligase which is deactivated before step (4) occurs.
48. The method as claimed in claim 33 , wherein the probes are molecular beacons.
49. The method as claimed in claim 33 , wherein in step (5) the detectable element is a fluorophore and fluorescence emitted by fluorophores is detected.
50. The method as claimed in claim 33 , wherein at least one of steps (1) to (5) is carried out in microdroplets.