Target Polynucleotide Detection and Sequencing by Incorporation of Modified Nucleotides for Nanaopore Analysis

1 . A method of determining the presence or absence of a target polynucleotide suspected of being present in a sample, comprising:

providing a sample suspected of comprising a target polynucleotide;

providing one or more modified nucleotides;

performing a template-driven polymerization reaction on said sample to incorporate said one or more modified nucleotides into a growing strand complementary to said target polynucleotide, if present, to generate an polynucleotide reaction product comprising said one or more modified nucleotides;

loading said sample into a device comprising a nanopore, wherein said nanopore separates an interior space of the device into two volumes;

configuring the device to pass the polynucleotide reaction product, if present, through said nanopore, wherein the device comprises a sensor configured to detect an electrical signal generated by objects passing through the nanopore; and

detecting the presence or absence of said target polynucleotide in said sample by determining whether a polynucleotide comprising said one or more modified nucleotides passed through the nanopore using said electrical signal from the sensor.

2 . The method of claim 1 , wherein said one or more modified nucleotides comprises a direct label modified nucleotide or an indirect label modified nucleotide.

3 . The method of claim 1 , wherein said one or more modified nucleotides comprises a charged chemical moiety, a neutral chemical moiety, a hydrophobic moiety, or a hydrophilic moiety.

4 . The method of claim 1 , wherein said one or more modified nucleotides comprises a linker capable of binding to a charged chemical moiety, a neutral chemical moiety, a hydrophobic moiety, or a hydrophilic moiety.

5 . The method of claim 1 , wherein said one or more modified nucleotides comprises a fluorescent dye.

6 . The method of claim 1 , wherein said one or more modified nucleotides comprises Atto488, Atto425, Atto532, Cy5, Texas Red, Fluorescein-12, Rhodamine-12, or aminomethylcoumarin-6.

7 . The method of claim 1 , wherein said modified nucleotide is a dUTP, dTTP, dCTP, dATP, or dGTP.

8 . The method of claim 1 , wherein said one or more modified nucleotides comprises a polyethylene glycol.

9 . The method of claim 1 , wherein said one or more modified nucleotides comprise a linker.

10 . The method of claim 9 , wherein said linker comprises bromo-2′-deoxyuridine-5′-triphosphate (BrdUTP), 5-aminoallyl-2′-deoxyuridine-5′-triphosphate, or 5-ethynyl-2′-deoxyuridine-5′-triphosphate (EdUTP), N6-(6-amino)hexyl-dATP (or dUTP or dCTP) and 7-propargylamino-7-deaza-dATP (or dUTP or dCTP).

11 . The method of any one of claim 9 or 10 , further comprising binding a detectable moiety to said linker, wherein said electrical signal is modified by the presence of said detectable moiety.

12 . The method of claim 11 , wherein said detectable moiety comprises an antibody.

13 . The method of claim 11 , wherein said detectable moiety comprises N-hydroxysuccinimide.

14 . The method of claim 11 , wherein said detectable moiety is azide-modified.

15 . The method of claim 1 , wherein the sensor measures an electrical signal that fluctuates upon translocation of said polynucleotide reaction product through said nanopore.

16 . The method of claim 1 , comprising detecting a plurality of target polynucleotides suspected of being present in the same sample by generating a plurality of distinct polynucleotide reaction products.

17 . The method of claim 16 , wherein said electrical signal is distinct for each of said plurality of target polynucleotides.

18 . The method of claim 17 , wherein said plurality of distinct polynucleotide reaction products are of different lengths.

19 . The method of claim 17 , wherein said plurality of distinct polynucleotide reaction products each comprise a unique modified nucleotide.

20 . The method of claim 17 , wherein said plurality of distinct polynucleotide reaction products comprise a unique modified nucleotide incorporation pattern.

21 . The method of claim 16 , wherein said template-driven polymerization reaction comprises a plurality of sequence-specific primers to enable multiplexed amplification and nanopore detection of more than one target.

22 . The method of claim 1 , wherein said polynucleotide is DNA or RNA.

23 . The method of claim 1 , wherein said template-driven polymerization reaction is an amplification reaction.

24 . The method of claim 23 , wherein said amplification reaction is a polymerase chain reaction or an isothermal reaction.

25 . The method of claim 1 , wherein said template-driven polymerization reaction comprises a polymerase.

26 . The method of claim 25 , wherein said polymerase is DNA polymerase or a reverse transcriptase.

27 . The method of claim 25 , wherein said polymerase is a thermostable DNA polymerase or a thermolabile DNA polymerase.

28 . The method of claim 1 , wherein the modified nucleotide comprises a plurality of labels.

29 . The method of claim 1 , wherein said polynucleotide reaction product is from 200 to 500 bases, from 100 to 2,000 bases, or from 50 to 10,000 bases in length.

30 . The method of claim 1 , wherein said polynucleotide reaction product is greater than 50 bases, greater than 100 bases, greater than 200 bases, greater than 300 bases, or greater than 400 bases in length.

31 . The method of claim 1 , wherein said polynucleotide reaction product is less than 50,000 bases, less than 10,000 bases, less than 5,000 bases, less than 1,000 bases, or less than 500 bases in length.

32 . A method of identifying a modified nucleotide present in a polynucleotide, comprising:

loading a sample suspected of comprising a polynucleotide comprising a plurality of modified nucleotides into a device comprising a nanopore, wherein said nanopore separates an interior space of the device into two volumes;

configuring the device to pass the polynucleotide, if present, through said nanopore, wherein the device comprises a sensor configured to detect an electrical signal generated by objects passing through the nanopore, and wherein said polynucleotide comprising said modified nucleotides generates a distinct electrical signal from a polynucleotide without said modified nucleotides; and

detecting the presence or absence of said polynucleotide in said sample by determining whether said polynucleotide comprising said plurality of modified nucleotides passed through the nanopore using said electrical signal detected by the sensor.

33 . The method of claim 32 , wherein said polynucleotide comprises at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, or at least 500 modified nucleotides.

34 . The method of claim 32 , wherein said plurality of modified nucleotides comprise a direct label modified nucleotide or an indirect label modified nucleotide.

35 . The method of claim 32 , wherein said plurality of modified nucleotides comprise a charged chemical moiety, a neutral chemical moiety, a hydrophobic moiety, or a hydrophilic moiety.

36 . The method of claim 32 , wherein said plurality of modified nucleotides comprise a linker capable of binding to a charged chemical moiety, a neutral chemical moiety, a hydrophobic moiety, or a hydrophilic moiety.

37 . The method of claim 32 , wherein said plurality of modified nucleotides comprise a fluorescent dye.

38 . The method of claim 32 , wherein said plurality of modified nucleotides comprise Atto488, Atto425, Atto532, Cy5, Texas Red, Fluorescein-12, Rhodamine-12, or aminomethylcoumarin-6.

39 . The method of claim 32 , wherein said plurality of modified nucleotides comprise dUTP, dTTP, dCTP, dATP, or dGTP.

40 . The method of claim 32 , wherein said plurality of modified nucleotides comprise a polyethylene glycol.

41 . The method of claim 32 , wherein said plurality of modified nucleotides comprise a linker.

42 . The method of claim 41 , wherein said linker comprises bromo-2′-deoxyuridine-5′-triphosphate (BrdUTP), 5-aminoallyl-2′-deoxyuridine-5′-triphosphate, or 5-ethynyl-2′-deoxyuridine-5′-triphosphate (EdUTP), N6-(6-amino)hexyl-dATP (or dUTP or dCTP) and 7-propargylamino-7-deaza-dATP (or dUTP or dCTP).

43 . The method of any one of claim 41 or 42 , further comprising binding a detectable moiety to said linker, wherein said electrical signal is modified by the presence of said detectable moiety.

44 . The method of claim 43 , wherein said detectable moiety comprises an antibody.

45 . The method of claim 43 , wherein said detectable moiety comprises N-hydroxysuccinimide.

46 . The method of claim 43 , wherein said detectable moiety is azide-modified.

47 . The method of claim 32 , wherein the sensor measures an electrical signal that fluctuates upon translocation of said polynucleotide reaction product through said nanopore.

48 . The method of claim 32 , comprising detecting a plurality of distinct polynucleotides comprising modified nucleotides.

49 . The method of claim 48 , wherein said electrical signal is distinct for each of said plurality of polynucleotides.

50 . The method of claim 48 , wherein said plurality of distinct polynucleotides are of different lengths.

51 . The method of claim 48 , wherein said plurality of distinct polynucleotides each comprise a unique modified nucleotide.

52 . The method of claim 48 , wherein said plurality of distinct polynucleotides have a distinct pattern of modified nucleotides along the length of each distinct polynucleotide.

53 . The method of claim 32 , wherein said polynucleotide is DNA or RNA.

54 . The method of claim 32 , wherein the modified nucleotide comprises a plurality of labels.

55 . The method of claim 32 , wherein said polynucleotide is from 200 to 500 bases, from 100 to 2,000 bases, or from 50 to 10,000 bases in length.

56 . The method of claim 32 , wherein said polynucleotide is greater than 50 bases, greater than 100 bases, greater than 200 bases, greater than 300 bases, or greater than 400 bases in length.

57 . The method of claim 32 , wherein said polynucleotide is less than 50,000 bases, less than 10,000 bases, less than 5,000 bases, less than 1,000 bases, or less than 500 bases in length.

58 . The method of claim 32 , wherein said modified nucleotides are individually incorporated into said polynucleotide by an amplification reaction.

59 . A system, comprising:

a device comprising a nanopore, wherein said nanopore separates an interior space of the device into two volumes, wherein the device comprises a sensor configured to detect an electrical signal generated by objects passing through the nanopore;

a polynucleotide comprising a modified nucleotide, wherein said polynucleotide is loaded into said device for detection by voltage-induced translocation through said nanopore; and

a module for analyzing said electrical signal to detect the presence or absence of said polynucleotide comprising said modified nucleotide.

60 . The system of claim 59 , wherein said polynucleotide comprises a plurality of modified nucleotides.

61 . The system of claim 59 , wherein said polynucleotide comprises at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, or at least 500 modified nucleotides.

62 . The system of claim 59 , wherein said modified nucleotide comprises a direct label modified nucleotide or an indirect label modified nucleotide.

63 . The system of claim 59 , wherein said modified nucleotide comprises a charged chemical moiety, a neutral chemical moiety, a hydrophobic moiety, or a hydrophilic moiety.

64 . The system of claim 59 , wherein said modified nucleotide comprises a linker capable of binding to a charged chemical moiety, a neutral chemical moiety, a hydrophobic moiety, or a hydrophilic moiety.

65 . The system of claim 59 , wherein said modified nucleotide comprises a fluorescent dye.

66 . The system of claim 59 , wherein said modified nucleotide comprises Atto488, Atto425, Atto532, Cy5, Texas Red, Fluorescein-12, Rhodamine-12, or aminomethylcoumarin-6.

67 . The system of claim 59 , wherein said plurality of modified nucleotide is dUTP, dTTP, dCTP, dATP, or dGTP.

68 . The system of claim 59 , wherein said modified nucleotide comprises a polyethylene glycol.

69 . The system of claim 59 , wherein said modified nucleotide comprise a linker.

70 . The system of claim 69 , wherein said linker comprises bromo-2′-deoxyuridine-5′-triphosphate (BrdUTP), 5-aminoallyl-2′-deoxyuridine-5′-triphosphate, or 5-ethynyl-2′-deoxyuridine-5′-triphosphate (EdUTP), N6-(6-amino)hexyl-dATP (or dUTP or dCTP) and 7-propargylamino-7-deaza-dATP (or dUTP or dCTP).

71 . The system of claim 69 or 70 , said linker is bound to a detectable moiety, and wherein said electrical signal is modified by the presence of said detectable moiety.

72 . The system of claim 71 , wherein said detectable moiety comprises an antibody.

73 . The system of claim 71 , wherein said detectable moiety comprises N-hydroxysuccinimide.

74 . The system of claim 71 , wherein said detectable moiety is azide-modified.

75 . The system of claim 59 , wherein the sensor is configured to measure an electrical signal that fluctuates upon translocation of said polynucleotide reaction product through said nanopore.

76 . The system of claim 59 , wherein said polynucleotide is DNA or RNA.

77 . The system of claim 59 , wherein the modified nucleotide comprises a plurality of labels.

78 . The system of claim 59 , wherein said polynucleotide is from 200 to 500 bases, from 100 to 2,000 bases, or from 50 to 10,000 bases in length.

79 . The system of claim 59 , wherein said polynucleotide is greater than 50 bases, greater than 100 bases, greater than 200 bases, greater than 300 bases, or greater than 400 bases in length.

80 . The system of claim 59 , wherein said polynucleotide is less than 50,000 bases, less than 10,000 bases, less than 5,000 bases, less than 1,000 bases, or less than 500 bases in length.