ION SENSOR DNA AND RNA SEQUENCING BY SYNTHESIS USING NUCLEOTIDE REVERSIBLE TERMINATORS

• US 20170101675A1
• Filed: 05/18/2015
• Published: 04/13/2017
• Est. Priority Date: 05/19/2014
• Status: Abandoned Application

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Yu, Lin, Ju, Jingyue, Russo, James J.
• Application Number:
  US15/312,130
• Publication Number:
  US 20170101675A1
• Time in Patent Office: Days
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2527/119 pH
• CPC Class Codes: C12Q 2535/113 Cycle sequencing
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode

Abstract

This disclosure is related to a method for determining the identity of a nucleotide residue of a single-stranded DNA or RNA, or sequencing DNA or RNA, in a solution using an ion-sensing field effect transistor and reversible nucleotide terminators.

Claims

1. A method for determining the identity of a nucleotide residue of a single-stranded DNA in a solution comprising:

(a) contacting the single-stranded DNA, having a primer hybridized to a portion thereof, with a DNA polymerase and a deoxyribonucleotide triphosphate (dNTP) analogue under conditions permitting the DNA polymerase to catalyze incorporation of the dNTP analogue into the primer if it is complementary to the nucleotide residue of the single-stranded DNA which is immediately 5′

to a nucleotide residue of the single-stranded DNA hybridized to the 3′

terminal nucleotide residue of the primer, so as to form a DNA extension product, wherein (1) the dNTP analogue has the structure;

3. The method of claim 1 or 2, wherein R′

is —

CH₂N₃;

wherein R′

is a substituted hydrocarbyl, and is a nitrobenzyl;

wherein R′

is a 2-nitrobenzyl;

or wherein R′

is a hydrocarbyl, and is allyl (—

CH₂—

CH═

CH₂).

4. The method of claim 1 or 2, wherein in each dNTP analogue, R′

has the structure;

5. The method of any one of claims 1-4, wherein the DNA is in a solution in a reaction chamber disposed on a sensor which is (i) formed in a semiconductor substrate and (ii) comprises a field-effect transistor or chemical field-effect transistor configured to provide at least one output signal in response to an increase in hydrogen ion concentration of the solution resulting from the formation of a phosphodiester bond between a nucleotide triphosphate or nucleotide triphosphate analogue and a primer or a DNA extension product.

6. The method of claim 5, wherein the reaction chamber is one of a plurality of reaction chambers disposed on a sensor array formed in a semiconductor substrate and comprised of a plurality of sensors, each reaction chamber being disposed on at least one sensor and each sensor of the array comprising a field-effect transistor, or a chemical field-effect transistor, configured to provide at least one output signal in response to an increase in hydrogen ion concentration of the solution resulting from the formation of a phosphodiester bond between a nucleotide triphosphate or nucleotide triphosphate analogue and a primer or a DNA extension product.

7. The method of claim 6, wherein said sensors of said array each occupy an area of 100 μ

m or less and have a pitch of 10 μ

m or less and wherein each of said reaction chambers has a volume in the range of from 1 μ

m³ to 1500 μ

m³;

or wherein each of said reaction chambers contains at least 10⁵ copies of the single-stranded DNA in the solution.

8. The method of any one of claims 6 and 7, wherein said plurality of said reaction chambers and said plurality of said sensors are each greater in number than 256,000.

9. The method of any one of claims 1-8, wherein single-stranded DNA(s) in the solution are attached to a solid substrate;

wherein a primer in the solution is attached to a solid substrate;

wherein the single-stranded DNA or primer is attached to a solid substrate via 1,3-dipolar azide-alkyne cycloaddition chemistry;

wherein the single-stranded DNA or primer is attached to a solid substrate via a polyethylene glycol molecule;

wherein the single-stranded DNA or primer is attached to a solid substrate via a polyethylene glycol molecule and is azide-functionalized;

wherein the DNA or primer is attached to a solid substrate via an azido linkage, an alkynyl linkage, or biotin-streptavidin interaction;

wherein the DNA or primer is alkyne-labeled;

wherein the DNA or primer is attached to a solid substrate which is in the form of a chip, a bead, a well, a capillary tube, a slide, a wafer, a filter, a fiber, a porous media, a matrix, a porous nanotube, or a column;

wherein the DNA or primer is attached to a solid substrate which is a metal, gold, silver, quartz, silica, a plastic, polypropylene, a glass, nylon, or diamond;

wherein the DNA or primer is attached to a solid substrate which is a porous non-metal substance to which is attached or impregnated a metal or combination of metals;

wherein the DNA or primer is attached to a solid substrate which is in turn attached to a second solid substrate;

or wherein the DNA or primer is attached to a solid substrate which is in turn attached to a second solid substrate which is a chip.

10. The method of any one of claims 1-9, wherein 1×

10⁹ or fewer copies of the DNA or primer are attached to a solid substrate;

wherein 1×

10⁸ or fewer copies of the DNA or primer are attached to a solid substrate;

wherein 2×

10⁷ or fewer copies of the DNA or primer are attached to a solid substrate;

wherein 1×

10⁷ or fewer copies of the DNA or primer are attached to a solid substrate;

wherein 1×

10⁸ or fewer copies of the DNA or primer are attached to a solid substrate;

wherein 1×

10⁴ or fewer copies of the DNA or primer are attached to a solid substrate;

or wherein 1,000 or fewer copies of the DNA or primer are attached to a solid substrate.

11. The method of any one of claims 1-9, wherein 10,000 or more copies of the DNA or primer are attached to a solid substrate;

wherein 1×

10⁷ or more copies of the DNA or primer are attached to a solid substrate;

wherein 1×

10⁸ or more copies of the DNA or primer are attached to a solid substrate;

or wherein 1×

10⁹ or more copies of the DNA or primer are attached to a solid substrate.

12. The method of any one of claims 1-11, wherein the DNA or primer are separated in discrete compartments, wells, or depressions on a solid surface.

13. The method of any one of claims 1-12 performed in parallel on a plurality of single-stranded DNAs;

and wherein optionally the single-stranded DNAs are templates having the same sequence.

14. The method of claim 13, further comprising contacting the plurality of single-stranded DNAs or templates after the residue of the nucleotide residue has been determined in step (b), or (c), as appropriate, with a dideoxynucleotide triphosphate which is complementary to the nucleotide residue which has been identified, so as to thereby permanently cap any unextended primers or unextended DNA extension products.

15. The method of any one of claim 13 or 14, wherein the single-stranded DNA is amplified from a sample of DNA prior to step (a);

and wherein optionally the single-stranded DNA is amplified by polymerase chain reaction.

16. The method of any one of claims 1-15, wherein UV light is used to treat the R′

group of a dNTP analogue incorporated into a primer or DNA extension product so as to photochemically cleave the moiety attached to the 3′

-O so as to replace the 3′

-O—

R′

with a 3′

-OH;

wherein the moiety is optionally a 2-nitrobenzyl moiety.

2. A method for determining the sequence of consecutive nucleotide residues in a single-stranded DNA in a solution comprising:

(a) contacting the single-stranded DNA, having a primer hybridized to a portion thereof, with a DNA polymerase and a deoxyribonucleotide triphosphate (dNTP) analogue under conditions permitting the DNA polymerase to catalyze incorporation of the dNTP analogue into the primer if it is complementary to the nucleotide residue of the single-stranded DNA which is immediately 5′

to a nucleotide residue of the single-stranded DNA hybridized to the 3′

terminal nucleotide residue of the primer, so as to form a DNA extension product, wherein (1) the dNTP analogue has the structure;

17. A method for determining the identity of a nucleotide residue of a single-stranded RNA in a solution comprising:

(a) contacting the single-stranded RNA, having an RNA primer hybridized to a portion thereof, with a polymerase and a ribonucleotide triphosphate (rNTP) analogue under conditions permitting the polymerase to catalyze incorporation of the rNTP analogue into the RNA primer if it is complementary to the nucleotide residue of the single-stranded RNA which is immediately 5′

to a nucleotide residue of the single-stranded RNA hybridized to the 3′

terminal nucleotide residue of the RNA primer, so as to form an RNA extension product, wherein (1) the rNTP analogue has the structure;

19. The method of claim 17 or 18, wherein R′

is —

CH₂N₃;

wherein R′

is a substituted hydrocarbyl, and is a nitrobenzyl;

wherein R′

is a 2-nitrobenzyl;

or wherein R′

is a hydrocarbyl, and is allyl (—

CH₂—

CH═

CH₂).

20. The method of claim 17 or 18, wherein in each rNTP analogue, R′

has the structure;

21. The method of any one of claims 17-20, wherein the RNA is in a solution in a reaction chamber disposed on a sensor which is (i) formed in a semiconductor substrate and (ii) comprises a field-effect transistor or chemical field-effect transistor configured to provide at least one output signal in response to an increase in hydrogen ion concentration of the solution resulting from the formation of a phosphodiester bond between a nucleotide triphosphate or nucleotide triphosphate analogue and a primer or an RNA extension product.

22. The method of claim 21, wherein the reaction chamber is one of a plurality of reaction chambers disposed on a sensor array formed in a semiconductor substrate and comprised of a plurality of sensors, each reaction chamber being disposed on at least one sensor and each sensor of the array comprising a field-effect transistor, or a chemical field-effect transistor, configured to provide at least one output signal in response to an increase in hydrogen ion concentration of the solution resulting from the formation of a phosphodiester bond between a nucleotide triphosphate or nucleotide triphosphate analogue and a primer or an RNA extension product.

23. The method of claim 22, wherein said sensors of said array each occupy an area of 100 μ

m or less and have a pitch of 10 μ

m or less and wherein each of said reaction chambers has a volume in the range of from 1 μ

m³ to 1500 μ

m³;

or wherein each of said reaction chambers contains at least 10⁵ copies of the single-stranded RNA in the solution.

24. The method of any one of claims 22 and 23, wherein said plurality of said reaction chambers and said plurality of said sensors are each greater in number than 256,000.

25. The method of any one of claims 17-24, wherein single-stranded RNA(s) in the solution are attached to a solid substrate;

wherein a primer in the solution is attached to a solid substrate;

wherein the single-stranded RNA or primer is attached to a solid substrate via 1,3-dipolar azide-alkyne cycloaddition chemistry;

wherein the single-stranded RNA or primer is attached to a solid substrate via a polyethylene glycol molecule;

wherein the single-stranded RNA or primer is attached to a solid substrate via a polyethylene glycol molecule and is azide-functionalized;

wherein the RNA or primer is attached to a solid substrate via an azido linkage, an alkynyl linkage, or biotin-streptavidin interaction;

wherein the RNA or primer is alkyne-labeled;

wherein the RNA or primer is attached to a solid substrate which is in the form of a chip, a bead, a well, a capillary tube, a slide, a wafer, a filter, a fiber, a porous media, a matrix, a porous nanotube, or a column;

wherein the RNA or primer is attached to a solid substrate which is a metal, gold, silver, quartz, silica, a plastic, polypropylene, a glass, nylon, or diamond;

wherein the RNA or primer is attached to a solid substrate which is a porous non-metal substance to which is attached or impregnated a metal or combination of metals;

wherein the RNA or primer is attached to a solid substrate which is in turn attached to a second solid substrate;

or wherein the RNA or primer is attached to a solid substrate which is in turn attached to a second solid substrate which is a chip.

26. The method of any one of claims 17-25, wherein 1×

10⁹ or fewer copies of the RNA or primer are attached to a solid substrate;

wherein 1×

10⁸ or fewer copies of the RNA or primer are attached to a solid substrate;

wherein 2×

10⁷ or fewer copies of the RNA or primer are attached to a solid substrate;

wherein 1×

10⁷ or fewer copies of the RNA or primer are attached to a solid substrate;

wherein 1×

10⁶ or fewer copies of the RNA or primer are attached to a solid substrate;

wherein 1×

10⁴ or fewer copies of the RNA or primer are attached to a solid substrate;

or wherein 1,000 or fewer copies of the RNA or primer are attached to a solid substrate.

27. The method of any one of claims 17-25, wherein 10,000 or more copies of the RNA or primer are attached to a solid substrate;

wherein 1×

10⁷ or more copies of the RNA or primer are attached to a solid substrate;

wherein 1×

10⁸ or more copies of the RNA or primer are attached to a solid substrate;

or wherein 1×

10⁹ or more copies of the RNA or primer are attached to a solid substrate.

28. The method of any one of claims 17-27, wherein the RNA or primer are separated in discrete compartments, wells, or depressions on a solid surface.

29. The method of any one of claims 17-28 performed in parallel on a plurality of single-stranded RNAs;

and wherein optionally the single-stranded RNAs are templates having the same sequence.

30. The method of claim 29, further comprising contacting the plurality of single-stranded RNAs or templates after the residue of the nucleotide residue has been determined in step (b), or (c), as appropriate, with a dideoxynucleotide triphosphate which is complementary to the nucleotide residue which has been identified, so as to thereby permanently cap any unextended primers or unextended RNA extension products.

31. The method of any one of claim 29 or 30, wherein the single-stranded RNA is amplified from a sample of RNA prior to step (a);

and wherein optionally the single-stranded RNA is amplified by polymerase chain reaction.

32. The method of any one of claims 17-31, wherein UV light is used to treat the R′

group of an rNTP analogue incorporated into a primer or RNA extension product so as to photochemically cleave the moiety attached to the 3′

-O so as to replace the 3′

-O—

R′

with a 3′

-OH;

wherein the moiety is optionally a 2-nitrobenzyl moiety.

18. A method for determining the sequence of consecutive nucleotide residues in a single-stranded RNA in a solution comprising:

(a) contacting the single-stranded RNA, having an RNA primer hybridized to a portion thereof, with a RNA polymerase and a ribonucleotide triphosphate (rNTP) analogue under conditions permitting the RNA polymerase to catalyze incorporation of the rNTP analogue into the RNA primer if it is complementary to the nucleotide residue of the single-stranded RNA which is immediately 5′

to a nucleotide residue of the single-stranded RNA hybridized to the 3′

terminal nucleotide residue of the RNA primer, so as to form an RNA extension product, wherein (1) the rNTP analogue has the structure;

33. A method for determining the identity of a nucleotide residue of a single-stranded RNA in a solution comprising:

(a) contacting the single-stranded RNA, having a DNA primer hybridized to a portion thereof, with a reverse transcriptase and a deoxyribonucleotide triphosphate (dNTP) analogue under conditions permitting the reverse transcriptase to catalyze incorporation of the dNTP analogue into the DNA primer if it is complementary to the nucleotide residue of the single-stranded RNA which is immediately 5′

to a nucleotide residue of the single-stranded RNA hybridized to the 3′

terminal nucleotide residue of the DNA primer, so as to form a DNA extension product, wherein (1) the dNTP analogue has the structure;

35. The method of claim 33 or 34, wherein R′

is —

CH₂N₃;

wherein R′

is a substituted hydrocarbyl, and is a nitrobenzyl;

wherein R′

is a 2-nitrobenzyl;

or wherein R′

is a hydrocarbyl, and is allyl (—

CH₂—

CH═

CH₂).

36. The method of claim 33 or 34, wherein in each dNTP analogue, R′

has the structure;

37. The method of any one of claims 33-36, wherein the RNA is in a solution in a reaction chamber disposed on a sensor which is (i) formed in a semiconductor substrate and (ii) comprises a field-effect transistor or chemical field-effect transistor configured to provide at least one output signal in response to an increase in hydrogen ion concentration of the solution resulting from the formation of a phosphodiester bond between a nucleotide triphosphate or nucleotide triphosphate analogue and a primer or a DNA extension product.

38. The method of claim 37, wherein the reaction chamber is one of a plurality of reaction chambers disposed on a sensor array formed in a semiconductor substrate and comprised of a plurality of sensors, each reaction chamber being disposed on at least one sensor and each sensor of the array comprising a field-effect transistor, or a chemical field-effect transistor, configured to provide at least one output signal in response to an increase in hydrogen ion concentration of the solution resulting from the formation of a phosphodiester bond between a nucleotide triphosphate or nucleotide triphosphate analogue and a primer or a DNA extension product.

39. The method of claim 38, wherein said sensors of said array each occupy an area of 100 μ

m or less and have a pitch of 10 μ

m or less and wherein each of said reaction chambers has a volume in the range of from 1 μ

m³ to 1500 μ

m³;

or wherein each of said reaction chambers contains at least 10⁵ copies of the single-stranded RNA in the solution.

40. The method of any one of claims 38 and 39, wherein said plurality of said reaction chambers and said plurality of said sensors are each greater in number than 256,000.

41. The method of any one of claims 33-40, wherein single-stranded RNA(s) in the solution are attached to a solid substrate;

wherein a primer in the solution is attached to a solid substrate;

wherein the single-stranded RNA or primer is attached to a solid substrate via 1,3-dipolar azide-alkyne cycloaddition chemistry;

wherein the single-stranded RNA or primer is attached to a solid substrate via a polyethylene glycol molecule;

wherein the single-stranded RNA or primer is attached to a solid substrate via a polyethylene glycol molecule and is azide-functionalized;

wherein the RNA or primer is attached to a solid substrate via an azido linkage, an alkynyl linkage, or biotin-streptavidin interaction;

wherein the RNA or primer is alkyne-labeled;

wherein the RNA or primer is attached to a solid substrate which is in the form of a chip, a bead, a well, a capillary tube, a slide, a wafer, a filter, a fiber, a porous media, a matrix, a porous nanotube, or a column;

wherein the RNA or primer is attached to a solid substrate which is a metal, gold, silver, quartz, silica, a plastic, polypropylene, a glass, nylon, or diamond;

wherein the RNA or primer is attached to a solid substrate which is a porous non-metal substance to which is attached or impregnated a metal or combination of metals;

wherein the RNA or primer is attached to a solid substrate which is in turn attached to a second solid substrate;

or wherein the RNA or primer is attached to a solid substrate which is in turn attached to a second solid substrate which is a chip.

42. The method of any one of claims 33-41, wherein 1×

10⁹ or fewer copies of the RNA or primer are attached to a solid substrate;

wherein 1×

10⁸ or fewer copies of the RNA or primer are attached to a solid substrate;

wherein 2×

10⁷ or fewer copies of the RNA or primer are attached to a solid substrate;

wherein 1×

10⁷ or fewer copies of the RNA or primer are attached to a solid substrate;

wherein 1×

10⁶ or fewer copies of the RNA or primer are attached to a solid substrate;

wherein 1×

10⁴ or fewer copies of the RNA or primer are attached to a solid substrate;

or wherein 1,000 or fewer copies of the RNA or primer are attached to a solid substrate.

43. The method of any one of claims 33-41, wherein 10,000 or more copies of the RNA or primer are attached to a solid substrate;

wherein 1×

10⁷ or more copies of the RNA or primer are attached to a solid substrate;

wherein 1×

10⁸ or more copies of the RNA or primer are attached to a solid substrate;

or wherein 1×

10⁹ or more copies of the RNA or primer are attached to a solid substrate.

44. The method of any one of claims 33-43, wherein the RNA or primer are separated in discrete compartments, wells, or depressions on a solid surface.

45. The method of any one of claims 33-44 performed in parallel on a plurality of single-stranded RNAs;

and wherein optionally the single-stranded RNAs are templates having the same sequence.

46. The method of claim 45, further comprising contacting the plurality of single-stranded RNAs or templates after the residue of the nucleotide residue has been determined in step (b), or (c), as appropriate, with a dideoxynucleotide triphosphate which is complementary to the nucleotide residue which has been identified, so as to thereby permanently cap any unextended primers or unextended DNA extension products.

47. The method of any one of claim 45 or 46, wherein the single-stranded RNA is amplified from a sample of RNA prior to step (a);

and wherein optionally the single-stranded RNA is amplified by polymerase chain reaction.

48. The method of any one of claims 33-47, wherein UV light is used to treat the R′

group of a dNTP analogue incorporated into a primer or DNA extension product so as to photochemically cleave the moiety attached to the 3′

-O so as to replace the 3′

-O—

R′

with a 3′

-OH;

wherein the moiety is optionally a 2-nitrobenzyl moiety.

34. A method for determining the sequence of consecutive nucleotide residues in a single-stranded RNA in a solution comprising:

(a) contacting the single-stranded RNA, having a DNA primer hybridized to a portion thereof, with a reverse transcriptase and a deoxyribonucleotide triphosphate (dNTP) analogue under conditions permitting the reverse transcriptase to catalyze incorporation of the dNTP analogue into the primer if it is complementary to the nucleotide residue of the single-stranded RNA which is immediately 5′

to a nucleotide residue of the single-stranded RNA hybridized to the 3′

terminal nucleotide residue of the DNA primer, so as to form a DNA extension product, wherein (1) the dNTP analogue has the structure;