Sequencing by Incorporation

• US 20060275817A1
• Filed: 08/11/2006
• Published: 12/07/2006
• Est. Priority Date: 02/23/1999
• Status: Active Grant

• Current Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Inventors: Chow, Andrea W., Parce, J. Wallace, Knapp, Michael R., Kopf-Sill, Anne R., Mehta, Tammy Burd, Nikiforov, Theo T.
• Granted Patent: US 7,344,865 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01F 33/30 Micromixers
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00459 Beads
• CPC Class Codes: B01J 2219/00468 by manipulation of individual beads
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/043 magnetic forces
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G01N 2035/00574 : Means for distributing beads
• CPC Class Codes: G01N 27/44756 : Apparatus specially adapted therefor
• CPC Class Codes: G01N 35/0098 : involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation magnetic particles used in immunoassays G01N33/54326; magnetic separation in general B03C
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]

Abstract

Nucleotides and nucleotide analogs are used in various sequencing by incorporation/sequencing by synthesis methods. Nucleotide analogs comprising 3′-blocking groups are used to provide reversible chain-termination for sequencing by synthesis. Typical blocking groups include phosphate groups and carbamate groups. Fluorescent nucleotides are used to perform sequencing by synthesis with detection by incorporation of the fluorescently labeled nucleotide, optionally followed by photobleaching and intercalating dyes are used to detect addition of a non-labeled nucleotide in sequencing by synthesis with detection by intercalation. Microfluidic devices, including particle arrays, are used in the sequencing methods.

Claims

1. A method of sequencing a nucleic acid, the method comprising:

(i) hybridizing a nucleic acid template and a primer, producing a hybridized nucleic acid comprising a double-stranded region;

(ii) incubating the hybridized nucleic acid with a polymerase and a series of nucleotides, thereby adding at least one nucleotide to the primer, resulting in an extended double-stranded region, wherein the incubating is performed in the presence of an intercalating dye, which intercalating dye intercalates into the extended double stranded region;

(iii) detecting the intercalating dye, thereby detecting the addition of the at least one nucleotide to the primer; and

,

(iv) repeating steps (ii) through (iv) thereby sequencing the nucleic acid.

2. The method of claim 1, wherein the nucleic acid is DNA or RNA.

3. The method of claim 1, wherein the series of nucleotides comprises one or more of:

deoxyadenosine 5′

-triphosphate, deoxyguanosine 5′

-triphosphate, deoxycytidine 5′

-triphosphate, deoxythymidine 5′

-triphosphate, deoxyuridine 5′

-triphosphate, adenosine 5′

-triphosphate, guanosine 5′

-triphosphate, cytidine 5′

-triphosphate, uridine 5′

-triphosphate, and an analog thereof.

4. The method of claim 1, wherein the intercalating dye comprises ethidium, ethidium bromide, an acridine dye, an intercalating nucleic acid stain, a cyanine dye, proflavin, propidium iodide, acriflavin, proflavin, actinomycin, anthracyclines, or nogalamycin.

5. The method of claim 1, wherein the intercalating dye intercalates into the double stranded region and into the extended double-stranded region.

6. The method of claim 1, wherein the detecting step comprises detecting a signal difference between the double stranded region and the extended double stranded region.

7. The method of claim 1, wherein the detecting step further comprises photobleaching the intercalating dye after detecting the intercalating dye or approximately concurrent with detecting the intercalating dye.

8. The method of claim 1, comprising performing the method in a microscale channel.

9. The method of claim 8, wherein the incubating step comprises:

(a) incubating the nucleic acid double stranded region with a first nucleotide;

(b) washing unincorporated nucleotides from the microscale channel and detecting the intercalating dye;

(c) repeating steps (a) and (b) for a second nucleotide, a third nucleotide, and a fourth nucleotide.

10. The method of claim 9, wherein the first nucleotide, the second, nucleotide, the third nucleotide and the fourth nucleotide each comprise a different nucleotide.

11. The method of claim 10, wherein the different nucleotide is selected from:

deoxyadenosine 5′

-triphosphate, deoxyguanosine 5′

-triphosphate, deoxycytidine 5′

-triphosphate, deoxythymidine 5′

-triphosphate, deoxyuridine 5′

-triphosphate, adenosine 5′

-triphosphate, guanosine 5′

-triphosphate, cytidine 5′

-triphosphate, uridine 5′

-triphosphate, and an analog thereof.

12. The method of claim 1, comprising providing a set of particles, which set of particles comprises one or more of:

the nucleic acid template and the primer.

13. The method of claim 12, wherein the set of particles comprises an ordered array.

14. The method of claim 12, wherein the set of particles comprises about 1 or more particles, about 10 or more particles, about 100 or more particles, about 1000 or more particles, or about 10,000 or more particles.

15. The method of claim 12, wherein the set of particles comprises a set of beads, which beads are selected from:

polymer beads, silica beads, ceramic beads, clay beads, glass beads, magnetic beads, metallic beads, paramagnetic beads, inorganic beads, and organic beads; and

wherein the beads have a shape, which shape is selected from one or more of;

spherical, helical, cylindrical, spheroid, irregular, rod shaped, cone shaped, cubic, and polyhedral.

16. The method of claim 12, comprising positioning the set of particles within a microscale channel.

17. The method of claim 12, wherein the incubating step comprises flowing the polymerase across the set of particles or flowing the set of particles through the polymerase.

18. A method of sequencing a nucleic acid, comprising:

(i) providing a first set of particles comprising at least one set of nucleic acid templates in a first microfluidic channel;

(ii) flowing a train of reagents comprising a plurality of sequencing reagents across the first set of particles, or flowing the first set of particles through the reagent train, thereby sequentially contacting the at least one set of nucleic acid templates with the plurality of sequencing reagents;

(iii) detecting a signal resulting from exposure of the first set of particles to the reagent train, thereby providing a portion of sequence of the nucleic acid template; and

, repeating steps (ii) and (iii), thereby sequencing the nucleic acid.

19. The method of claim 18, wherein the nucleic acid templates comprise DNA or RNA.

20. The method of claim 18, wherein the train of reagents comprises one or more of:

a template, a primer, a polymerase, a sufurylase, an apyrase, an inorganic phosphate, ATP, a thermostable polymerase, luciferin, luciferase, an endonuclease, an exonuclease, Mg⁺⁺, a molecular crowding agent, a buffer, a dNTP, a salt, a phosphatase, a reducing agent, a modified dNTP, a nucleotide, a nucleotide analog, a nucleotide analog comprising a 3′

-blocking group, a nucleotide analog comprising a 3′

-phosphate group, a nucleotide analog comprising a 3′

-carbamate group, a chain-terminating nucleotide analog, a reversible chain terminating nucleotide analog, a fluorescently labeled nucleotide, and an intercalating dye.

21. The method of claim 18, wherein the plurality of sequencing reagents comprises at least one of:

a compound having formula (I), and a compound having formula (II);

22. The method of claim 21, wherein the linker moiety comprises an acyl, an S-acyl, an alkyl, an aromatic, or an heteroaromatic group.

23. The method of claim 21, wherein the nitrogenous base comprises adenine, guanine, thymine, cytosine, or uracil.

24. The method of claim 18, wherein step (ii) comprises:

(a) flowing at least one set of nucleic acid primers across the set of particles or flowing the set of particles through at least one set of nucleic acid primers, thereby contacting the nucleic acid templates with the nucleic acid primers and forming a double-stranded region; and

,

(b) flowing a polymerase and one or more nucleotides or nucleotide analogs across the set of particles or flowing the set of particles through the polymerase and the one or more nucleotides or nucleotide analogs, thereby adding at least one of the one or more nucleotides or nucleotide analogs to at least one of the nucleic acid primers, resulting in an extended double-stranded region.

25. The method of claim 24, wherein the one or more nucleotide analogs comprise chain-terminating nucleotide analogs.

26. The method of claim 24, wherein the chain-terminating nucleotide analogs comprise reversible chain-terminating nucleotide analogs.

27. The method of claim 24, wherein the one or more nucleotide analogs comprise a 3′

-blocking group.

28. The method of claim 27, wherein the 3′

-blocking group comprises a carbamate group or a phosphate group.

29. The method of claim 27, the method further comprising removing the 3′

-blocking group from the extended double-stranded region, resulting in an extendable double-stranded region.

30. The method of claim 29, wherein the removing the 3′

-blocking group comprises flowing a reducing agent and a phosphatase across the set of particles or flowing the set of particles through the reducing agent and the phosphatase.

31. The method of claim 30, wherein the reducing agent is selected from diborane, dithiothreitol, glutathione, TCEP, and disulfide reductase.

32. The method of claim 24, wherein the one or more nucleotides comprise a series of nucleoside 5′

-triphosphates and the method further comprises flowing an intercalating dye across the set of particles or flowing the set of particles through an intercalating dye.

33. The method of claim 32, wherein detecting the signal comprises detecting the intercalating dye.

34. The method of claim 32, the method further comprising photobleaching the intercalating dye after detecting the intercalating dye or concurrent with detecting the intercalating dye.

35. The method of claim 18, wherein step (iii) comprises photobleaching the at least one of the one or more nucleotides or nucleotide analogs.

36. The method of claim 18, wherein one or more of:

the first set of particles and the reagent train, is flowed through the microscale channel by one or more of;

pressure, centripetal force, centrifugal force, a moving magnetic field, and an electrokinetic force.

37. The method of claim 18, wherein the first set of particles comprises an ordered array.

38. The method of claim 18, wherein the first set of particles comprises about 1 or more particles, about 10 or more particles, about 100 or more particles, about 1000 or more particles, or about 10,000 or more particles.

39. The method of claim 18, wherein the first set of particles comprises a set of beads, which beads are selected from:

polymer beads, silica beads, ceramic beads, clay beads, glass beads, magnetic beads, metallic beads, paramagnetic beads, inorganic beads, and organic beads; and

wherein the beads have a shape, which shape is selected from one or more of;

spherical, helical, cylindrical, spheroid, irregular, rod-shaped, cone-shaped, cubic, and polyhedral.

40. The method of claim 18, wherein the train of reagents comprises reagents for sequencing the nucleic acid templates by pyrosequencing, by incorporation, by synthesis, by photobleaching, or by intercalation.

41. The method of claim 18, further comprising reiteratively repeating steps (ii) and (iii) until at least one reagent in the reagent train becomes depleted, the method further comprising flowing a second train of reagents comprising a plurality of sequencing reagents across the first set of particles, or flowing the first set of particles through the second reagent train.

42. The method of claim 18, wherein the first set of particles is present in an array of particle sets, the method further comprising flowing the train of reagents across the array of particle sets, or flowing the array of particle sets through the reagent train.