Sequencing by incorporation

• US 20030215862A1
• Filed: 04/14/2003
• Published: 11/20/2003
• Est. Priority Date: 02/23/1999
• Status: Active Grant

• Current Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Caliper Technologies Corp (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 7,105,300 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 for sequencing a nucleic acid, the method comprising:

(i) providing a nucleic acid template and a primer;

(ii) incubating the nucleic acid template and the primer with a polymerase and one or more nucleotide analogs having formula (III),

thereby adding at least one of the one or more nucleotide analogs to the primer, resulting in an extended primer, wherein R¹ comprises a nucleoside, a nucleotide, a nucleotide analog, or a nucleoside analog, R² comprises a blocking group, which blocking group comprises a detectable label, and R³ comprises a hydrogen or a negative charge;

(iii) detecting the detectable label, thereby detecting the at least one of the one or more nucleotide analogs and removing the blocking group from the extended primer;

or, removing the blocking group from the extended primer and detecting the detectable label, thereby detecting the at least one of the one or more nucleotide analogs;

(iv) removing a 3′

-phosphate from the extended primer, thereby producing an extendable primer; and

,

(v) repeating steps (ii) through (v), thereby sequencing the nucleic acid template.

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

3. The method of claim 1, wherein the detectable label is a fluorescent moiety or a chemiluminescent moiety.

4. The method of claim 1, R¹ comprising a nucleoside 5′

-triphosphate.

5. The method of claim 4, wherein the nucleoside 5′

-triphosphate comprises 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, or an analog thereof.

6. The method of claim 1, R¹ comprising a compound having formula (IV)

7. The method of claim 6, wherein the nitrogenous base comprises adenine, guanine, thymine, cytosine, or uracil.

8. The method of claim 1, R² comprising a chain terminating blocking group.

9. The method of claim 8, wherein the chain-terminating blocking group comprises a reversible chain-terminating blocking group.

10. The method of claim 8, R² comprising a compound having formula (V) or formula (VI)

11. The method of claim 10, comprising providing the linker moiety to comprise an acyl, an S-acyl, an alkyl, an aromatic, or an acetyl group.

12. The method of claim 10, wherein removing the blocking group from the extended primer comprises contacting the extended primer with a reducing agent.

13. The method of claim 12, wherein removing the blocking group from the extended primer comprises contacting the extended primer with one or more of:

diborane, dithiothreitol, glutathione, TCEP, and disulfide reductase.

14. The method of claim 1, wherein removing the blocking group from the extended primer produces a compound having formula (VII):

15. The method of claim 14, wherein the compound having formula (VII) self-cleaves to produce a compound having formula (VIII):

16. The method of claim 1, comprising detecting the detectable label after removing the blocking group from the extended primer.

17. The method of claim 1, comprising detecting the detectable label prior to removing the blocking group from the extended primer.

18. The method of claim 1, comprising removing the blocking group from the extended primer concurrently with removing the 3′

-phosphate from the extended primer.

19. The method of claim 1, wherein step (iv) comprises enzymatically or chemically removing the 3′

-phosphate from the extended primer.

20. The method of claim 1, wherein step (iv) comprises incubating the extended primer with a phosphatase.

21. The method of claim 20, wherein the phosphatase is an alkaline phosphatase.

22. The method of claim 1 comprising performing the method in a microfluidic device.

23. 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.