3′-OH unblocked, fast photocleavable terminating nucleotides and methods for nucleic acid sequencing

• US 9,399,798 B2
• Filed: 10/01/2014
• Issued: 07/26/2016
• Est. Priority Date: 09/13/2011
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: LaserGen, Inc. (Agilent Technologies, Inc.)
• Inventors: Stupi, Brian P., Li, Hong, Wu, Weidong, Hersh, Megan N., Hertzog, David, Morris, Sidney E., Metzker, Michael L.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/504,093
• Publication Number:
  US 20150176074A1
• Time in Patent Office: 664 Days
• Field of Search: 435/6.12, 536/22.1, 536/25.32, 536/26.1, 536/26.2
• US Class Current: 1/1
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/168 Specific optical properties, e.g. reflective coatings
• 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: C07H 19/073 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C07H 19/173 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

The present invention relates generally to 3′-OH unblocked nucleotides and nucleosides labeled and unlabeled with 5-methoxy-substituted nitrobenzyl-based photocleavable terminating groups for use in methods and systems related to DNA and RNA sequencing and analysis. These compounds may be used as reversible terminators as they exhibit fast nucleotide incorporation kinetics, single-base termination, high nucleotide selectivity, and rapid terminating group cleavage that results in a naturally occurring nucleotide.

Claims

1. A method of identifying a plurality of bases in a target nucleic acid comprising the following steps:

(i) attaching the 5′

-end of a primer to a solid surface;

(ii) hybridizing a target nucleic acid to the primer attached to the solid surface to form a hybridized primer/target nucleic acid complex;

(iii) obtaining a polymerase and a compound of formulas I-VII, with the proviso that compounds of different formulas I-VII have different reporters;

2. The method of claim 1, wherein step (vi) is conducted in the presence of sodium acetate.

3. The method of claim 1, wherein steps (v) or (vi) is conducted in the presence of thiourea.

4. The method of claim 1, wherein step (vi) is conducted in the presence of dithiothreitol (DTT).

7. The method of claim 1, wherein the incorporation of at least one compound according to step (iv) occurs at about 70% to about 100% of the efficiency of incorporation of its natural nucleotide counterpart.

8. The method of claim 7, wherein the incorporation efficiency occurs at about 85% to about 100%.

9. The method of claim 1, wherein the polymerase is selected from the group consisting of reverse transcriptase, terminal transferase, and DNA polymerase.

10. The method of claim 1, wherein about 85% to about 100% of the photocleavable terminating moieties are removed by exposure to a light source in step (vi).

11. The method of claim 1, wherein incorporation of at least one compound according to step (iv) is followed by termination of strand growth at an efficiency of from about 90% to about 100%.

12. The method of claim 1, further comprising prior to step (iv) capping any primers or growing primer strands that did not react in step (iv).

13. The method of claim 1, further comprising sequencing multiple target nucleic acids synchronistically.

14. The method of claim 1, wherein R₁ is a triphosphate.

15. The method of claim 1, wherein R₁ is an α

-thiotriphosphate.

16. The method of claim 1, wherein R₃ is alkyl_(C≦

8).

17. The method of claim 16, wherein R₃ is alkyl_(C3-4).

18. The method of claim 17, wherein R₃ is tert-butyl.

19. The method of claim 1, wherein R₄ is hydrogen.

20. The method of claim 1, wherein the linker of the -linker-reporter is:

21. The method of claim 20, wherein X is —

C≡

C—

.

22. The method of claim 20, wherein n is zero.

23. The method of claim 1, wherein the dye is a zanthene, fluorescein, rhodamine, BODIPY, cyanine, coumarin, pyrene, phthalocyanine, phycobiliprotein, or a squaraine dye.

24. The method of claim 1, wherein the reporter is:

25. The method of claim 1, wherein R₆ is hydrogen.

26. The method of claim 1, wherein the starred carbon atom is in the S configuration.

5. A method of identifying a plurality of bases in a target nucleic acid comprising the following steps:

(i) attaching the 5′

-end of a target nucleic acid to a solid surface;

(ii) hybridizing a primer to the nucleic acid attached to the solid surface to form a hybridized primer/target nucleic acid complex;

(iii) obtaining a polymerase and a compound of formulas I-VII, with the proviso that compounds of different formulas I-VII have different reporters;

6. The method of claim 5, wherein step (vi) is conducted in the presence of dithiothreitol (DTT).