Modified nucleotide linkers

• US 10,190,157 B2
• Filed: 08/07/2015
• Issued: 01/29/2019
• Est. Priority Date: 08/08/2014
• Status: Active Grant

• Current Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Inventors: Wu, Xiaolin, Liu, Xiaohai
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/821,251
• Publication Number:
  US 20160040225A1
• Time in Patent Office: 1,271 Days
• Field of Search: 435 61, 536 41
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

Some embodiments of the present application relate to novel modified nucleotide linkers for increasing the efficiency of nucleotide incorporation in Sequencing by Synthesis applications. Methods of preparing these modified nucleotide linkers are also provided herewith.

Claims

1. A nucleoside or nucleotide covalently attached to a fluorophore through a linker, wherein said linker comprises a structure of formula (I) or (II), or combination of both:

2. The nucleoside or nucleotide of claim 1, wherein R¹ is methyl.

3. The nucleoside or nucleotide of claim 1, wherein R² is hydrogen or methyl.

4. The nucleoside or nucleotide of claim 1, wherein m is 0 or 1.

5. The nucleoside or nucleotide of claim 1, wherein n is 1.

6. The nucleoside or nucleotide of claim 1, wherein the structure of formula (I) is also represented by formula (Ia):

7. The nucleoside or nucleotide of claim 1, wherein R³ is methyl.

8. The nucleoside or nucleotide of claim 1, wherein R³ is —

NH—

C(═

O)CH₃.

9. The nucleoside or nucleotide of claim 1, wherein R³ is —

NH—

C(═

O)Ot-butyl.

10. The nucleoside or nucleotide of claim 1, wherein R⁴ is hydrogen or methyl.

11. The nucleoside or nucleotide of claim 1, wherein X is methylene.

12. The nucleoside or nucleotide of claim 1, wherein p is 1 or 2.

13. The nucleoside or nucleotide of claim 1, wherein the structure of formula (II) is also represented by formula (IIb), (IIc), (IId), (IIe) or (llf):

14. A kit comprising a nucleoside or nucleotide according to claim 1.

15. A reagent for modifying a nucleoside or a nucleotide, comprising a fluorophore and a linker according to claim 1.

16. A method for detecting a nucleoside or nucleotide that has been incorporated into a polynucleotide, comprising:

(a) incorporating a nucleoside or nucleotide according to claim 1 into a polynucleotide; and

(b) detecting a fluorescent signal from said nucleoside or nucleotide that was incorporated in step (a).

17. The method of claim 16, further comprising providing a template nucleic acid strand and a partially hybridized nucleic acid strand, wherein step (a) incorporates into the hybridized strand at least one nucleoside or nucleotide that is complementary to a nucleoside or nucleotide at the corresponding position of the template strand, and wherein step (b) identifies the base of the incorporated nucleoside or nucleotide, thereby indicating the identity of the complementary nucleoside or nucleotide of the template strand.

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

incorporating one or more labeled nucleotides into a strand of nucleic acid complementary to the template nucleic acid;

determining the identity of the base present in one or more incorporated labeled nucleotides in order to determine the sequence of the template nucleic acid molecule;

wherein the identity of the base present in said one or more labeled nucleotides is determined by detecting a fluorescent signal produced by said labeled nucleotides; and

wherein at least one incorporated labeled nucleotide is a nucleotide according to claim 1.

19. The method of claim 18, wherein the identity of the base present in said one or more nucleotides is determined after each nucleotide incorporation step.

20. The nucleoside or nucleotide of claim 9, wherein R⁴ is hydrogen or methyl.

21. The nucleoside or nucleotide of claim 20, wherein R⁴ is hydrogen.

22. The nucleoside or nucleotide of claim 21, wherein X is methylene.

23. The nucleoside or nucleotide of claim 1, wherein said linker comprises a structure of (II), and wherein R³ is —

NR⁷—

C(═

O)—

OR⁸.

24. The nucleoside or nucleotide of claim 23, wherein X is methylene.

25. The nucleoside or nucleotide of claim 24, wherein p is 2.

26. The nucleoside or nucleotide of claim 25, wherein the linker comprises the following structure:

27. The kit of claim 14, comprising a nucleotide.

28. The kit of claim 27, wherein said linker comprises a structure of (II), and wherein R³ is —

NR⁷—

C(═

O)—

OR⁸.

29. The kit of claim 28, wherein X is methylene.

30. The kit of claim 29, wherein p is 2.

31. The kit of claim 30, wherein the linker comprises the following structure: