Unnatural polymerase substrates that can sustain enzymatic synthesis of double stranded nucleic acids from a nucleic acid template and methods of use

• US 8,058,414 B2
• Filed: 01/16/2009
• Issued: 11/15/2011
• Est. Priority Date: 04/29/2008
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Chen, Jer-Kang, Lam, Joe Y., Menchen, Steven, Khan, Shoeb, Kenney, Paul, Huang, Boli, Rosenblum, Barnett, Ma, Zhaochun
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US12/355,487
• Publication Number:
  US 20090269759A1
• Time in Patent Office: 1,033 Days
• Field of Search: 536/4.1, 536/23.1, 536/24.3, 536/24.33, 536/26.6, 435/6, 435/91.1
• US Class Current: 536/4.1
• CPC Class Codes: C07H 19/073 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 19/173 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety

Abstract

Nucleotide analogs that can sustain the enzymatic synthesis of double-stranded nucleic acid from a nucleic template are described. The nucleotide analogs include: (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine, uracil and their analogs; (ii) a label attached to the base or analog of the base via a cleavable linker; (iii) a deoxyribose; and (iv) one or more phosphate groups. The linker and/or the label inhibits template directed polymerase incorporation of a further nucleotide substrate onto an extended primer strand. In addition, cleavage of the linker leaves a residue attached to the base which is not present in the natural nucleotide and which does not inhibit extension of the primer strand. The nucleotide analogs can therefore be used as reversible terminators in sequencing by synthesis methods without blocking the 3′ hydroxyl group. Methods of sequencing DNA using the substrates are also described.

Claims

1. A compound having a structure of formula (I) or formula (II):

2. The compound of claim 1 where R₁ is an organic substituent selected from the group consisting of neutral forms of substituted or un-substituted alkyls, substituted or un-substituted aromatics, substituted or un-substituted heterocycles, substituted or un-substituted aromatic heterocycles, or anionic or cationic forms thereof.

3. The compound of claim 2, wherein cleavage of the chemically labile group L₂ leaves a linker residue moiety attached to the base having a terminal group F.

4. The compound of claim 3, wherein the terminal group F is not an amino group.

5. The compound of claim 3, wherein the terminal group F is an hydroxyl group.

6. The compound of claim 1, wherein 1 is 2 or 3.

7. The compound of claim 1, wherein the compound has a structure represented by the following formula (II):

8. The compound of claim 1, wherein L₂ comprises a chemically labile group selected from the group consisting of:

—

C(O)—

S—

;

—

C(O)O—

;

—

CH—

(SCH₃)-Ph- and [RO]₂P(O)S—

;

wherein Ph is a substituted phenyl group or a substituted aromatic ring; and

wherein R₁ is an organic substituent selected from the group consisting of neutral forms of substituted or un-substituted alkyls, substituted or un-substituted aromatics, substituted or un-substituted heterocycles, substituted or un-substituted aromatic heterocycles, or anionic or cationic forms thereof.

9. The compound of claim 2, wherein L₁ forms a permanent linkage between the base and the heteroatom F, and L₂ forms a transient linkage between the heteroatom F and R₂.

10. The compound of claim 9, wherein the heteroatom is O, S, or N.

11. The compound of claim 9, wherein L₁ comprises a moiety represented by the formula:

12. The compound of claim 9, wherein L₁ comprises a moiety represented by the formula:

13. The compound of claim 9, wherein L₂-R₂ comprises a moiety represented by the formula:

14. The compound of claim 1, wherein the compound has a structure represented by the following formula (VI):

15. The compound of claim 1, wherein the chemically labile group L₂ is cleavable by Ag⁺ between the dye and the base ₂.

16. The compound of claim 1, wherein F-L₂ comprises a moiety selected from the group consisting of:

17. The compound of claim 1, wherein L₂ comprises a moiety of the following formula:

18. The compound of claim 1, wherein R₁ is a group that suppresses the template directed polymerase incorporation rate of the nucleoside triphosphate onto the 3′

end of an extending oligonucleotide prior to cleavage of L₂-R₂ from the previous incorporation event.

19. The compound of claim 18, wherein R₁ is a reporter or quencher.

20. The compound of claim 1, wherein the compound has a structure represented by the following formula (VI):

21. The compound of claim 20, wherein R₁

is an organic substituent selected from the group consisting of neutral forms of substituted or un-substituted alkyls, substituted or un-substituted aromatics, substituted or un-substituted heterocycles, substituted or un-substituted aromatic heterocycles, or anionic or cationic forms thereof.

22. The compound of claim 7, wherein R₁ is not H.

23. The compound of claim 22, wherein R₁ is an organic substituent selected from the group consisting of neutral forms of substituted or un-substituted alkyls, substituted or un-substituted aromatics, substituted or un-substituted heterocycles, substituted or un-substituted aromatic heterocycles, or anionic or cationic forms thereof.

24. The compound of claim 1, wherein the compound has a structure of formula (I):

25. The compound of claim 17, wherein the L₂ benzene group to which R₂ is attached is further substituted.