METHOD OF SEQUENCING NUCLEIC ACIDS USING ELABORATED NUCLEOTIDE PHOSPHOROTIOLATE COMPOUNDS

• US 20090171078A1
• Filed: 11/20/2008
• Published: 07/02/2009
• Est. Priority Date: 11/20/2007
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Straus, Neil A., LAO, Kai Qin
• Application Number:
  US12/275,176
• Publication Number:
  US 20090171078A1
• Time in Patent Office: Days
• US Class Current: 536/26.100
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material

Abstract

The present teachings provide methods, compositions, and kits for synthesizing and sequencing nucleic acids. In some embodiments, elaborated nucleotide phosphorothiolate compounds are employed along with efficient cleaving reactions. Improved sequencing efficiency is achieved by the rapid polymerase-mediated incorporation of elaborated nucleotide phosphorothiolate compounds. Increased sequencing efficiency is also achieved by the ability of the cleaving reactions to restore the incorporated nucleotides to their natural structure prior to subsequent elongation.

Claims

1. A method of synthesizing a nucleic acid comprising;

hybridizing a primer to a template;

polymerase extending the primer with an elaborated mono-nucleotide phosphorothiolate compound to form an extension product, wherein the elaborated mono-nucleotide phosphorothiolate compound comprises a 3′

C—

O—

PO₂—

S—

X group;

cleaving the 3′

C—

O—

PO₂—

S—

X group with a phosphorothiolate cleaving agent, wherein the S—

X are removed from the extension product, to leave a 3′

PO₄ group;

hydrolyzing the 3′

PO₄ group with a phosphate removing agent to leave a 3′

OH; and

,

repeating (b)-(d) to synthesize the nucleic acid.

2. The method according to claim 1 wherein the elaborated mono-nucleotide phosphorothiolate compound comprises a first nucleotide and a second nucleotide, and wherein the 3′

C—

O—

PO₂—

S—

X group is attached to the 3′

carbon of the first nucleotide.

3. The method according to claim 1 wherein X of the elaborated mono-nucleotide phosphorothiolate compound is selected from the group consisting of a universal base, CH₂(CH₂O)n(CH2)m, (CH2)_aCO—

NH—

(CH2)_b—

O—

(CH2)_c—

NH, glycerol, an ether group, an ester group, a carbohydrate, a substituted carbohydrate, a carbamate, or a phosphoamidite.

4. The method according to claim 3 wherein n is 0 to 5 and m is 0 to 10.

5. The method according to claim 1 wherein the phosphorothiolate cleaving agent is a metal compound selected from the group consisting of Au, Ag, Hg, Cu, Mn, Zn, or Cd, or is a halide compound selected from the group consisting of iodine or bromine.

6. The method according to claim 1 wherein the phosphate removing agent is selected from the group consisting of a phosphatase and a reversible kinase.

7. A method of sequencing a nucleic acid comprising;

hybridizing a primer to a template;

polymerase extending the primer with an elaborated nucleotide phosphorothiolate compound to form an extension product, wherein the elaborated nucleotide phosphorothiolate compound comprises a 3′

C—

O—

PO₂—

S—

X, wherein X further comprises a label;

detecting the label to determine base identity or to determine probe family identity;

cleaving the 3′

C—

O—

PO₂—

S—

X group with a phosphorothiolate cleaving agent, wherein the S—

X are removed from the elongated strand, to leave a 3′

PO₄ group on a terminal nucleotide;

cleaving the 3′

PO₄ with a phosphate removing agent to leave a 3′

OH on the terminal nucleotide; and

repeating (b)-(e) to sequence the nucleic acid.

8. The method according to claim 7 wherein the elaborated nucleotide phosphorothiolate compound comprises an elaborated di-nucleotide, wherein the elaborated di-nucleotide comprises a first nucleotide and a second nucleotide, and wherein the 3′

C—

O—

PO₂—

S—

X group is attached to the 3′

carbon of the second nucleotide.

9. The method according to claim 8 wherein X is a nucleotide containing a universal base, wherein the universal base comprises a blocking moiety at its 3′

carbon.

10. The method according to claim 9 wherein the universal base of the nucleotide comprises the label, and wherein the label is attached to either the universal base or to the blocking moiety at the 3′

carbon of the universal base.

11. The method according to claim 7 wherein X is selected from the group consisting of CH₂(CH₂O)n(CH2)m, (CH2)_aCO—

NH—

(CH2)_b—

O—

(CH2)_c—

NH, glycerol, ether, ester, carbohydrate, substituted carbohydrate, carbamate, or phosphoamidite.

12. The method according to claim 11 wherein n is 0 to 5 and wherein m is 0 to 10.

13. The method according to claim 7 wherein the elaborated nucleotide phosphorothiolate compound comprises an elaborated mono-nucleotide, wherein the elaborated mono-nucleotide comprises a first nucleotide, and wherein the 3′

C—

O—

PO₂—

S—

X group is attached to the 3′

carbon of the first nucleotide.

14. The method according to claim 13 wherein X is a nucleotide containing a universal base, wherein the universal base comprises a blocking moiety at its 3′

carbon.

15. The method according to claim 14 wherein the universal base of the nucleotide comprises the label, and wherein the label is attached to either the base or to the blocking moiety at the 3′

carbon.

16. The method according to claim 7 wherein the phosphorothiolate cleaving agent is a metal compound selected from the group consisting of Au, Ag, Hg, Cu, Mn, Zn, or Cd, or is a halide compound selected from the group consisting of iodine or bromine.

17. The method according to claim 7 wherein the phosphate removing agent is selected from the group consisting of a phosphatase and a reversible kinase.

18. An elaborated nucleotide phosphorothiolate compound consisting essentially of an elaborated mono-nucleotide, wherein the elaborated mono-nucleotide comprises;

a first nucleotide, wherein the 3′

carbon of the first nucleotide is connected to a 3′

C—

O—

PO₂—

S—

X group.

19. The compound according to claim 18 wherein X comprises a blocking moiety selected from the group consisting of a universal nucleotide base, CH₂(CH₂O)n(CH2)m, (CH2)_aCO—

NH—

(CH2)_b—

O—

(CH2)_c—

NH, glycerol, ether, ester, carbohydrate, substituted carbohydrate, carbamate, or phosphoamidite, and wherein X further comprises a label.

20. The composition according to claim 19 wherein n is 0 to 5 and m is 0 to 10.

21. An elaborated nucleotide phosphorothiolate compound comprising an elaborated di-nucleotide, wherein the elaborated di-nucleotide comprises;

a first nucleotide and a second nucleotide, wherein the 3′

carbon of the second nucleotide comprises a 3′

C—

O—

PO₂—

S—

X group.

22. The compound according to claim 21 wherein X comprises a blocking moiety selected from the group consisting of a universal nucleotide base, CH₂(CH₂O)n(CH2)m, (CH2)_aCO—

NH—

(CH2)_b—

O—

(CH2)_c—

NH, glycerol, ether, ester, carbohydrate, substituted carbohydrate, carbamate, or phosphoamidite, and wherein X further comprises a label.

23. The composition according to claim 22 wherein n is 0 to 5 and m is 0 to 5.

24. A kit for sequencing a template comprising;

(b) at least four elaborated nucleotide phosphorothiolate compounds, each elaborated nucleotide phosphorothiolate compound comprising a 3′

C—

O—

PO₂—

S—

X group, wherein X of the at least four elaborated nucleotide phosphorothiolate compounds comprises a blocking moiety and a distinguishable label; and

,

(c) a suitable polymerase.

25. The kit according to claim 24 wherein the at least four elaborated nucleotide phosphorothiolate compounds are elaborated mono-nucleotide phosphorothiolate compounds.