Labeled nucleotides and nucleosides and methods for their use in DNA sequencing

• US 20080131952A1
• Filed: 12/05/2006
• Published: 06/05/2008
• Est. Priority Date: 12/05/2006
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: LaserGen, Inc. (Agilent Technologies, Inc.)
• Inventors: Stupi, Brian P., Wu, Weidong, Metzker, Michael L., Litosh, Vladislav A.
• Granted Patent: US 7,893,227 B2
• Time in Patent Office: Days
• US Class Current: 435/199
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

Provided are novel nucleotides, nucleoside, and their derivatives described herein, that can be used in DNA sequencing technology and other types of DNA analysis. In one embodiment, the nucleotide or nucleoside with an unprotected 3′-OH group is derivatized at the nucleobase to include a fluorescent dye attached via a linker to a non-cleavable terminating group. The non-cleavable-fluorescent group is designed to terminate DNA synthesis so that DNA oligomers can be sequenced efficiently in a parallel format. These reagents and methods will lead to more accurate identification of polymorphisms and other valuable genetic information.

Claims

1. A compound according to the following formula:

2. A compound according to claim 1, wherein the noncleavable terminating moiety is attached to the base through a linkage selected from the group consisting of benzyl amine, benzyl ether, carbamate, carbonate, 2-(o-nitrophenyl)ethyl carbamate, and 2-(o-nitrophenyl)ethyl carbonate.

3. A compound according to claim 1, wherein the compound is selected from the group consisting of:

4. A compound according to claim 3, wherein R₃ and R₄ are selected from the group consisting of —

CH₃, —

CH₂CH₃, —

CH₂CH₂CH₃, isopropyl, tert-butyl and phenyl.

5. A compound according to claim 3, wherein R₃ and R₄ are selected from the group consisting of alkyl and aromatic groups optionally containing at least one heteroatom in the alkyl or aromatic groups, and further wherein the aromatic group may optionally be an aryl or polycyclic group.

6. A compound according to claim 3, wherein R₅, R₆, R₇, and R₈ are selected from an aromatic group consisting of aryl and polycyclic groups.

7. A compound according to claim 3, wherein the fluorophore is selected from the group consisting of of BODIPY, fluorescein, rhodamine, coumarin, xanthene, cyanine, pyrene, phthalocyanine, phycobiliprotein, alexa, squarene dye, combinations resulting in energy transfer dyes, and derivatives thereof.

8. A compound according to claim 3, wherein the compound is selected from the group consisting of

9. A method of determining the sequence of a target nucleic acid comprising

(i) adding a target nucleic acid to a Sanger or Sanger-type sequencing apparatus,

(ii) adding one or more compounds according to claim 1 to the sequencing apparatus, with the proviso that where more than one type of base is present, each base is attached to a different fluorophore;

(iii) adding a complementary primer and a polymerase enzyme,

(iv) performing a polymerase reaction to incorporate at least one of the compounds of step (ii) into a growing nucleic acid strand, and

(v) analyzing the result of the Sanger sequencing reaction with fluorescence sequencing instrumentation or by pulsed multiline excitation fluorescence,

wherein steps (i)-(iii) can be performed in any order.

10. The method according to claim 9, 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%.

11. The method according to claim 9, 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 a native substrate with the same base in the polymerase reaction.

12. The method according to claim 11, wherein the incorporation efficiency occurs at about 85% to about 100%.

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

14. The method according to claim 9, wherein the polymerase is selected from the group consisting of Taq DNA polymerase, Klenow(exo-) DNA polymerase, Bst DNA polymerase, Vent(-exo-) DNA polymerase, Pfu(exo-) DNA polymerase, and DeepVent(exo-) DNA polymerase.

15. The method according to claim 9, wherein the polymerase is a modified polymerase selected from the group consisting of TaqFS DNA polymerase, ThermoSequenase DNA polymerase, ThermoSequenase II DNA polymerase, Therminator DNA polymerase, Therminator II DNA polymerase, and Vent(exo-) A488L DNA polymerase.

16. A method of incorporating a non-naturally occurring component into a nucleic acid comprising:

(i) adding a target nucleic acid to a sequencing apparatus;

(ii) adding one or more compounds according to claim 1 to the sequencing apparatus, with the proviso that where more than one type of base is present, each base is attached to a different fluorophore;

(iii) adding a polymerase enzyme; and

(iv) performing a polymerase reaction to incorporate at least one of the compounds of step (ii) into a growing nucleic acid strand,

wherein steps (i)-(iii) can be performed in any order.

17. The method according to claim 16, further comprising

(v) analyzing the result of the polymerase chain reaction for incorporation of at least one compound from step (ii).

18. The method according to claim 16, 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%.

19. The method according to claim 16, 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 native substrate with the same base in the polymerase reaction.

20. A method of terminating nucleic acid synthesis comprising the step of placing a 3′

-OH unprotected nucleotide or nucleoside according to claim 1 in the environment of a polymerase and allowing incorporation of the 3′

-OH unprotected nucleotide or nucleoside into a nucleic acid.

21. The method according to claim 20 wherein the efficiency of termination upon incorporation of the 3′

-OH unprotected nucleotide or nucleoside ranges from about 90% to about 100%.

22. The method according to claim 20 wherein the efficiency of incorporation of the 3′

-OH unprotected nucleotide or nucleoside ranges from about 70% to about 100% compared to the efficiency of incorporation of a naturally-occurring nucleotide or nucleoside with the same base.

23. A method of performing Sanger or Sanger-type sequencing comprising addition of a compound according to claim 1 to a Sanger or Sanger-type sequencing method.

24. A method of performing mini-sequencing or minisequencing-type sequencing comprising addition of a compound according to claim 1 to a mini-sequencing or minisequencing-type sequencing method.