3'-OH unblocked uncleotides and nucleosides base modified with non-cleavable, terminating groups and methods for their use in DNA squencing

• US 20110200988A1
• Filed: 01/07/2011
• Published: 08/18/2011
• 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 8,198,029 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• 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

1-8. -8. (canceled)

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 of the formula:

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.

14-15. -15. (canceled)

20. A method of terminating nucleic acid synthesis comprising the step of placing a compound of the formula:

21. The method according to claim 20 wherein the efficiency of termination upon incorporation of the compound ranges from about 90% to about 100%.

22. The method according to claim 20 wherein the efficiency of incorporation of the compound 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 of the formula:

24. A method of performing mini-sequencing or minisequencing-type sequencing comprising addition of a compound of the formula: