Method of DNA sequencing using cleavable tags

• US 20040033522A1
• Filed: 06/30/2003
• Published: 02/19/2004
• Est. Priority Date: 06/29/2001
• Status: Active Grant

• Current Assignee: Steven M. Fischer
• Original Assignee: Steven M. Fischer
• Inventors: Fischer, Steven M.
• Granted Patent: US 7,816,079 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety

Abstract

The present invention provides novel systems for sequencing nucleic acid molecules using dNTPs that are 3′ end labeled with cleavable tags that block further extension and uniquely identify the bases to which they are attached. Removal of the tags liberates the 3′ ends of the extension products for further extension. In related embodiments, oligonucleotides containing sequence-related cleavable tags are employed in a ligation reaction to determine the sequence of a particular DNA sample.

Claims

1. A method of determining the sequence of a nucleic acid, comprising steps of:

(a) hybridizing an oligonucleotide to a single stranded DNA, wherein the oligonucleotide is complementary to at least a portion of the single stranded DNA;

(b) providing a DNA polymerase and four deoxynucleotide triphosphates (dNTPs) comprising dATP, dGTP, dCTP, and dTTP, wherein each dNTP is 3′

-end labeled with a cleavable tag (cdNTP) that distinguishes it from the other cdNTPs;

(c) extending the single stranded DNA hybridized to the oligonucleotide by one complementary end-labeled cdNTP in a polymerase extension reaction, wherein the tag on the extended cdNTP blocks further extension by the DNA polymerase;

(d) cleaving the tag from the complementary cdNTP; and

(e) detecting the tag, thereby identifying the complementary dNTP.

2. The method of claim 1, further comprising the step of removing excess cdNTPs that are not extended onto the single stranded DNA.

3. The method of claim 1, further comprising the step of repeating steps (a) through (e) on the sample of single stranded DNA.

4. The method of claim 1, wherein the cleavable tags are cleavable by chemical cleavage.

5. The method of claim 4, wherein the cleavable tags are acid cleavable tags.

6. The method of claim 4, wherein the cleavable tags are base cleavable.

7. The method of claim 1, wherein the tags are photocleavable.

8. The method of claim 1, wherein the tag is a fluorescent tag.

9. The method of claim 1, wherein the tag is a mass tag.

10. A method of determining the sequence of a nucleic acid, comprising steps of:

(a) hybridizing a complementary oligonucleotide to a single stranded DNA, wherein the oligonucleotide is 3′

-end labeled with one or more cleavable tags that distinguishes it from other oligonucleotides;

(b) cleaving the one or more tags from the ligated complementary oligonucleotide, and

(c) detecting the one or more tags.

11. The method of claim 10, wherein the hybridizing of the complementary oligonucleotide occurs adjacent to a primer.

12. The method of claim 11, further comprising ligating the hybridized oligonucleotide to the primer.

13. The method of claim 12, further comprising the step of removing excess oligonucleotides that are not ligated onto the single stranded DNA.

14. The method of claim 12, further comprising the step of repeating steps (a) through (c) on the single stranded DNA.

15. The method of claim 10, wherein one or more tags are cleaved by chemical cleavage.

16. The method of claim 15, wherein the cleavable tags are acid cleavable tags.

17. The method of claim 15, wherein the cleavable tags are base cleavable.

18. The method of claim 10, wherein the tags are photocleavable.

19. The method of claim 10, wherein the tag is a fluorescent tag.

20. The method of claim 10, wherein the tag is a mass tag.