Method of DNA sequencing using cleavable tags

• US 6,613,523 B2
• Filed: 06/29/2001
• Issued: 09/02/2003
• Est. Priority Date: 06/29/2001
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Fischer, Steven M.
• Primary Examiner(s): Whisenant, Ethan
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US09/896,299
• Publication Number:
  US 20030008285A1
• Time in Patent Office: 795 Days
• Field of Search: 435/6, 435/91.2, 435/77, 435/78, 536/24.33, 536/25.3, 536/27
• US Class Current: 435/6.12
• 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 a nucleic acid sequence, comprising:

(a) hybridizing an oligonucleotide to a single stranded DNA within a chamber, wherein a filtration membrane is attached to the chamber, wherein the oligonucleotide is complementary to at least a portion of the single stranded DNA, and wherein the single stranded DNA is retained within the chamber by the filtration membrane;

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

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

(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) washing the cdNTPs that are not extended onto the single stranded DNA through the filtration membrane, wherein the filtration membrane allows cdNTPs to pass through the membrane and retains the single stranded DNA in the chamber;

(e) cleaving the tag from the complementary cdNTP;

(f) washing the cleaved tag through the filtration membrane; and

(g) detecting the cleaved tag that is washed through the filtration membrane of step (f), thereby identifying the complementary dNTP.

2. The method of claim 1, further comprising the step of repeating steps (b) through (g) on the sample of single stranded DNA.

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

4. The method of claim 3, wherein the cleavable tags are acid cleavable tags.

5. The method of claim 3, wherein the cleavable tags are base cleavable.

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

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

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

9. A method of determining a nucleic acid sequence, comprising:

(a) hybridizing a complementary oligonucleotide to a single stranded DNA, wherein the single stranded DNA is retained within a chamber by a filtration membrane that is attached to the chamber, wherein the single stranded DNA is not attached to the filtration membrane, wherein the complementary oligonucleotide is hybridized adjacent to a primer that is hybridized to the single stranded DNA, wherein the complementary oligonucleotide is 3′

-end labeled with at least one cleavable tag that distinguishes it from other complementary oligonucleotides;

(b) ligating the hybridized complementary oligonucleotide to the adjacent primer to generate a ligated complementary oligonucleotide;

(c) cleaving the tag from the ligated complementary oligonucleotide;

(d) washing the cleaved tag through the filtration membrane that allows the cleaved tag to pass through the membrane; and

(e) detecting the cleaved tag that distinguishes the complementary oligonucleotide that is washed through the filtration membrane of step (e).

10. The method of claim 9, further comprising the step of repeating steps (a) through (e) on the single stranded DNA.

11. The method of claim 9, wherein one or more tags are cleaved by chemical cleavage.

12. The method of claim 11, wherein the cleavable tags are acid cleavable tags.

13. The method of claim 11, wherein the cleavable tags are base cleavable.

14. The method of claim 9, wherein the tags are photocleavable.

15. The method of claim 9, wherein the tag is a fluorescent tag.

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