Method of DNA sequencing using cleavable tags

• US 7,816,079 B2
• Filed: 06/30/2003
• Issued: 10/19/2010
• 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): Horlick; Kenneth R.
• Assistant Examiner(s): Tung; Joyce
• Application Number:
  US10/611,409
• Publication Number:
  US 20040033522A1
• Time in Patent Office: 2,668 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, said method comprising:

(a) hybridizing a primer nucleic acid to a single stranded template nucleic acid;

(b) extending said primer nucleic acid by at least one complementary nucleotide to produce an extension product that includes a 3′

cleavable tag, wherein said at least one complementary nucleotide includes a 3′

cleavable tag;

(c) cleaving said 3′

cleavable tag from said extension product to produce a cleaved tag, not bound to said at least one complementary nucleotide, and an extension product that includes said at least one complementary nucleotide hybridized to said template nucleic acid sequence; and

(d) detecting said cleaved tag away from said extension product to determine said nucleic acid sequence.

2. The method according to claim 1, wherein said primer nucleic acid is extended by a single deoxynucleotide triphosphate labeled with a cleavable tag (cdNTP) to produce said extension product.

3. The method according to claim 2, wherein said extension product is produced by a polymerase in the presence of four distinguishable cdNTPs.

4. The method according to claim 3, wherein said four distinguishable cdNTPs each include a distinguishable cleavable tag.

5. The method according to claim 1, wherein said primer nucleic acid is extended by an oligonucleotide of at least two nucleotides in length that includes a cleavable tag.

6. The method according to claim 5, wherein said cleavable tag is a 3′

cleavable tag.

7. The method according to claim 5, wherein said extension product is produced by a ligase in the presence of said oligonucleotide.

8. The method according to claim 1, wherein said cleavable tag is cleavable by chemical cleavage.

9. The method according to claim 8, wherein said cleavable tag is an acid cleavable tag.

10. The method according to claim 8, wherein said cleavable tag is a base cleavable tag.

11. The method according to claim 1, wherein said cleavable tag is a photocleavable tag.

12. The method according to claim 1, wherein said cleavable tag is a fluorescent tag.

13. The method according to claim 1, wherein said cleavable tag is a mass tag.

14. The method according to claim 1, wherein said steps (a) to (d) are repeated at least once.

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

(a) hybridizing a primer nucleic acid to a single stranded template nucleic acid;

(b) extending said primer nucleic acid by at least one complementary nucleotide to produce a single extension product that includes a 3′

cleavable tag, wherein said at least one complementary nucleotide includes a 3′

cleavable tag;

(c) cleaving said 3′

cleavable tag from said single extension product to produce a cleaved tag not bound to said at least one complementary nucleotide, and an extension product that includes said at least one complementary nucleotide hybridized to said template nucleic acid sequence;

(d) detecting said cleaved tag away from said extension product;

(e) repeating steps (b) to (d) and thereby determining said nucleic acid sequence.

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

(a) hybridizing a primer nucleic acid to a single stranded template nucleic acid in a sample;

(b) extending said primer nucleic acid by a single complementary nucleotide to produce an extension product that includes a 3′

cleavable tag, wherein said extension product is produced by a polymerase in the presence of four distinguishable nucleotides, each labeled with a distinguishable 3′

cleavable tag;

(c) cleaving said 3′

cleavable tag from said extension product to produce a cleaved tag not bound to said at least one complementary nucleotide and an extension product not separated from said single stranded template nucleic acid; and

(d) detecting said cleaved tag away from said extension product.

17. The method of claim 16, comprising repeating steps (b) to (d) on said extension product produced in step (c).