Modified nucleotides and methods useful for nucleic acid sequencing

• US 6,579,704 B2
• Filed: 05/08/2001
• Issued: 06/17/2003
• Est. Priority Date: 03/23/1998
• Status: Expired due to Fees

• Current Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Short, Jay M.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/851,542
• Publication Number:
  US 20020034750A1
• Time in Patent Office: 770 Days
• Field of Search: 435/91.1, 435/91.2
• US Class Current: 435/91.1
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer

Abstract

The present invention relates to a method for sequencing nucleic acids. In particular, such method includes incorporating certain modified nucleotides referred to as Simtides into nucleic acid strands. These Simtides are complexes comprising a nucleotide base (or analog thereof), a linker, and a label capable of generating a detectable signal for sequencing. A Simtide may be incorporated into a sequencing fragment as a primer component, as a chain elongator, or as a chain terminator.

Claims

1. A method for determining the nucleotide sequence of a target nucleic acid, the method comprising:

a) generating labeled nucleic acid fragments which are complementary to a portion of the target nucleic acid;

b) incorporating into the nucleic acid fragments by a nucleic acid polymerase, two or more complexes comprising a detectable label molecule, a reversible linker, and a nucleotide base or analog thereof, wherein the reversible linker for a first complex is phenylboronic acid or a derivative thereof, and the reversible linker for a second complex comprises a disulfide bond, and wherein each labeled nucleic acid fragment comprises at least one complex;

c) separating the labeled fragments by size separation; and

d) detecting the labeled nucleic acid fragments, thereby determining the nucleotide sequence of the target nucleic acid.

2. The method of claim 1, wherein the complexes are incorporated into the nucleic acid fragments as a primer.

3. The method of claim 1, wherein the complexes are incorporated into the nucleic acid fragments as a chain elongator.

4. The method of claim 1, wherein the complexes are incorporated into the nucleic acid fragments as a chain terminator.

5. The method of claim 4, wherein the nucleotide base is a dideoxynucleotide.

6. The method of claim 1, wherein the complexes comprise two or more label molecules.

7. The method of claim 1, wherein the label molecule is selected from the group consisting of fluorescent labels, chemiluminescent labels, colorimetric labels, radioactive labels and infrared labels.

8. The method of claim 1, wherein the complexes are incorporated into the nucleic acid fragments as a complete complex.

9. The method of claim 1, wherein the complexes are sequentially incorporated by first incorporating a partial complex into the nucleic acid and subsequently associating with the partial complex the label molecule prior to detecting the label molecule.

10. The method of claim 1, wherein the disulfide bond is generated by reacting a primary amine and a thiol-containing compound, wherein either the primary amine or the thiol-containing compound comprises a nucleotide trisphosphate and the other comprises the detectable label molecule.

11. The method of claim 10, wherein the primary amine comprises a nucleotide trisphosphate and the thiol-containing compound comprises the detectable label molecule.

12. The method of claim 1, wherein the two or more complexes includes four different complexes each having a different combination of reversible linker and detectable label molecule, and each binding to one of the four different nucleotides of the target nucleic acid.

13. A method for determining the nucleotide sequence of a target nucleic acid, the method comprising:

a) generating labeled nucleic acid fragments which are complementary to a portion of the target nucleic acid;

b) incorporating by a nucleic acid polymerase into the nucleic acid fragments as a chain terminator, at least one of four or more complexes comprising a detectable label molecule, a reversible linker, and a nucleotide base or analog thereof, wherein the four or more complexes each comprise a dideoxyribonucleotide base or analog thereof that binds to a different nucleotide, wherein the reversible linker for at least one complex is phenylboronic acid or a derivative thereof, and wherein a combination of detectable label molecule and reversible linker is correlated with the dideoxyribonucleotide base;

c) separating the labeled fragments by size separation; and

d) detecting the labeled nucleic acid fragments, thereby determining the nucleotide sequence of the target nucleic acid.

14. The method of claim 13, wherein at least one complex comprises a reversible linker comprising a disulfide bond.

15. The method of claim 13, wherein the method is performed in a single reaction.

16. An isolated dideoxynucleotide complex comprising a reversible phenylboronic acid linker bound to a dideoxynucleotide or analog thereof.

17. The isolated dideoxynucleotide complex of claim 16, further comprising a detectable label molecule bound to the reversible phenylboronic acid linker.

18. The isolated dideoxynucleotide complex of claim 17, wherein the detectable label molecule is selected from the group consisting of fluorescent labels, chemiluminescent labels, colorimetric labels, radioactive labels and infrared labels.