Methods useful for nucleic acid sequencing using modified nucleotides comprising phenylboronic acid

• US 6,232,103 B1
• Filed: 03/23/1999
• Issued: 05/15/2001
• Est. Priority Date: 03/23/1998
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Short, Jay M.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/274,711
• Time in Patent Office: 784 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 selected target nucleic acid molecule;

b) incorporating into the nucleic acid fragments by a nucleic acid polymerase, a complex comprising a detectable label molecule, a reversible linker, and a nucleotide base or analog thereof;

c) separating the labeled fragments by size separation; and

d) detecting the labeled molecule,

wherein the reversible linker is phenylboronic acid or a derivative thereof.

2. The method of claim 1 wherein the complex is incorporated into the nucleic acid fragments as a primer.

3. The method of claim 1 wherein the complex is incorporated into the nucleic acid fragments as a chain elongator.

4. The method of claim 1 wherein the complex is 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 complex comprises 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 complex is incorporated into the nucleic acid fragments as a complete complex.

9. The method of claim 1 wherein the complex is 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.