Method of determining the nucleotide sequence of oligonucleotides and DNA molecules

• US 7,037,687 B2
• Filed: 08/28/2002
• Issued: 05/02/2006
• Est. Priority Date: 05/01/1998
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: University of Alberta, Arizona Board of Regents (University of Arizona)
• Inventors: Williams, Peter, Rose, Seth D., Pizziconi, Vincent B., Hayes, Mark A., Bloom, Linda B., Reha-Krantz, Linda J.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US10/229,997
• Publication Number:
  US 20030138809A1
• Time in Patent Office: 1,343 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/22.1, 536/23.1
• US Class Current: 435/91.1
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2563/103 luminescence
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions.

Claims

1. A method of nucleic acid sequencing, the method comprising the steps of:

(a) providing a nucleic acid template/primer system comprising individual template/primer duplexes immobilized on a surface;

(b) exposing said individual template/primer duplexes to a polymerase and one or more type of nucleotide comprising an optically-detectable label, wherein said optically-detectable label is not attached to the 3′

position of the sugar moiety of said nucleotide;

(c) removing nucleotide that is not incorporated into a primer;

(d) detecting incorporated nucleotide by the presence of said label;

(e) removing said label from said incorporated nucleotide;

(f) repeating steps (b) through (e) at least once with a different type of nucleotide comprising an optically-detectable label for incorporation into said primer; and

(g) determining a nucleic acid sequence based upon said incorporated nucleotides.

2. The method of claim 1, wherein said label is a fluorescent label.

3. The method of claim 2, wherein said fluorescent label is selected from the group consisting of fluorescein, rhodamine and BODIPY.

4. The method of claim 2, wherein said detecting step comprises sensing said label.

5. The method of claim 1, wherein said detecting step comprises resolving an image of said duplex.