Method of determining the nucleotide sequence of oligonucleotides and DNA molecules

• US 7,645,596 B2
• Filed: 05/05/2004
• Issued: 01/12/2010
• Est. Priority Date: 05/01/1998
• Status: Expired due to Fees

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Arizona Board of Regents (University of Arizona)
• Inventors: Hayes, Mark A., Taylor, Thomas J., Gould, Ian, Williams, Peter, Williams, Daniel J. B.
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US10/709,436
• Publication Number:
  US 20050032076A1
• Time in Patent Office: 2,078 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3
• US Class Current: 435/91.1
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2533/101 Primer extension
• 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 poly-merase-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 for sequencing nucleic acid, the method comprising the steps of:

hybridizing nucleic acid to a complementary nucleic acid sequence to form one or more duplexes;

adding at least one nucleotide comprising a detectable label to said duplex, wherein said nucleotide is not a dideoxy nucleotide and wherein said nucleotide has a free 3′

hydroxyl when added to said duplex;

identifying said nucleotide comprising said detectable label; and

repeating said adding and identifying steps, thereby to determine the sequence of said nucleic acid.

2. The method of claim 1, wherein said identifying step comprises detecting pyrophosphate production.

3. The method of claim 1, further comprising amplifying nucleic acid prior to said hybridizing step.

4. The method of claim 1, further comprising immobilizing said one or more duplexes on a substrate.

5. The method of claim 4, wherein said one or more duplexes are immobilized such that said one or more duplexes are individually optically resolvable on said substrate.

6. The method of claim 1, wherein said detectable label is an optically-detectable label.

7. The method of claim 6, wherein said optically-detectable label is a fluorescent label.

8. The method of claim 6, wherein said optically-detectable label is a chemiluminescent label.