Method of determining the nucleotide sequence of oligonucleotides and DNA molecules

• US 8,263,364 B2
• Filed: 01/18/2011
• Issued: 09/11/2012
• 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: Williams, Peter, Taylor, Thomas J., Williams, Daniel J. B., Gould, Ian, Hayes, Mark A.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/008,303
• Publication Number:
  US 20110183322A1
• Time in Patent Office: 602 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3, 536/26.6
• US Class Current: 435/91.1
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• 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 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes
• CPC Class Codes: G01N 33/6863 Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors

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) hybridizing a template to a universal primer that comprises a homopolymer region to form a primer/template system;

(b) on a solid phase, catalyzing incorporation of a nucleotide to said primer/template system with a polymerase by exposing said primer/template system to said polymerase and one or more types of nucleotides comprising an optically-detectable label, wherein said optically-detectable label is not attached to the 3′

hydroxyl of the sugar moiety of said nucleotide;

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

(d) removing said label from said incorporated nucleotide;

(e) permitting steps (b) through (d) to occur multiple times so as to determine a nucleic acid sequence based upon said incorporated nucleotides.

2. The method of claim 1, wherein said template or primer is covalently attached to said solid phase.

3. The method of claim 1, wherein a single nucleotide type is exposed to said primer/template in step (b).

4. The method of claim 1, wherein said polymerase is a mutant polymerase.

5. The method of claim 1, wherein said label is attached to said nucleotide via a chemically-cleavable linker.

6. The method of claim 1, wherein said label is removed at the completion of each of said multiple times.

7. The method of claim 1, wherein said nucleotides comprising an optically-detectable label are present in excess concentration relative to said template.

8. The method of claim 1, wherein said detecting step comprises real-time detection of polymerase-catalyzed incorporation of said incorporated nucleotide.

9. The method of claim 8, wherein said real-time detection comprises detecting said incorporated nucleotide while an incorporation reaction is occurring.

10. The method of claim 1, wherein said removing step comprises cleaving a chemically cleavable linker.

11. The method of claim 1, wherein said template is an unknown sequence.

12. The method of claim 1, wherein said template is generated via PCR amplification.

13. The method of claim 1, wherein steps (b) through (d) are repeated to determine an entire sequence of said template.

14. The method of claim 1, wherein said template is directly attached to said solid phase.

15. The method of claim 1, wherein said template is indirectly attached to said solid phase.

16. The method of claim 1, wherein said primer/template system is immobilized such that said primer/template system is individually optically resolvable on said solid phase.

17. The method of claim 1, wherein said solid phase is in a microfluidic system.

18. The method of claim 1, wherein excess unincorporated nucleotide is removed prior to detecting said label.