Method of determining the nucleotide sequence of oligonucleotides and DNA molecules

• US 8,263,365 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:
  US13/008,392
• Publication Number:
  US 20110183323A1
• Time in Patent Office: 602 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3, 536/24.33, 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 DNA sequencing comprising:

providing a) a DNA template system comprising a nucleic acid molecule hybridized to a universal primer oligonucleotide and a DNA polymerase enzyme, and b) at least one deoxyribonucleotide comprising a detectable fluorescent label that is not attached to the 3′

hydroxyl;

extending under conditions wherein said DNA polymerase extends said primer oligonucleotide by at least one nucleotide base to form an extended primer;

identifying said at least one nucleotide base on said extended primer by observing the DNA template system; and

repeating said extending and identifying steps at least once to determine the nucleotide sequence of the nucleic acid molecule.

2. The method of claim 1, wherein said at least one deoxyribonucleotide comprises a single type of deoxyribonucleotide.

3. The method of claim 1, wherein said identifying comprising detecting said detectable fluorescent label on said extended primer.

4. The method of claim 1, further comprising a step wherein excess deoxyribonucleotide is removed after said extending and prior to said identifying.

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

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

7. The method of claim 1, wherein said nucleotides comprising the labels are present in excess concentration relative to said template.

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

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

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

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

14. 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.

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