Method of determining the nucleotide sequence of oligonucleotides and DNA molecules

• US 9,957,561 B2
• Filed: 07/21/2017
• Issued: 05/01/2018
• Est. Priority Date: 05/01/1998
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Williams, Peter
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US15/656,129
• Publication Number:
  US 20170321269A1
• Time in Patent Office: 284 Days
• Field of Search: 435 61, 435 911, 435 912
• 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 for sequencing DNA, comprising:

a) providing a primer/template system comprising a template sequence hybridized to a primer oligonucleotide and a DNA polymerase;

b) contacting the primer/template system with a single type of deoxyribonucleotide under conditions that produce a detectable signal when the DNA polymerase incorporates a deoxyribonucleotide onto the 3′

end of the primer oligonucleotide, wherein the single type of deoxyribonucleotide is an unlabeled and unblocked deoxyribonucleotide, and wherein the contacting occurs in a reaction chamber;

c) converting, with a device, the detectable signal into an electrical signal based on an electrical potential generated across the device by the detectable signal, wherein the converting occurs in the reaction chamber;

d) detecting the electrical signal generated by the detectable signal produced in the reaction chamber; and

e) identifying the deoxyribonucleotide that is incorporated onto the 3′

end of the primer oligonucleotide.

2. The method of claim 1, further comprising flushing the reaction chamber with a buffer that is free of deoxyribonucleotides.

3. The method of claim 1, wherein step (b) further comprises:

introducing into, and evacuating from, the reaction chamber at least one reagent selected from the group consisting of;

buffers, electrolytes, DNA template, DNA primer, deoxyribonucleotides, and polymerase enzymes.

4. The method of claim 1, further comprising repeating steps (b) through (e).

5. The method of claim 1, further comprising repeating steps (b) through (e) until the complete nucleotide sequence of the template sequence is determined.

6. The method of claim 1, wherein the amplitude of the electrical signal in step (c) corresponds to the number of nucleotides incorporated onto the 3′

end of the primer oligonucleotide by the DNA polymerase.

7. The method of claim 1, wherein the electrical signal in step (d) is measured with a voltmeter.

8. The method of claim 1, wherein the electrical signal provides real-time detection of incorporation of the deoxyribonucleotide monophosphate onto the primer strand by the DNA polymerase.

9. The method of claim 1, wherein the deoxyribonucleotide is not chemically modified.

10. The method of claim 1, wherein the template sequence is tethered to a solid support.

11. The method of claim 10, wherein the template sequence comprises a linker moiety that is tethered to the solid support.

12. The method of claim 1, wherein the reaction chamber further includes a solid support having the template sequence tethered thereon.