Method of determining the nucleotide sequence of Oligonucleotides and DNA molecules

• US 8,216,514 B2
• Filed: 06/08/2011
• Issued: 07/10/2012
• 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:
  US13/156,117
• Publication Number:
  US 20120015353A1
• Time in Patent Office: 398 Days
• Field of Search: 422/68.1, 435/6.1, 435/91.1, 536/26.6
• US Class Current: 422/68.1
• 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. An apparatus for DNA sequencing using chemically-modified dNTPs comprising:

(a) at least one reaction chamber comprising a surface, wherein said reaction chamber comprises;

i) a primer/template system comprising a template sequence hybridized to a universal primer, wherein said primer/template system is tethered to said surface;

ii) an excess of chemically modified dNTPs, wherein said chemically modified dNTPs each comprise;

A) a dNTP, b) a fluorescent label, and c) a chemically-cleavable linker between said dNTP and said fluorescent label; and

iii) a polymerase mutant, wherein said polymerase mutant is capable of more efficiently incorporating said chemically modified dNTPs into said primer-template system than the corresponding wild-type enzyme;

(b) a component for introducing into, and evacuating from, said reaction chamber at least one reagent selected from the group consisting of;

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

(c) a component for illuminating said chemically modified dNTPs with optical radiation at a wavelength absorbed by said fluorescent label, and

(d) a device capable of sensing fluorescence from said chemically modified dNTPs.

2. A method of DNA sequencing comprising:

(a) providing a primer/template system comprising a template sequence hybridized to a primer oligonucleotide,

(b) contacting said primer/template system with a polymerase mutant and an excess of chemically-modified dNTPs under conditions such that a chemically modified dNTP is incorporated onto the 3′

end of said primer oligonucleotide,

wherein said chemically modified dNTPs each comprise;

A) a dNTP, b) a fluorescent label, and c) a chemically-cleavable linker between said dNTP and said fluorescent label; and

wherein said polymerase mutant is capable of more efficiently incorporating said chemically modified dNTPs into said primer-template system than the corresponding wild-type enzyme;

(c) illuminating said chemically modified dNTP that is incorporated into said primer with optical radiation at a wavelength absorbed by said fluorescent label;

(d) detecting said chemically modified dNTP; and

(e) treating said chemically modified dNTP that is incorporated into said primer oligonucleotide such that said fluorescent label, but not said linker, is cleaved off.

3. The method of claim 2, further providing a reaction chamber, wherein said primer/template system is present in said reaction chamber.

4. The method of claim 3, further comprising (f) flushing said reaction chamber with dNTP free buffer.

5. The method of claim 4, further comprising repeating steps (b) through (f).

6. The method of claim 4, further comprising repeating steps (b) through (f) until the complete nucleotide sequence of said template sequence is determined.