Method of determining the nucleotide sequence of oligonucleotides and DNA molecules

• US 10,208,341 B2
• Filed: 05/02/2014
• Issued: 02/19/2019
• 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, Williams, Daniel J. B., Bloom, Linda, Taylor, Thomas J.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/268,254
• Publication Number:
  US 20140342365A1
• Time in Patent Office: 1,754 Days
• Field of Search: 435 61, 435 911
• 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 phase error compensation, comprising:

providing a plurality of template nucleic acid strands;

adding, in an order, individual types of deoxynucleoside triphosphates serially to the plurality of template nucleic acid strands to perform a series of nucleotide incorporation reactions;

after each nucleotide incorporation reaction of the series, detecting a signal transmitted from incorporation of nucleotides resulting from the adding of the deoxynucleoside triphosphates, the signal having an amplitude corresponding to a number of nucleotide incorporations; and

altering the order of the addition of the individual types of deoxynucleoside triphosphates to perform one or more subsequent nucleotide incorporation reactions, thereby compensating for phasing errors due to one or more out-of-phase template nucleic acid strand populations.

2. The method of claim 1, wherein the plurality of template nucleic acid strands are hybridized to a primer.

3. The method of claim 1, wherein the one or more out-of-phase template nucleic acid strand populations comprises a trailing fraction of the template nucleic acid strands that are out of phase relative to an in-phase fraction of the template nucleic acid strands because of extension failures.

4. The method of claim 3, further comprising monitoring the trailing fraction of the template nucleic acid strands to determine where and how to alter the order of addition of the individual types of deoxynucleoside triphosphates so as selectively to extend the trailing fraction of the template nucleic acid strands to bring them back into phase with the in-phase fraction of the template nucleic acid strands.

5. The method of claim 3, wherein altering the order of addition of the individual types of deoxynucleoside triphosphates comprises reversing the order of addition of the individual types of deoxynucleoside triphosphates at arbitrary intervals to bring back into phase about one-third of the trailing fraction of the template nucleic acid strands.

6. The method of claim 1, wherein the one or more out-of-phase template nucleic acid strand populations comprise a leading fraction of the template nucleic acid strands that are out of phase relative to an in-phase fraction of the template nucleic acid strands because of incorrect extensions.

7. The method of claim 6, wherein altering the order of addition of the individual types of deoxynucleoside triphosphates comprises reversing the order of addition of the individual types of deoxynucleoside triphosphates at arbitrary intervals to bring back into phase about two-thirds of the leading fraction of the template nucleic acid strands.

8. The method of claim 3, wherein the one or more out-of-phase template nucleic acid strand populations further comprise a leading fraction of the template nucleic acid strands that are out of phase relative to an in-phase fraction of the template nucleic acid strands because of incorrect extensions.

9. The method of claim 8, wherein altering the order of addition of the individual types of deoxynucleoside triphosphates comprises reversing the order of addition of the deoxynucleoside triphosphates at arbitrary intervals to bring back into phase about two-thirds of the leading fraction of the template nucleic acid strands.

10. A method for phase error compensation, comprising:

providing a plurality of template nucleic acid strands;

adding, in an order, individual types of deoxynucleoside triphosphates serially to the plurality of template nucleic acid strands to perform a series of nucleotide incorporation reactions;

after each nucleotide incorporation reaction of the series, detecting a signal transmitted from incorporation of nucleotides resulting from the adding of the deoxynucleoside triphosphates, the signal having an amplitude corresponding to a number of nucleotide incorporations; and

altering the order of the addition of the individual types of deoxynucleoside triphosphates to perform one or more subsequent nucleotide incorporation reactions, thereby compensating for phasing errors due to one or more out-of-phase template nucleic acid strand populations,

wherein the one or more out-of-phase template nucleic acid strand populations comprises a trailing fraction of the template nucleic acid strands that are out of phase relative to an in-phase fraction of the template nucleic acid strands because of extension failures, and

wherein altering the order of the addition of the individual types of deoxynucleoside triphosphates is based on monitoring the trailing fraction of the template nucleic acid strands to determine where to alter the order of addition of the deoxynucleoside triphosphates so as selectively to extend the trailing fraction of the template nucleic acid strands to bring them back into phase with the in-phase fraction of the template nucleic acid strands.

11. The method of claim 10, wherein the plurality of template nucleic acid strands are hybridized to a primer.

12. The method of claim 10, wherein altering the order of the addition of the individual types of deoxynucleoside triphosphates comprises reversing the order of addition of the individual types of deoxynucleoside triphosphates at arbitrary intervals to bring back into phase about one-third of the trailing fraction of template nucleic acid strands.

13. A method for phase error compensation, comprising:

providing a plurality of template nucleic acid strands;

adding, in an order, individual types of deoxynucleoside triphosphates serially to the plurality of template nucleic acid strands to perform a series of nucleotide incorporation reactions;

after each nucleotide incorporation reaction of the series, detecting a signal transmitted from incorporation of nucleotides resulting from the adding of the deoxynucleoside triphosphates, the signal having an amplitude corresponding to a number of nucleotide incorporations; and

altering the order of the addition of the individual types of deoxynucleoside triphosphates to perform one or more subsequent nucleotide incorporation reactions, thereby compensating for phasing errors due to one or more out-of-phase template nucleic acid strand populations,

wherein the one or more out-of-phase template nucleic acid strand populations comprise a leading fraction of the template nucleic acid strands that are out of phase relative to an in-phase fraction of the template nucleic acid strands because of incorrect extensions; and

wherein altering the order of addition of the individual types of deoxynucleoside triphosphates comprises reversing the order of addition of the individual types of deoxynucleoside triphosphates at arbitrary intervals.

14. The method of claim 13, wherein the plurality of template nucleic acid strands are hybridized to a primer.