Intermittent detection during analytical reactions

• US 9,057,102 B2
• Filed: 11/27/2013
• Issued: 06/16/2015
• Est. Priority Date: 03/28/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Turner, Stephen, Sorenson, Jon, Maxham, Kenneth Mark, Eid, John
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US14/091,961
• Publication Number:
  US 20140134629A1
• Time in Patent Office: 566 Days
• Field of Search: 435/91.2
• US Class Current: 1/1
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search
• CPC Class Codes: G16B 30/20 Sequence assembly

Abstract

Methods, devices, and systems for performing intermittent detection during analytical reactions are provided. Such methods facilitate collection of reaction data from disparate reaction times. Further, such methods are useful for reducing photo-induced damage of one or more reactants in an illuminated analytical reaction at a given reaction time. In preferred embodiments, the reaction mixture is subjected to at least one illuminated and non-illuminated period and allowed to proceed such that the time in which the reaction mixture is illuminated is less than a photo-induced damage threshold period.

Claims

1. A method of performing a nucleic acid sequencing reaction, comprising:

a) preparing a reaction mixture comprising components of the nucleic acid sequencing reaction including a polynucleotide to be sequenced;

b) introducing the reaction mixture to a membrane comprising a nanopore;

c) initiating the nucleic acid sequencing reaction in the reaction mixture to begin progression of the nucleic acid sequencing reaction, wherein the nucleic acid sequencing reaction comprises passage of the polynucleotide or a nascent strand complementary thereto through the nanopore and monitoring the nanopore as voltage is applied across the membrane; and

d) maintaining conditions that allow the nucleic acid sequencing reaction to proceed while subjecting the reaction mixture to at least two detection periods and at least one non-detection period during the progression of the nucleic acid sequencing reaction, wherein the progress of the polynucleotide or the nascent strand through the nanopore is slowed during the detection periods to allow sequence determination, and further wherein the progress of the polynucleotide or the nascent strand through the nanopore is sped up during the non-detection period, thereby performing the nucleic acid sequencing reaction.

2. The method of claim 1, wherein the polynucleotide or the nascent strand is drawn through the nanopore by an electrophoretic method.

3. The method of claim 2, wherein the progress of the polynucleotide or the nascent strand through the nanopore is sped up during the non-detection period by increasing an electrophoretic field.

4. The method of claim 1, wherein the polynucleotide or the nascent strand is drawn through the nanopore by an enzymatic method.

5. The method of claim 4, wherein the enzymatic method comprises a protein chaperone that directs the polynucleotide or the nascent strand through the nanopore.

6. The method of claim 5, wherein the progress of the polynucleotide or the nascent strand through the nanopore is sped up by increasing the speed at which the protein chaperone directs the polynucleotide or the nascent strand through the nanopore.

7. The method of claim 5, wherein the protein chaperone is a polymerase enzyme.

8. The method of claim 1, wherein the nucleic acid sequencing reaction generates a plurality of noncontiguous sequence reads from the single nucleic acid template.

9. The method of claim 1, wherein the monitoring comprises detecting changes in ionic current through the nanopore.

10. The method of claim 1, wherein the membrane is a lipid bilayer.

11. The method of claim 1, wherein the membrane is a solid-state membrane.

12. The method of claim 1, wherein the nanopore comprises a protein channel.

13. A method of performing a nucleic acid sequencing reaction, comprising:

a) preparing a reaction mixture comprising components of the nucleic acid sequencing reaction including a polynucleotide to be sequenced;

b) introducing the reaction mixture to a membrane comprising a nanopore;

c) initiating the nucleic acid sequencing reaction in the reaction mixture to begin progression of the nucleic acid sequencing reaction, wherein the nucleic acid sequencing reaction comprises cleavage of the polynucleotide, passage of nucleotides cleaved from the polynucleotide through the nanopore, and monitoring the nanopore as voltage is applied across the membrane; and

d) maintaining conditions that allow the nucleic acid sequencing reaction to proceed while subjecting the reaction mixture to at least two detection periods and at least one non-detection period during the progression of the nucleic acid sequencing reaction, wherein the progress of the nucleotides through the nanopore is slowed during the detection periods to allow sequence determination, and further wherein the progress of the nucleotides through the nanopore is sped up during the non-detection period, thereby performing the nucleic acid sequencing reaction.

14. The method of claim 13, wherein the cleavage is catalyzed by an exonuclease.

15. The method of claim 14, wherein the progress of the nucleotides through the nanopore is sped up by increasing the speed at which the exonuclease cleaves the polynucleotide.

16. The method of claim 13, wherein the nucleic acid sequencing reaction generates a plurality of noncontiguous sequence reads from the single nucleic acid template.

17. The method of claim 13, wherein the monitoring comprises detecting changes in ionic current through the nanopore.

18. The method of claim 13, wherein the membrane is a lipid bilayer.

19. The method of claim 13, wherein the membrane is a solid-state membrane.

20. The method of claim 13, wherein the nanopore comprises a protein channel.