Nucleic acid sequence analysis

• US 9,738,929 B2
• Filed: 12/19/2016
• Issued: 08/22/2017
• 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, Heiner, Cheryl, Travers, Kevin
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US15/383,965
• Publication Number:
  US 20170121764A1
• Time in Patent Office: 246 Days
• Field of Search: 435 61
• 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 determining a nucleotide sequence of a region of interest in a polynucleotide, the method comprising:

introducing a polynucleotide comprising a region of interest to a sequence analysis system comprising a nanopore in a membrane, wherein the polynucleotide comprises a double-stranded portion comprising complementary strands of the region of interest;

applying a voltage across the membrane;

monitoring variations in ionic current through the nanopore of the sequence analysis system during enzyme chaperone-regulated passage of the polynucleotide through the nanopore;

analyzing the monitored variations in ionic current to obtain nucleotide sequence information for the polynucleotide, wherein the nucleotide sequence information comprises redundant sequence information for the region of interest, wherein the redundant sequence information comprises the nucleotide sequence of the complementary strands; and

determining a consensus sequence for the region of interest based on the redundant sequence information.

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

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

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

5. The method of claim 1, further comprising changing reaction conditions to alter the rate of enzyme chaperone regulated passage of the polynucleotide through the nanopore.

6. The method of claim 1, wherein the polynucleotide is greater than 75% double-stranded DNA.

7. The method of claim 1, wherein the polynucleotide is greater than 90% double-stranded DNA.

8. The method of claim 1, wherein the complementary strands are linked.

9. The method of claim 1, wherein the polynucleotide comprises multiple repeats of the region of interest, wherein the redundant sequence information further comprises the nucleotide sequence of the multiple repeats.

10. The method of claim 8, wherein the complementary strands are linked by a linker comprising a nucleotide.

11. The method of claim 10, wherein the linker comprises an oligonucleotide.

12. The method of claim 11, wherein the oligonucleotide comprises a registration sequence.

13. The method of claim 11, wherein the linker comprises a nick.

14. The method of claim 8, wherein the complementary strands are linked by a synthetic linker.

15. The method of claim 14, wherein the synthetic linker is a carbon-based linker.

16. The method of claim 5, wherein the monitoring comprises a detection period and a non-detection period, wherein the rate of passage of the polynucleotide through the nanopore by the enzyme chaperone is sped up during the non-detection period and slowed during the detection period, wherein the monitored variations in ionic current through the nanopore in the detection period is subjected to the analyzing and determining steps.

17. The method of claim 16, wherein the monitoring comprises multiple detection periods.