Nucleic acid sequence analysis

Patent metadata

Field	Value
Publication number	US-9738929-B2
Application number	US-201615383965-A
Country	US
Kind code	B2
Filing date	Dec 19, 2016
Priority date	Mar 28, 2008
Publication date	Aug 22, 2017
Grant date	Aug 22, 2017

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.

First claim

What is claimed is:

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.

Assignees

• Pacific Biosciences California Inc

Inventors

• Turner Stephen
• Sorenson Jon
• Maxham Kenneth Mark
• Eid John
• Heiner Cheryl
• Travers Kevin

Classifications

• G01N33/48721

  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 title

• G01N21/6428

  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 title

• G01N21/6486

  Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence) · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12N9/1252

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title