Intermittent detection during analytical reactions

Patent metadata

Field	Value
Publication number	US-11214830-B2
Application number	US-201916658576-A
Country	US
Kind code	B2
Filing date	Oct 21, 2019
Priority date	Sep 24, 2008
Publication date	Jan 4, 2022
Grant date	Jan 4, 2022

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 performing an analytical reaction on a polynucleotide, the method comprising:
introducing a polynucleotide to a sequence analysis system comprising a nanopore in an electrically insulating membrane;
applying a voltage across the membrane;
monitoring variations in ionic current through the nanopore of the sequence analysis system during passage of the polynucleotide through the nanopore, wherein the monitoring comprises a detection period and a non-detection period, wherein the rate of passage of the polynucleotide through the nanopore is sped up during the non-detection period and slowed during the detection period; and
analyzing the monitored variations in ionic current of the detection period to obtain nucleotide sequence information for a region of interest in the polynucleotide.

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 , wherein the rate of passage of the polynucleotide through the nanopore is regulated by an enzyme chaperone.

6. The method of claim 5 , wherein the rate of enzyme chaperone regulated passage of the polynucleotide through the nanopore is changed by adjusting reaction conditions.

7. The method of claim 1 , wherein the polynucleotide comprises a double-stranded portion comprising complementary strands of the region of interest.

8. The method of claim 7 , wherein the polynucleotide is greater than 75% double-stranded DNA.

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

10. The method of claim 7 , wherein the complementary strands are linked.

11. The method of claim 7 , wherein the nucleotide sequence information comprises redundant sequence information for the region of interest, wherein the redundant sequence information comprises the nucleotide sequence information from the complementary strands, the method further comprising determining a consensus sequence for the region of interest based on the redundant sequence information.

12. The method of claim 7 , wherein the complementary strands are linked by a linker comprising a nucleotide.

13. The method of claim 12 , wherein the linker comprises an oligonucleotide.

14. The method of claim 13 , wherein the oligonucleotide comprises a registration sequence.

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

16. The method of claim 7 , wherein the complementary strands are linked by a synthetic linker.

17. The method of claim 16 , wherein the synthetic linker is a carbon-based linker.

18. The method of claim 1 , wherein the polynucleotide comprises multiple repeats of the region of interest, wherein the nucleic acid sequence information comprises redundant sequence information comprising nucleotide sequence information from the multiple repeats, the method further comprising determining a consensus sequence for the region of interest based on the redundant sequence information.

19. The method of claim 1 , wherein the monitoring comprises multiple detection periods.

Assignees

• Pacific Biosciences California Inc

Inventors

• Turner Stephen
• Sorenson Jon
• Maxham Kenneth Mark
• Eid John

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

• G01N21/6486

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

• G16B30/00

  ICT specially adapted for sequence analysis involving nucleotides or amino acids · CPC title

• C12N9/1252

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

• G16B30/20

  Sequence assembly · CPC title