Intermittent detection during analytical reactions

Patent metadata

Field	Value
Publication number	US-10563255-B2
Application number	US-201715631602-A
Country	US
Kind code	B2
Filing date	Jun 23, 2017
Priority date	Sep 24, 2008
Publication date	Feb 18, 2020
Grant date	Feb 18, 2020

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 a sequencing reaction, comprising:
a) preparing a sequencing reaction mixture comprising:
a single polymerase;
a single circular nucleic acid template comprising a region of interest;
a single sequencing primer; and
nucleotides, nucleotide analogs, or a combination thereof, wherein at least one or more of the nucleotides or nucleotide analogs is detectable;

b) initiating a sequencing reaction in the sequencing reaction mixture during a non-detection period during which the single circular template is completely replicated at least one time; and
c) maintaining conditions that allow the sequencing reaction to proceed while subjecting the sequencing reaction mixture to at least one detection period after the non-detection period, wherein the sequencing reaction generates at least one sequence read in the region of interest during the at least one detection period by detecting the at least one detectable nucleotide or nucleotide analog, and wherein the sequencing reaction does not generate a sequence read for the region of interest during the first non-detection period, thereby performing the sequencing reaction.

2. The method of claim 1 , wherein the at least one detection period is an illuminated period and the first non-detection period is a non-illuminated period, the method further comprising collecting illumination data during the illuminated period using an optical system.

3. The method of claim 2 , further comprising collecting non-illumination data during the non-illuminated period, wherein the optical system is used in collecting the illumination data but is not used in collecting the non-illumination data.

4. The method of claim 3 , wherein the sequencing reaction comprises:
collecting detection data in real time during the at least one detection period;
collecting non-detection data in real time during the non-detection period; and
combining the detection data and the non-detection data to perform the sequencing reaction.

5. The method of claim 1 , wherein subjecting the sequencing reaction mixture to the non-detection period results in a change in one or more aspects of the sequencing reaction, said aspects selected from the group consisting of processivity, fidelity, rate, and duration.

6. The method of claim 1 , wherein the sequencing reaction comprises at least two non-detection periods and at least two detection periods and generates a plurality of noncontiguous sequence reads for the region of interest from the single circular nucleic acid template.

7. The method of claim 6 , wherein the plurality of non-contiguous sequence reads comprises at least three noncontiguous sequence reads.

8. The method of claim 1 , wherein the single nucleic acid template comprises multiple repeat sequences.

9. The method of claim 1 , wherein the detectable nucleotide or nucleotide analog comprises a detectable label.

10. The method of claim 9 , wherein the detectable label is a luminescent, fluorescent, or fluorogenic label.

11. The method of claim 1 , wherein a concentration of the detectable nucleotide or nucleotide analog in the reaction mixture is greater than a concentration of any unlabeled nucleotide or nucleotide analog present in the reaction mixture throughout the progression of the analytical reaction.

12. The method of claim 1 , wherein the at least one detection period and the non-detection period are created without any substitution of components in the sequencing reaction during progression of the sequencing reaction.

13. The method of claim 1 , wherein a plurality of sequencing reactions are performed, and wherein the plurality of sequencing reactions are disposed on a solid support.

14. The method of claim 1 , wherein during the at least one detection period the single circular nucleic acid template is replicated multiple times to generate multiple complete sequence reads for the region of interest.

15. The method of claim 14 , wherein during the at least one detection period at least one incomplete sequence read is generated for the region of interest.

16. The method of claim 15 , wherein the at least one incomplete sequence read can be used to construct a scaffold for assembly of the multiple complete sequence reads.

17. The method of claim 1 , wherein the single circular nucleic acid template is a single-stranded circular nucleic acid template.

18. The method of claim 17 , wherein the single-stranded circular nucleic acid template comprises a double-stranded region and two hairpins joining the 3′ end of each complementary strand of the double-stranded region to the 5′ end of the other complementary strand.

19. The method of claim 18 , wherein the at least one sequence read comprises sequences from both of the complementary strands of the double-stranded region.

20. The method of claim 1 , wherein the polymerase is a strand-displacing polymerase.

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

• 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

• 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