INTERMITTENT DETECTION DURING ANALYTICAL REACTIONS

• US 20100075309A1
• Filed: 03/27/2009
• Published: 03/25/2010
• Est. Priority Date: 09/24/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Sorenson, Jon, Eid, John, Marks, Patrick, Travers, Kevin, Maxham, Kenneth Mark
• Granted Patent: US 8,143,030 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2525/307 Circular 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 2021/7786 Fluorescence
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

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 an analytical reaction, comprising:

a) preparing a reaction mixture containing components of the analytical reaction, wherein at least one of the components is a detectable component;

b) initiating the analytical reaction in the reaction mixture to begin progression of the analytical reaction; and

c) maintaining conditions that allow the analytical reaction to proceed while subjecting the reaction mixture to at least one detection period and at least one non-detection period during the progression of the analytical reaction, wherein the detectable component is present during both said detection period and said non-detection period, thereby performing the analytical reaction.

2. The method of claim 1, wherein the detection period is an illuminated period, the non-detection period is a non-illuminated period, further comprising collecting illumination data during the illuminated period, collecting non-illumination data during the non-illuminated period, and further wherein an optical system is used in collecting the illumination data but is not used in collecting the non-illumination data.

3. The method of claim 1, wherein the analytical reaction comprises an enzyme selected from the group consisting of a polymerase, a ligase, a ribosome, a nuclease, and a kinase.

4. The method of claim 3, wherein the enzyme is a polymerase and the method further comprises pause or stop points to control the activity of the polymerase during the analytical reaction.

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

6. The method of claim 1, wherein the analytical reaction is a sequencing reaction comprising a single nucleic acid template, wherein the sequencing reaction generates sequence reads during the detection period by detecting the detectable component, and does not generate sequence reads during the non-detection period by suspending detection of the detectable component.

7. The method of claim 6, wherein the sequencing reaction comprises at least two detection periods and generates a plurality of noncontiguous sequence reads from the single nucleic acid template.

8. The method of claim 7, wherein the plurality comprises at least three noncontiguous sequence reads.

9. The method of claim 6, wherein the single nucleic acid template comprises multiple repeat sequences.

10. The method of claim 6, wherein the sequencing reaction comprises passage of the single nucleic acid template through a nanopore.

11. The method of claim 6, wherein the sequencing reaction comprises primer extension by a polymerase enzyme and the detectable component is a nucleotide or nucleotide analog.

12. The method of claim 1, wherein the analytical reaction is a processive reaction.

13. The method of claim 12, further comprising collecting detection data in real time during the 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 characterize the analytical reaction.

14. The method of claim 1, wherein the detectable component has a detectable label.

15. The method of claim 14, wherein the detectable label is a luminescent, fluorescent, or fluorogenic label.

16. The method of claim 1, wherein the detectable component is a labeled nucleotide or nucleotide analog, and further wherein a concentration of the labeled 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.

17. The method of claim 1, wherein the detection period and the non-detection period are created without any substitution of components in the analytical reaction during progression of the analytical reaction.

18. The method of claim 1, wherein a plurality of analytical reactions are characterized, and further wherein the plurality of analytical reactions are disposed on a solid support.

19. A method of mitigating photo-induced damage during an illuminated reaction, comprising:

a) preparing a reaction mixture comprising a first reactant and a second reactant, wherein interaction of the first reactant with the second reactant under excitation illumination causes an amount of photo-induced damage to the first reactant; and

b) subjecting the illuminated reaction to intermittent excitation illumination, wherein the intermittent excitation illumination reduces the amount of photo-induced damage to the first reactant during the illuminated reaction as compared to the illuminated reaction under constant excitation illumination, thereby mitigating photo-induced damage to the first reactant.

20. The method of claim 19, wherein said illuminated reaction is a base extension reaction.

21. The method of claim 19, wherein said first reactant is a polymerase enzyme.

22. The method of claim 19, wherein said second reactant comprises a fluorescent or fluorogenic molecule.

23. The method of claim 19, wherein a length of time during which the illuminated reaction is subjected to the excitation illumination is less than a photo-induced damage threshold period.

24. A method of generating a plurality of noncontiguous sequence reads from a single nucleic acid template molecule, comprising:

a) preparing a reaction mixture comprising the single template nucleic acid molecule, a polymerase enzyme, and a set of labeled nucleotides or nucleotide analogs, wherein the set comprises at least one type of labeled nucleotide or nucleotide analog for each of the natural nucleobases (A, G, T, and C), and further wherein each type of labeled nucleotide or nucleotide analog in the set comprises a detectable label that distinguishes it from every other type in the set;

b) initiating the polymerization reaction to begin a first processive incorporation of a plurality of the labeled nucleotides or nucleotide analogs into a nascent nucleic acid strand complementary to the single template nucleic acid molecule;

c) detecting the first processive incorporation by optical means, thereby generating one of the plurality of noncontiguous sequence reads from the single template nucleic acid template molecule;

d) performing a buffer exchange to substitute the labeled nucleotides or nucleotide analogs with unlabeled nucleotides or nucleotide analogs;

e) allowing the polymerization reaction to begin a second processive incorporation of the unlabeled nucleotides or nucleotide analogs without detecting the second processive incorporation of the unlabeled nucleotides or nucleotide analogs;

f) performing a buffer exchange to substitute the unlabeled nucleotides or nucleotide analogs with the labeled nucleotides or nucleotide analogs;

g) allowing the polymerization reaction to initiate a third processive incorporation of a plurality of the labeled nucleotides or nucleotide analogs;

h) detecting the third processive incorporation by optical means, thereby generating a second of the plurality of noncontiguous sequence reads from the single template nucleic acid molecule.

25. The method of claim 24 further comprising repeating steps d-g, wherein steps d-g are repeated n times to generate (n+2) noncontiguous sequence reads.

26. A device comprising:

a) a substrate having an observation region;

b) a first reactant immobilized within the observation region;

c) a second reactant disposed within the observation region; and

d) a means for subjecting the observation region to at least one illuminated period and at least one non-illuminated period.

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37. A system for performing intermittent detection of an analytical reaction, comprising:

a) a solid support having reagents for the analytical reaction disposed thereon;

b) a mounting stage configured to receive the solid support;

c) an optical train positioned to be in optical communication with at least a portion of the solid support to detect signals emanating therefrom;

d) a means for subjecting the portion of the solid support to at least one detection period and at least one non-detection period;

e) a translation system operably coupled to the mounting stage or the optical train for moving one of the optical train and the solid support relative to the other; and

f) a data processing system operably coupled to the optical train.

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43. A machine-implemented method for transforming nucleotide sequence read data into consensus sequence data, wherein the nucleotide sequence read data is representative of a sequence of bases detected when sequencing a target region of a template nucleic acid multiple times, and the consensus sequence data is representative of a most likely actual sequence of the template nucleic acid, the machine-implemented method comprising:

a) mapping the nucleotide sequence data to a target sequence using a local alignment method that produces a set of local alignments comprising an optimal local alignment and sub-optimal local alignments;

b) enumerate the set of local alignments;

c) construct a weighted directed graph wherein each local alignment in the set of local alignments is represented as a node, thereby generating a set of nodes in the weighted directed graph;

d) draw edges between pairs of nodes in the weighted directed graph if the pair represents a potential reconstruction of the template nucleic acid;

e) assign weights to the edges drawn in step d, wherein a given weight for a given edge represents the log-likelihood that a given pair of nodes connected by the given edge is truly a reconstruction of the template nucleic acid;

f) find the shortest path to each node in the weighted directed graph, thereby generating a set of shortest paths for the weighted directed graph;

g) rank the set of shortest paths to determine the best assignment; and

h) storing the results of steps a-g on a machine-readable medium.

44. The method of claim 43, wherein steps a-h are performed via a user interface implemented in a machine that comprises instructions stored in machine-readable medium and a processor that executes the instructions.

45. A computer program product comprising a computer usable medium having

a) a computer readable program code embodied therein, said computer readable program code adapted to be executed to implement the method of claim 43;

and

b) the machine-readable medium on which the results of steps a-g are stored.