Intermittent detection during analytical reactions
First Claim
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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, wherein the analytical reaction comprises an enzyme selected from the group consisting of a polymerase, a ligase, a ribosome, a nuclease, and a kinase; 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 detection period is an illuminated period and the non-detection period is a non-illuminated period, and further wherein the detectable component is present during both said detection period and said non-detection period, thereby performing the analytical reaction.
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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.
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Citations
19 Claims
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1. A method of performing an analytical reaction, comprising:
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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, wherein the analytical reaction comprises an enzyme selected from the group consisting of a polymerase, a ligase, a ribosome, a nuclease, and a kinase; 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 detection period is an illuminated period and the non-detection period is a non-illuminated period, and further wherein the detectable component is present during both said detection period and said non-detection period, thereby performing the analytical reaction. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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18. A method of generating a plurality of noncontiguous sequence reads from a single nucleic acid template molecule, comprising:
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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; and 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. - View Dependent Claims (19)
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Specification