Intermittent detection during analytical reactions
First Claim
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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.
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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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20 Claims
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1. A method of performing a sequencing reaction, comprising:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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Specification