Mitigation of photodamage in analytical reactions
First Claim
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1. A composition, comprising:
- a first reactant, wherein the first reactant comprises a polymerase;
a second reactant, wherein the second reactant comprises a fluorescent or fluorogenic substrate for the polymerase; and
more than one different photodamage mitigating agent present in a concentration sufficient to reduce photodamage to the polymerase when the first and second reactant are subjected to an excitation illumination for the second reactant;
wherein interaction of the first reactant with the second reactant under excitation illumination causes photodamage to the first reactant in the absence of the more than one different photodamage mitigating agent.
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Abstract
Compositions, devices, systems and methods for reducing and/or preventing photodamage of one or more reactants in illuminated analytical reactions by one or more of incorporating photodamage mitigating agents within the reaction mixture and/or interrogating different observation regions of the reaction mixture for a period that is less than a photodamage threshold period.
61 Citations
40 Claims
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1. A composition, comprising:
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a first reactant, wherein the first reactant comprises a polymerase; a second reactant, wherein the second reactant comprises a fluorescent or fluorogenic substrate for the polymerase; and
more than one different photodamage mitigating agent present in a concentration sufficient to reduce photodamage to the polymerase when the first and second reactant are subjected to an excitation illumination for the second reactant;
wherein interaction of the first reactant with the second reactant under excitation illumination causes photodamage to the first reactant in the absence of the more than one different photodamage mitigating agent. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
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27. A composition, comprising:
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a confined polymerase; a fluorescent or fluorogenic substrate for said polymerase and photodamage mitigating agents comprising more than one different triplet state quencher present in a concentration sufficient to reduce photodamage to the polymerase when the polymerase and the fluorescent or fluorogenic substrate are subjected to excitation illumination for the fluorescent or fluorogenic substrate wherein interaction of the enzyme with the fluorescent or fluorogenic substrate under excitation illumination for the fluorescent or fluorogenic substrate causes photodamage to the polymerase in the absence of the photodamage mitigating agents. - View Dependent Claims (28, 29, 30, 31, 32, 33, 34, 35)
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36. A device, comprising:
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a substrate having an observation region; a first reactant immobilized within the observation region, wherein the first reactant comprises a polymerase; a second reactant comprising a fluorescent or fluorogenic substrate for the polymerase disposed within the observation region, wherein interaction between the first and second reactants under excitation illumination causes photodamage to both the fluorescent of fluorogenic substrate and the first reactant; and more than one different photodamage mitigating agent that are present in a concentration sufficient to reduce photodamage to the polymerase when the first and second reactant are subjected to an excitation illumination wavelength for the second reactant disposed within the observation region. - View Dependent Claims (37, 38, 39, 40)
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Specification