Mitigation of photodamage in analytical reactions
First Claim
Patent Images
1. A composition, comprising:
- a first reactant;
a second reactant; and
a photodamage mitigating agent;
wherein interaction of the first reactant with the second reactant under excitation illumination causes photodamage to the first reactant in the absence of the 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.
116 Citations
38 Claims
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1. A composition, comprising:
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a first reactant;
a second reactant; and
a photodamage mitigating agent;
wherein interaction of the first reactant with the second reactant under excitation illumination causes photodamage to the first reactant in the absence of the photodamage mitigating agent. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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20. A composition, comprising:
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a confined enzyme;
a substrate for said enzyme; and
a photodamage mitigating agent;
wherein interaction of the enzyme with the substrate under excitation illumination causes photodamage to the enzyme in the absence of the photodamage mitigating agent. - View Dependent Claims (21, 22, 23, 24, 25, 26)
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27. A composition, comprising:
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a confined enzyme;
a substrate for said enzyme, wherein interaction of the enzyme with the substrate under excitation illumination causes photodamage to the first reactant; and
a photodamage mitigating agent.
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28. A device, comprising:
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a substrate having an observation region;
a first reactant immobilized within the observation region;
a second reactant disposed within the observation region, wherein interaction between the first and second reactants under excitation illumination causes photodamage to the first reactant; and
a photodamage mitigating agent disposed within the observation region
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29. A method of performing an illuminated reaction, comprising:
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providing a substrate having a reaction mixture disposed thereon, wherein the reaction mixture comprises a first reactant, a second reactant and a photodamage mitigating agent, wherein the photodamage mitigating agent reduces an amount of photodamage to the first reactant resulting from interaction of the first reactant with the second reactant under excitation illumination that would occur in the absence of the photodamage mitigating agent; and
illuminating the reaction mixture on the substrate, with an excitation illumination. - View Dependent Claims (30)
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31. A method of performing an enzyme reaction, comprising:
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providing an enzyme within a first observation region;
contacting the enzyme with a fluorescent or fluorogenic substrate for the enzyme; and
directing an excitation radiation at and detecting signals from the first observation region for a period that is less than a photodamage threshold period. - View Dependent Claims (32)
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33. A method of monitoring a base extension reaction, comprising:
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providing a polymerase enzyme within a first observation region;
contacting the polymerase with at least a first fluorescent or fluorogenic nucleotide analog; and
monitoring a fluorescent signal emitted from the first observation region in response to illumination with excitation radiation for a period that is less than a photodamage threshold period.
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34. A system for analyzing an illuminated reaction that is susceptible to photodamage when illuminated for a period longer than an photodamage threshold period, comprising:
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a substrate having reagents for the reaction disposed thereon;
a mounting stage supporting the substrate and configured to receive the substrate;
an optical train positioned to be in optical communication with at least a portion of the substrate to illuminate the portion of the substrate and detect signals emanating therefrom;
a translation system operably coupled to the mounting stage or the optical train for moving one of the optical train and the substrate relative to the other.
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35. A method of performing an enzyme reaction, comprising:
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providing an enzyme within an observation region;
contacting the enzyme with a fluorescent or fluorogenic substrate for the enzyme under excitation illumination, in the presence of at least a first photodamage mitigating agent.
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36. A method of monitoring a base extension reaction, comprising:
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providing a polymerase enzyme within an observation region;
contacting the polymerase with at least a first fluorescent or fluorogenic nucleotide analog in the presence of at least a first photodamage mitigating agent; and
monitoring a fluorescent signal emitted from the observation region in response to illumination with excitation radiation.
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- 37. A method of monitoring a reaction mixture comprising at least a first enzyme and a fluorescent or fluorogenic substrate for the first enzyme, comprising directing an excitation radiation at a first observation region for a first period that is less than a photodamage threshold period.
Specification