Mitigation of photodamage in analytical reactions
First Claim
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1. A method of performing an illuminated reaction, comprising:
- providing a substrate having an observation region and a reaction mixture disposed thereon, wherein the reaction mixture comprisesa) a single molecule of a polymerase confined within the observation region,b) a plurality of second reactants comprising substrates for the polymerase, each of which comprises a fluorescent label,c) and photodamage mitigating agents which are present in a concentration sufficient to reduce photodamage to the polymerase comprising more than one different triplet state quencher wherein the photodamage mitigating agents reduce an amount of photodamage to the polymerase during the interaction between the single molecule of the polymerase with one of the second reactants under excitation illumination of the second reactants that would occur in the absence of the photodamage mitigating agents; and
illuminating the reaction mixture on the substrate having an observation region with the excitation illumination of the second reactants.
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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.
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Citations
21 Claims
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1. A method of performing an illuminated reaction, comprising:
- providing a substrate having an observation region and a reaction mixture disposed thereon, wherein the reaction mixture comprises
a) a single molecule of a polymerase confined within the observation region, b) a plurality of second reactants comprising substrates for the polymerase, each of which comprises a fluorescent label, c) and photodamage mitigating agents which are present in a concentration sufficient to reduce photodamage to the polymerase comprising more than one different triplet state quencher wherein the photodamage mitigating agents reduce an amount of photodamage to the polymerase during the interaction between the single molecule of the polymerase with one of the second reactants under excitation illumination of the second reactants that would occur in the absence of the photodamage mitigating agents; and illuminating the reaction mixture on the substrate having an observation region with the excitation illumination of the second reactants. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 14)
- providing a substrate having an observation region and a reaction mixture disposed thereon, wherein the reaction mixture comprises
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10. A method of performing an enzyme reaction, comprising:
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providing a single polymerase within an observation region; contacting the single polymerase with a reaction mixture comprising a fluorescent or fluorogenic substrate for the polymerase under an excitation illumination for the fluorescent or fluorogenic substrate, in the presence of photodamage mitigating agents which are present in a concentration sufficient to reduce photodamage to the polymerase, and further wherein the photodamage mitigating agents comprise at least one triplet state quencher selected from the group consisting of ascorbic acid, dithiothreitol, mercaptoethylamine, beta-mercaptoethanol, n-propyl gallate, p-phenylenediamine, hydroquinone, sodium azide and diazobicyclooctane and at least one singlet oxygen scavenger or singlet oxygen quencher selected from the group consisting of superoxide dismutase, glucose oxidase/catalase, glutathione peroxidase, ergothionine, methionine, cysteine, beta-dimethyl cysteine, mercaptopropionylglycine, 2-mercaptoethane sulfonate sodium, glutathione, N-acetyl cysteine, captopril, lycopene, gamma-carotene, astazanthin, canthazanthin, alpha-carotene, beta-carotene, bixin, zeaxanthin, lutein, bilirubin, biliverdin, tocopherol, polyene dialdehydes, melatonin, alpha-tocopheryl succinate, pyridoxinel, hydrazine, sodium sulfite and hydroxylamine, and further wherein only a portion of the reaction mixture within the observation region is exposed to the excitation illumination. - View Dependent Claims (11, 12, 13, 15)
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16. A method of monitoring a base extension reaction, comprising:
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providing a single polymerase within an observation region; contacting the single polymerase with a reaction mixture comprising at least a first fluorescent or fluorogenic nucleotide analog under an excitation illumination for the fluorescent or fluorogenic nucleotide analog in the presence of more than one different photodamage mitigating agent, which are present in a concentration sufficient to reduce photodamage to the polymerase, and further wherein the fluorescent or fluorogenic nucleotide analog is a phosphate labeled nucleoside polyphosphate; and monitoring a fluorescent signal emitted from the observation region in response to illumination with the excitation radiation of the fluorescent or fluorogenic nucleotide analog, wherein the excitation radiation does not excite a subset of additional fluorescent or fluorogenic nucleotide analogs that are within the reaction mixture but outside of the observation region. - View Dependent Claims (17, 18, 19, 20, 21)
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Specification