Method to improve single molecule analyses
First Claim
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1. An analytical method comprising;
- localizing a plurality of active molecules into optical confinements on a substrate whereby a plurality of the optical confinements comprise a single active molecule;
exposing the single active molecules in the optical confinements to a reagent solution, whereby either the single active molecules or a reagent in the reagent solution or both comprise one or more fluorescent labels;
initiating a reaction of the single active molecules;
measuring fluorescence from the plurality of optical confinements over time to monitor the reaction and to obtain reaction data from each of the plurality of confinements;
halting the reaction;
measuring fluorescence from the plurality of optical confinements over time while the reaction is halted to obtain sticking data; and
combining the sticking data with reaction data to provide a more accurate measurement of the reaction than if the sticking data was not used.
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Abstract
The quality of information from single molecule analyses is improved by employing a method in which a single molecule reaction carried out within an optical confinement is monitored, the single molecule reaction is halted, and data from the optical confinement is obtained while the reaction is not occurring. Characteristic optical behavior observed while the reaction is halted is used to improve the quality of information obtained during the single molecule reaction, for example, by correcting the reaction data, excluding the reaction data, or providing a confidence level to the reaction data.
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Citations
22 Claims
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1. An analytical method comprising;
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localizing a plurality of active molecules into optical confinements on a substrate whereby a plurality of the optical confinements comprise a single active molecule; exposing the single active molecules in the optical confinements to a reagent solution, whereby either the single active molecules or a reagent in the reagent solution or both comprise one or more fluorescent labels; initiating a reaction of the single active molecules; measuring fluorescence from the plurality of optical confinements over time to monitor the reaction and to obtain reaction data from each of the plurality of confinements; halting the reaction; measuring fluorescence from the plurality of optical confinements over time while the reaction is halted to obtain sticking data; and combining the sticking data with reaction data to provide a more accurate measurement of the reaction than if the sticking data was not used. - View Dependent Claims (2, 3, 4, 5, 6)
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7. A method of improving accuracy in single molecule sequencing comprising:
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localizing a plurality of polymerase enzyme complexes into optical confinements whereby a plurality of the optical confinements comprise a single active polymerase enzyme complex; exposing the polymerase enzyme complexes to a reagent solution comprising the components necessary for polymerase activity including a plurality of types of labeled nucleotides or nucleotide analogs, each type comprising a different label; initiating a sequencing reaction; measuring fluorescence from the plurality of optical confinements over time during the sequencing reaction to obtain sequencing data; halting the sequencing reaction; measuring fluorescence from the plurality of optical confinements over time while the sequencing reaction is halted to obtain sticking data; and using both the sticking data and the sequencing data to provide sequencing information that is more accurate than if the sticking data was not used. - View Dependent Claims (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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Specification