Fluorescent Nucleotide Imaging Reagents to Reduce Photo-Bleaching
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Solution Overview
Problem
Existing nucleic acid sequencing methods suffer from significant photo-bleaching of fluorescently-labeled nucleotides, which degrades the imaging quality and accuracy of sequencing results.
Innovation Solution
The use of imaging reagents containing ascorbic acid, such as sodium ascorbate, in conjunction with other components like Tris-HCl, EDTA, and non-catalytic divalent cations, helps reduce photo-bleaching by forming stable binding complexes and maintaining fluorescent signal integrity during imaging.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescently-labeled nucleotide conjugates are used for nucleic acid sequencing, then detection capability is improved, but photo-bleaching occurs which degrades imaging quality over time
Solution Approach 1:
The patent introduces an imaging reagent as an intermediary substance that mediates between the fluorescently-labeled nucleotide conjugates and the imaging process. This reagent contains compounds that reduce photo-bleaching of the fluorophores, thereby protecting the fluorescent labels while maintaining their detection capability. The imaging reagent acts as a protective intermediary that extends the stability and reliability of the fluorescent signals during imaging.
2Loss of information
If imaging is performed to obtain sequencing data, then information is obtained, but fluorescent signals degrade due to photo-bleaching
Solution Approach 1:
The patent applies preliminary action by introducing the imaging reagent before imaging begins and maintaining it throughout the imaging process. This reagent pre-establishes a protective environment that prevents photo-bleaching before it can occur during data acquisition. By having the protective reagent in place beforehand, the fluorescent signals remain stable throughout the sequencing imaging process, preventing signal degradation.
3Measurement precision
If extended imaging is performed to improve sequencing accuracy, then measurement precision is improved, but photo-bleaching increases reducing signal reliability
Solution Approach 1:
The patent ensures continuous protection against photo-bleaching by maintaining the imaging reagent throughout the entire imaging process. The reagent continuously acts to reduce photo-bleaching, allowing extended imaging to be performed without progressive signal degradation. This continuous protective action enables prolonged data acquisition while maintaining signal reliability and fluorescence intensity throughout the extended imaging duration.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach significantly reduces the risk of photo-bleaching, enhancing the imaging quality and accuracy of nucleic acid sequencing by preserving the fluorescent signals over time.
Implementation Method 1
obtaining an image of the binding complex in a presence of the imaging reagent, thereby reducing a risk of photo-bleaching of the fluorescently-labeled nucleotide conjugate as compared to a risk of photo-bleaching under like conditions in absence of the imaging reagent
Data Source
AI summary
Provided herein are fluorescently-labeled nucleotide conjugates for nucleic acid analysis. Also provided are reagents used for forming binding complexes between a fluorescently-labeled nucleotide conjugate and a target nucleic acid sequence in the presence of one or more reagents disclosed herein. Binding complexes can be detected in the presence of the one or more reagents. For example, the one or more reagents may contain a photobleaching reducing agent configured to reduce photobleaching resulting from use of the fluorescently-labeled nucleotide conjugate to form the binding complex in a nucleic acid analysis. Such nucleic acid analysis may be used to identify sites of nucleobase binding or incorporation between the target nucleic acid sequence and one or more nucleotide moieties of the fluorescently-labeled nucleotide conjugate in a nucleic acid sequence reaction.