Bead-Labeled Nucleotides for Sequencing Signal Brightness
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Solution Overview
Problem
Traditional methods of labeling nucleotides for nucleic acid analyses face issues with label brightness, photodamage, and instability due to photobleaching, requiring expensive equipment like high-power lasers and electron multiplying CCD cameras.
Innovation Solution
Nucleotides labeled with beads that produce a brighter signal, featuring multiple fluorophores to enhance brightness and stability, reducing the effects of photobleaching and allowing for 'recycling' of nucleoside polyphosphates, thereby improving signal-to-noise ratios and reducing background noise.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If traditional fluorophore-labeled nucleotides are used, then nucleic acid analysis can be performed, but the label brightness is insufficient and photodamage occurs
Solution Approach 1:
The patent divides the labeling approach by attaching multiple fluorophores to a single nucleotide molecule through a bead structure, rather than using a single fluorophore per nucleotide. This segmentation of the labeling function increases brightness while reducing the need for high-power lasers that cause photodamage.
Solution Approach 2:
The patent creates a composite labeled nucleotide structure consisting of a nucleotide base, a bead, and multiple fluorophores integrated into a single functional unit. This composite structure provides enhanced brightness and reduced photodamage compared to traditional single-fluorophore labels.
2Reliability
If traditional fluorophore-labeled nucleotides are used, then sequencing can proceed, but the label is unstable due to photobleaching
Solution Approach 1:
The patent segments the fluorophore loading by distributing multiple fluorophores across a bead structure attached to each nucleotide. This segmentation provides redundancy where if some fluorophores bleach, others remain functional, extending the label's durable action period.
Solution Approach 2:
The patent changes the physical and chemical parameters of the labeling system by using a bead-based structure with multiple fluorophores instead of a single small-molecule fluorophore. This parameter change increases the statistical probability that at least one fluorophore remains unbleached throughout the sequencing process.
3Measurement precision
If traditional fluorophore-labeled nucleotides are used, then sequencing analysis can be performed, but expensive equipment such as high power lasers and electron multiplying CCD cameras is required
Solution Approach 1:
The patent merges multiple fluorophores into a single bead-labeled nucleotide structure, consolidating the signal source. This merging increases the total signal intensity per nucleotide, allowing detection with less sensitive and less expensive equipment while maintaining measurement precision.
Solution Approach 2:
The patent changes the signal intensity parameter by attaching multiple fluorophores to each nucleotide, thereby increasing the photons emitted per detection event. This parameter change enables the use of lower-cost, less sensitive detection equipment while maintaining adequate measurement precision.
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
The use of bead-labeled nucleotides provides a significantly brighter and more robust signal, reducing false positives and increasing the accuracy and efficiency of nucleic acid sequencing applications, while also reducing the need for expensive equipment and minimizing photodamage to polymerases.
Implementation Method 1
L is a bead comprising a fluorophore
Data Source
AI summary
The present invention provides labeled phospholink nucleotides that can be used in place of naturally occurring nucleotide triphosphates or other analogs in template directed nucleic acid synthesis reactions and other nucleic acid reactions and various analyses based thereon, including DNA sequencing, single base identification, hybridization assays, and others.