Dendritic Molecule Capture for Monoclonal Polynucleotide Amplification
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Solution Overview
Problem
Current methods for capturing and amplifying polynucleotides, such as cluster amplification, face challenges in achieving monoclonality and efficient signal-to-noise ratios, particularly in sequencing applications.
Innovation Solution
The use of dendritic molecules with a dendritic core, a seeding primer, and multiple dendrons, each with an inert elongated polymer, to capture and amplify polynucleotides within a flowcell. These molecules sterically exclude other molecules from the same well, ensuring monoclonal clusters are generated.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If cluster amplification is performed on a substrate surface with primers coupled to the surface, then polynucleotide amplification is achieved, but monoclonality of clusters cannot be ensured due to random seeding locations
Solution Approach 1:
The invention segments the amplification process by using individual dendritic molecules as isolated reaction chambers. Each dendritic molecule contains a single primer and undergoes amplification independently, eliminating the need for complex substrate patterning while ensuring monoclonality. The dendritic structure itself (core with multiple arms) provides the segmentation function that previously required external substrate features.
Solution Approach 2:
Dendritic molecules serve as intermediary carriers that bring the primer and reaction environment together in a single mobile unit. Instead of primers being fixed on a patterned substrate, the dendritic molecule acts as a self-contained intermediary that can be flowed through the system and selectively captured, simplifying the substrate requirements while maintaining amplification functionality.
2Measurement precision
If multiple primers are coupled to substrate surface at random locations, then amplification can occur, but signal-to-noise ratio deteriorates due to polyclonal clusters
Solution Approach 1:
By segmenting the system into individual dendritic molecules, each containing a single primer, the invention ensures that amplification occurs in isolated units. This segmentation prevents cross-contamination between different polynucleotide targets, thereby improving signal-to-noise ratio and maintaining cluster uniformity without requiring complex substrate patterns.
Solution Approach 2:
The invention changes the physical state and mobility parameters of the primer system. Instead of fixed primers on a substrate, primers are attached to mobile dendritic molecules that can be controlled through flow conditions. This parameter change allows selective capture of single dendritic molecules in each well, ensuring uniform monoclonal clusters and improved measurement precision.
3Reliability
If dendritic molecules with hydrodynamic diameter matching well diameter are used, then single molecule per well is achieved, but device complexity increases due to precise size control requirements
Solution Approach 1:
The invention utilizes parameter changes in the dendritic molecule design, specifically adjusting the hydrodynamic diameter to match the well diameter. This parameter matching ensures that only one dendritic molecule can fit in each well, guaranteeing single target capture. The dendritic structure's inherent geometry and the ability to control arm length and core size provide a straightforward method to achieve the required size control without excessive manufacturing complexity.
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 enables the generation of substantially monoclonal clusters, improving signal-to-noise ratios and enhancing the quality of sequencing data by ensuring that each well contains a single captured target polynucleotide.
Implementation Method 1
These molecules sterically exclude other molecules from the same well, ensuring monoclonal clusters are generated.
Implementation Method 2
The second functional group is to react with one of the first functional groups to form a covalent bond.
Implementation Method 3
a polynucleotide including a seeding adapter, wherein the seeding adapter is hybridized to the seeding primer
Data Source
AI summary
In some examples, a dendritic molecule may include a dendritic core; a seeding primer coupled to the dendritic core; and a plurality of dendrons, each of the dendrons comprising an inert, elongated polymer comprising a first end coupled to the dendritic core and a second end coupled to a first functional group, wherein the first functional group is to react with a second functional group to form a covalent bond. In other examples, a dendritic molecule may include a dendritic core; a single-stranded polynucleotide covalently bonded to the dendritic core; and a plurality of dendrons, each of the dendrons comprising an inert, elongated polymer comprising a first end coupled to the dendritic core and a second end.