Nucleic Acid Polymer Particles for High-Density Amplicon Loading
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for generating signal in genomics and biomedical research, such as hybrid selection, in-gel PCR, and emulsion PCR, face limitations including low fragment density, labor-intensive emulsion reactions, and unsuitable particulate supports for exacting applications like large-scale DNA sequencing.
Innovation Solution
The development of nucleic acid polymer particles with non-nucleosidic polymer networks that have high primer concentrations and controlled size distributions, allowing for enhanced analyte binding and amplification reactions without the need for emulsion reactions, providing higher fragment concentrations and uniformity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If traditional bead supports are used for amplicon attachment, then spatial concentration of amplicons is achieved, but fragment density and signal-to-noise ratio are insufficient
Solution Approach 1:
The patent employs porous polymer particles with controlled pore sizes (5-50 nm) that allow high-density attachment of amplicons throughout the particle volume. The porous structure provides extensive internal surface area while maintaining small external dimensions (1-30 μm), enabling high fragment density without increasing particle size, thus improving signal-to-noise ratio.
Solution Approach 2:
The invention transitions from two-dimensional surface attachment on traditional beads to three-dimensional volume-based attachment in porous particles. Amplicons can be attached throughout the entire particle volume including internal pores, effectively utilizing three-dimensional space to increase fragment density while maintaining small particle dimensions for high signal-to-noise ratio.
2Reliability
If emulsion PCR is used to obtain clonal populations, then clonal amplification is achieved, but the process becomes labor intensive and costly
Solution Approach 1:
The patent extracts the essential function of emulsion PCR (clonal amplification) and implements it through a simplified solid-phase bridge amplification process on porous particles. By removing the complex emulsion formation and manipulation steps while retaining the clonal amplification capability through controlled primer attachment and amplification conditions, the process becomes less labor-intensive and more cost-effective.
Solution Approach 2:
The porous polymer particles serve as intermediaries that facilitate clonal amplification without requiring emulsion conditions. The particles provide a solid-phase support with controlled primer distribution that enables clonal amplification through bridge PCR, acting as a mediator between the template DNA and the amplification reaction, thereby eliminating the need for complex emulsion handling.
3Ease of operation
If gel particulates with varying size distributions are used, then analytical reagent access is improved, but suitability for exacting applications like DNA sequencing is reduced
Solution Approach 1:
The patent employs controlled polymerization conditions including specific monomer concentrations, crosslinker ratios, and polymerization temperatures to produce porous particles with narrow size distributions (coefficient of variation <10%). This precise control of size parameters maintains manufacturing precision while the porous structure ensures adequate reagent access throughout the particle volume.
Solution Approach 2:
The invention creates particles with non-uniform internal structure (porous) combined with uniform external dimensions. The local porosity provides reagent access throughout the particle volume while the uniform external size ensures manufacturing precision and consistency for exacting applications like DNA sequencing. Different regions of the particle have different properties: uniform size externally, porous internally.
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 enhances the sensitivity and efficiency of nucleic acid analysis by creating monodisperse populations of gel-based particulate supports, improving signal-to-noise ratios and reducing costs through easier handling and reagent access.
Implementation Method 1
each such particle comprising a non-nucleosidic polymer network having attached throughout its volume a primer at a concentration of at least 1 × 10^5 primers per particle
Data Source
Figure 1A~1B
Figure 2A
Figure 2B
AI summary
The invention provides particle compositions having applications in nucleic acid analysis. Nucleic acid polymer particles of the invention allow polynucleotides to be attached throughout their volumes for higher loading capacities than those achievable solely with surface attachment. In one aspect, nucleic acid polymer particles of the invention comprise polyacrylamide particles with uniform size distributions having low coefficients of variations, which result in reduced particle-to-particle variation in analytical assays. Such particle compositions are used in various amplification reactions to make amplicon libraries from nucleic acid fragment libraries.