Masked Binding Moiety Amplicons for Emulsion PCR Efficiency
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Solution Overview
Problem
Current methods for clonally amplifying target polynucleotides using amplification primers attached to surfaces are inefficient compared to amplification reactions performed entirely in solution, necessitating compositions and methods that enable efficient clonal amplification without surface-bound primers.
Innovation Solution
The use of masked or caged binding moieties incorporated into amplicons during amplification reactions, allowing for clonal amplification entirely in solution and subsequent attachment to surfaces via unmasking and binding to their partners, such as caged biotin or fluorescein, using techniques like PCR in inverse emulsions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If amplification primers are attached to surfaces to facilitate clonal amplification, then parallel amplification of large numbers of target polynucleotides is enabled, but amplification efficiency decreases compared to solution-based reactions
Solution Approach 1:
The system segments the amplification process into two distinct phases: (1) clonal amplification in solution using free primers, and (2) surface attachment after amplification. This segmentation allows each phase to optimize for its specific function - solution-based amplification maximizes efficiency while surface attachment enables parallel processing and detection
Solution Approach 2:
The invention performs clonal amplification as a preliminary action before surface attachment. By completing the amplification reaction in solution first, the system ensures high amplification efficiency is achieved before the amplicons are transferred to the surface for parallel analysis
2Productivity
If amplification is performed entirely in solution without surface-bound primers, then amplification efficiency increases, but the ability to analyze large numbers of amplicons in parallel is reduced
Solution Approach 1:
The workflow separates amplification from analysis by using solution-based amplification followed by surface attachment, allowing the system to achieve both high amplification efficiency and the capacity to analyze large numbers of amplicons in parallel through the segmented approach
Solution Approach 2:
The invention uses an intermediary attachment step where amplified amplicons are bound to surfaces via biotin-streptavidin or similar interactions. This intermediary step bridges the gap between solution-based amplification and surface-based parallel analysis, enabling both high efficiency and high throughput
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 efficiency and yield of amplification products by allowing clonal amplification in solution and subsequent surface attachment, enabling the analysis of millions of amplicons in parallel through detection, sequencing, and other methods.
Implementation Method 1
clonal amplification reactions can include one or more amplification primers
Implementation Method 2
a masked binding moiety can be a caged biotin and the binding partner can be an anti-biotin antibody or an avidin (e.g., avidin, streptavidin, NEUTRAVIDIN®)
Implementation Method 3
The caged biotin or fluorescein can be uncaged or activated by various methods such as irradiation with light of an appropriate wavelength
Implementation Method 4
isolating a target polynucleotide in various types of reaction vessels, including but not limited to, a well of multiwell plate or a hydrophilic compartment of an inverse emulsion
Data Source
AI summary
Compositions and methods of use are disclosed for clonally amplifying target polynucleotide sequences in solution and attaching the amplicons to a surface by activation of a masked binding moiety. In an embodiment, the amplicons comprise the masked binding moiety and the surface comprises a binding partner of the binding moiety. Upon activation of the binding moiety, the amplicons bind to the binding partner on the surface. In a non-limiting example, the masked binding moiety is caged biotin or caged fluorescein, while the corresponding binding partner is avidin or an anti-fluorescein antibody.