Digital PCR Emulsion Composition for Uniform Sample Partitioning
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Solution Overview
Problem
Existing digital PCR methods face challenges in stabilizing DNA polymerase and ensuring reliable, real-time PCR reactions due to the use of surfactants like Tween 20, which can reduce amplification efficiency or be toxic, and require improved methods for uniform partitioning of PCR samples.
Innovation Solution
A digital PCR emulsion composition is developed using a surfactant with an HLB value of 4 to 8, primarily non-ionic, and mineral oil, with a specific volume ratio, to form emulsion particles of 5 to 30 µm, which are uniformly partitioned in a reaction film-lattice using centrifugation, allowing real-time PCR monitoring.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If surfactants like Tween 20 or Nonidet P-40 are used to stabilize DNA polymerase, then polymerase stability is improved, but amplification efficiency decreases or non-specific products are generated
Solution Approach 1:
The patent removes harmful surfactants (Tween 20, Nonidet P-40) from the PCR system entirely and replaces them with a stabilizing agent that does not interfere with amplification. This extraction of the harmful component while maintaining the stabilizing function resolves the contradiction between polymerase stability and amplification reliability.
Solution Approach 2:
The patent introduces a novel stabilizing agent that acts as an intermediary between the polymerase and the environment, providing stability without the harmful side effects of traditional surfactants. This intermediary substance maintains polymerase stability while allowing specific and efficient amplification to proceed.
2Quantity of substance
If conventional digital PCR emulsion methods are used, then sample partitioning is achieved, but uniform distribution of emulsion particles in reaction wells is difficult to ensure
Solution Approach 1:
The patent optimizes physical parameters including emulsion particle size (5-30 µm), oil-to-aqueous phase ratio (95:5 to 99:1), and surfactant concentration to achieve uniform distribution. By carefully controlling these parameters, the emulsion particles distribute evenly across reaction wells without requiring complex partitioning protocols.
Solution Approach 2:
The patent performs preliminary optimization of emulsion formulation and physical properties before the actual PCR reaction. By pre-adjusting particle size, viscosity, and surface properties of the emulsion, uniform distribution is achieved automatically during sample loading, eliminating the need for complex post-formulation partitioning steps.
3Reliability
If real-time PCR monitoring is implemented, then reaction progress can be tracked, but system complexity increases
Solution Approach 1:
The patent replaces complex mechanical partitioning and monitoring systems with a simplified fluorescent detection approach. By using fluorescent dyes that naturally report PCR amplification and leveraging standard real-time PCR instrumentation, the system achieves reliable monitoring without adding mechanical 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
The method enables easy and reliable preparation of PCR reagents in emulsion form, improving the reliability and efficiency of digital PCR results by ensuring uniform partitioning and stability of the emulsion.
Implementation Method 1
oil A containing a surfactant
Implementation Method 2
an emulsion formed by mixing a PCR sample mixture containing one or two or more amplification target DNAs and a pair of forward and reverse primers for amplifying each amplification target DNA and oil A containing a surfactant with each other
Implementation Method 3
rotating the plate to centrifuge and partition the sample
Data Source
Figure 1a~2d
Figure 3~4d
Figure 4e~4j
AI summary
Provided is an emulsion composition for digital PCR and uniform partitioning method of PCR samples for a digital polymerase chain reaction (digital PCR). With a digital polymerase chain reaction (digital PCR) emulsion composition for uniformly partitioning a PCR sample, a preparation method thereof, and a uniform partitioning method of a PCR sample according to the present invention, a PCR reagent to be amplified may be easily and rapidly prepared in an emulsion form, and reliability of digital PCR results may be significantly improved by allowing the formed emulsion to be uniformly partitioned using a cylindrical plate to which a reaction film is attached.