Digital PCR Well Array for Nucleic Acid Quantification
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Solution Overview
Problem
Conventional digital PCR methods cannot distinguish between single-stranded and double-stranded nucleic acids, leading to inaccurate quantification of these molecules in biological samples.
Innovation Solution
A detection method involving a well array device where target nucleic acids are introduced into individual wells, sealed to prevent mixing, and amplified using specific binding substances that emit distinct luminescence signals for single-stranded and double-stranded nucleic acids, allowing for accurate counting and quantification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional digital PCR methods are used to detect target nucleic acids, then detection sensitivity is improved, but the ability to distinguish between single-stranded and double-stranded nucleic acids is lost
Solution Approach 1:
The detection method segments the detection process into two distinct phases: first, sealing wells while nucleic acids are in double-stranded form to preserve structural information, then denaturing to single-stranded form for signal amplification. This segmentation allows both structural information preservation and detection sensitivity to be achieved.
Solution Approach 2:
The method performs preliminary sealing of wells before denaturation occurs. By sealing the wells while nucleic acids are still in their double-stranded state, the invention preserves structural information beforehand, then proceeds with denaturation for sensitive detection without losing the structural context.
2Measurement precision
If nucleic acids are denatured before well sealing to improve detection, then signal amplification is enhanced, but the original nucleic acid structure information is lost
Solution Approach 1:
The invention performs the sealing action preliminarily before denaturation occurs. This ensures that the original double-stranded structure information is captured and preserved in the well assignment, while the subsequent denaturation step still provides enhanced signal amplification for detection.
Solution Approach 2:
Instead of the conventional approach of denaturing first then sealing, the invention inverts the sequence by sealing first while nucleic acids are double-stranded, then denaturing. This inversion preserves structural information while still achieving sensitive detection.
3Ease of operation
If single-stranded and double-stranded nucleic acids are detected together using conventional methods, then detection process is simplified, but quantification accuracy of individual nucleic acid types is reduced
Solution Approach 1:
The method applies local quality differentiation by using two distinct detection channels: one for detecting double-stranded nucleic acids (before denaturation) and another for detecting single-stranded nucleic acids (after denaturation). This allows individual quantification of each nucleic acid type while maintaining a relatively simple overall process.
Solution Approach 2:
The invention uses different fluorescence signals (analogous to color changes) to distinguish between double-stranded and single-stranded nucleic acid detection channels. This enables simultaneous detection and individual quantification of different nucleic acid types through signal differentiation rather than complex procedural changes.
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
Enables precise individual quantification of single-stranded and double-stranded nucleic acids, improving the accuracy of molecular analysis in biological samples.
Implementation Method 1
amplifying, in the well, a signal derived from the target nucleic acid; and detecting the signal emitted from the well, wherein the target nucleic acid includes a first nucleic acid, a second nucleic acid that is complementary to the first nucleic acid, and a double-stranded nucleic acid resulting from complimentary binding of the first nucleic acid and the second nucleic acid, in the amplifying of the signal, a first signal is emitted as a result of a first specific binding substance being bound to the first nucleic acid, and a second signal is emitted as a result of a second specific binding substance being bound to the second nucleic acid
Implementation Method 2
a double-stranded nucleic acid resulting from complimentary binding of the first nucleic acid and the second nucleic acid
Data Source
Figure 1~2
Figure 3~4
Figure 5
AI summary
This detection method includes: introducing a target nucleic acid into a well of a device including a plurality of wells in a manner to cause at most one molecule to be included per well; sealing the well to prevent the target nucleic acid from moving between the plurality of wells; amplifying, in the well, a signal derived from the target nucleic acid; and detecting the signal emitted from the well. The target nucleic acid includes a first nucleic acid, a second nucleic acid that is complementary to the first nucleic acid, and a double-stranded nucleic acid resulting from complementary binding of the first nucleic acid and the second nucleic acid. In the sealing, the double-stranded nucleic acid has a double-strand when sealed within the well. In the amplifying of the signal, a first signal is emitted as a result of a first specific binding substance being bound to the first nucleic acid, and a second signal is emitted as a result of a second specific binding substance being bound to the second nucleic acid. The first signal and the second signal are different.