Nucleic Acid Analysis via Droplet Segmentation and Reversible Fluorescence
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Solution Overview
Problem
Current gene analysis methods using nucleic acid amplification face challenges with specificity, quantitativity, and sensitivity due to amplification bias, particularly in detecting small amounts of template nucleic acid, necessitating a method with higher accuracy.
Innovation Solution
A method involving fractionation of a sample into multiple nucleic acid fractions, followed by amplification and detection of target sequences using a primer set and signal-generating substances that produce reversible signal generation or quenching, allowing for accurate discrimination of amplified fractions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If nucleic acid amplification is performed using conventional methods, then the target sequence can be detected, but amplification bias occurs causing reduced specificity and quantitativity
Solution Approach 1:
The sample is divided into multiple discrete droplets or microwell partitions, each containing template nucleic acid and amplification reagents. This segmentation isolates amplification reactions in individual compartments, preventing cross-contamination and reducing amplification bias from non-specific binding, thereby improving both detection accuracy and amplification specificity
Solution Approach 2:
Different regions or droplets are assigned different probe sequences or amplification conditions optimized for specific target sequences. This allows local optimization of amplification parameters for different genomic regions, reducing overall amplification bias and improving quantitative accuracy across diverse target sequences
2Measurement precision
If conventional amplification methods are used to detect small amounts of template nucleic acid, then detection is possible, but sensitivity and accuracy are insufficient
Solution Approach 1:
By partitioning the sample into many individual droplets, the method enables digital counting of positive amplification events. Even trace amounts of template nucleic acid produce detectable signal in a subset of droplets, allowing sensitive detection and quantitative analysis through statistical analysis of positive droplet frequency
Solution Approach 2:
Fluorogenic probes emit detectable fluorescence signals upon hybridization to amplified target sequences. This optical signal change provides high-contrast detection that significantly enhances sensitivity for detecting small amounts of template nucleic acid, allowing accurate measurement even at low concentrations
3Ease of manufacture
If amplification reagents are used without fractionation, then the process is simpler, but amplification bias reduces quantitativity
Solution Approach 1:
The automated droplet generation system partitions template and reagents into discrete units in a single integrated process. This segmentation enables accurate quantification by counting positive droplets while maintaining process simplicity through automation, resolving the contradiction between ease of manufacture and quantitative accuracy
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 accuracy of nucleic acid analysis by enriching template nucleic acid in specific fractions and utilizing fluorogenic probes for sensitive and specific detection, even at low concentrations.
Implementation Method 1
a signal generating substance that generates or quenches a signal in response to the amplification, and the signal generating substance generates a signal in a state where it is bound sequence-dependently and quenches a signal in a state where it is not bound
Data Source
AI summary
The present invention provides a method for analyzing a template nucleic acid, a method for analyzing a target substance, an analysis kit for a template nucleic acid or a target substance, and an analyzer for a template nucleic acid or a target substance, which are excellent in accuracy. The method for analyzing a template nucleic acid of the present invention includes the steps of: fractionating a sample containing a template nucleic acid into a plurality of template nucleic acid fractions; amplifying a target sequence and its complementary sequence in the template nucleic acid with respect to each of the plurality of template nucleic acid fractions in the presence of a nucleic acid amplification reagent; detecting generation or quenching of a signal that shows an amplification of the target sequence or the complementary sequence with respect to each of the plurality of template nucleic acid fractions after the amplification step; and discriminating a template nucleic acid fraction in which the generation or quenching of a signal that shows the amplification has been detected among the plurality of template nucleic acid fractions as an amplified fraction in which the target sequence or the complementary sequence has been amplified, wherein the nucleic acid amplification reagent contains a primer set that amplifies the target sequence and the complementary sequence and a signal generating substance that generates or quenches a signal in response to the amplification, and the signal generating substance generates a signal in a state where it is bound sequence-dependently and quenches a signal in a state where it is not bound or quenches a signal in a state where it is bound sequence-dependently and generates a signal in a state where it is not bound, and generation and quenching of a signal are reversible.