Nucleic Acid Analysis via Single-Molecule Fluorescence Detection
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Solution Overview
Problem
Current nucleic acid analysis methods, such as microarrays and next-generation sequencing, face limitations in global identification performance and dynamic range, particularly for analyzing untranslated RNAs and microRNAs, requiring costly and time-consuming processes with potential biases due to amplification inefficiencies.
Innovation Solution
A method involving the spatial fixation of nucleic acid molecules on a support substrate, hybridization with labeled molecules, and fluorescence imaging to analyze types and expression levels at single-molecule sensitivity and resolution without amplification reactions, enabling high global identification and quantitative performance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If microarray method is used for global identification, then the number of genes that can be collectively analyzed increases, but the quantitative performance deteriorates with only 2- to 3.5-digit dynamic range
Solution Approach 1:
The invention segments the detection process by fixing individual nucleic acid molecules at spatially separated positions on a support substrate, allowing each molecule to be detected independently. This segmentation enables single-molecule resolution while maintaining the ability to analyze thousands of molecules simultaneously, thus achieving both high global identification performance and high quantitative performance with 4-or-more-digit dynamic range without requiring amplification reactions.
2Measurement precision
If quantitative PCR method is used for high quantitative performance, then the dynamic range improves to 6- to 7-digit, but the number of genes that can be collectively analyzed decreases to about 400
Solution Approach 1:
The invention merges the advantages of single-molecule detection (high quantitative performance) with parallel analysis of multiple targets (high global identification performance). By fixing multiple individual nucleic acid molecules at spatially separated positions on a single support substrate and detecting them simultaneously using fluorescence imaging, the method achieves both 4-or-more-digit dynamic range and the ability to analyze several thousand types of nucleic acids in a single experiment, eliminating the need for two-step experiments.
3Measurement precision
If next-generation sequencing is used for comprehensive expression analysis, then the dynamic range increases to 8 or more digits, but the running costs and analysis time increase significantly
Solution Approach 1:
The invention extracts and eliminates the PCR amplification step from the nucleic acid analysis process. By directly fixing and detecting individual nucleic acid molecules without amplification, the method achieves high dynamic range (4-or-more-digit) while dramatically reducing analysis time (from several tens of hours to much shorter duration) and running costs (by eliminating expensive reagents required for amplification reactions), making it highly effective for analyzing untranslated RNAs and microRNAs.
4Measurement precision
If PCR amplification is performed for nucleic acid analysis, then the sensitivity improves, but amplification efficiency varies due to base sequences causing biased nucleic acid population
Solution Approach 1:
The invention inverts the conventional approach by detecting native nucleic acid molecules directly without amplification. Instead of amplifying nucleic acids and then detecting them (which introduces bias), the method fixes and detects individual nucleic acid molecules in their native state, ensuring that the detected population accurately reflects the original sample composition without amplification bias, while maintaining single-molecule sensitivity through direct fluorescence detection.
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 allows for rapid and convenient analysis of nucleic acids and biomolecules with high sensitivity and resolution, overcoming the limitations of existing methods by eliminating the need for amplification reactions and improving the analysis of low-abundance nucleic acids.
Implementation Method 1
fixing the nucleic acid molecules of samples at the positions spatially separated from each other one at a time
Implementation Method 2
hybridizing the nucleic acids, which have known base sequences and have been labeled with a fluorescence substance, with a group of the nucleic acid molecules of the samples
Implementation Method 3
labeled with a fluorescence substance... to capture fluorescent images
Data Source
AI summary
A convenient method for nucleic acid analysis is provided, which enables 1000 or more types of nucleic acid to be analyzed collectively with high comprehensiveness and with a dynamic range of at least four digits. In particular, provided is a very effective analytical method especially for untranslated RNAs and microRNAs, of which the types of target nucleic acids is 10000 or lower. Nucleic acids can be analyzed conveniently and rapidly with high comprehensiveness and quantitative performance at single-molecule sensitivity and resolution by following the steps of: preparing a group of target nucleic acid fragments one molecule at a time and hybridizing the nucleic acid molecules, which have known base sequences and have been labeled with the fluorescence substances, with the group of the target nucleic acid fragments to detect the fluorescence substances labeling the hybridized nucleic acid molecules.