Scanning Molecule Counting for Nucleic Acid Polymorphism Detection
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Solution Overview
Problem
Current methods for identifying nucleic acid polymorphisms, such as somatic mutations or single nucleotide polymorphisms, face challenges in sensitivity and specificity, particularly when dealing with low concentrations of target nucleic acid molecules, and often require complex statistical processing to detect fluorescence intensity fluctuations.
Innovation Solution
The method employs a scanning molecule counting technique using a confocal microscope or multiphoton microscope, where a nucleic acid probe specifically hybridizes with target nucleic acid molecules, and the hybrid is detected using a photodetection system that moves across the sample solution, allowing for individual detection and counting of fluorescent signals without the need for statistical processing of fluorescence intensity fluctuations, and includes the use of decoy nucleotides to suppress non-specific hybridization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional fluorescence intensity measurement methods are used to detect nucleic acid polymorphisms, then detection can be performed, but sensitivity and specificity deteriorate when dealing with low concentrations of target nucleic acid molecules
Solution Approach 1:
The patent segments the continuous fluorescence intensity signal into discrete individual molecular detection events. By using a confocal microscope to detect single molecules passing through a small observation volume, the method divides the detection problem into individual molecular events rather than measuring bulk fluorescence intensity, thereby achieving high sensitivity even at low concentrations.
Solution Approach 2:
The patent replaces the conventional statistical processing of fluorescence intensity fluctuations with a direct counting method. Instead of analyzing intensity variations that require complex statistical treatment, the system directly counts individual hybrid molecules based on their characteristic fluorescence signals, simplifying the measurement process while improving precision.
2Measurement precision
If conventional fluorescence detection methods are used, then nucleic acid polymorphisms can be identified, but complex statistical processing is required to detect fluorescence intensity fluctuations
Solution Approach 1:
The patent replaces complex statistical processing of fluorescence intensity with direct molecular counting. By detecting individual hybrid molecules through a confocal microscope's small observation volume, the system obtains discrete counting data that directly reflects the presence and type of polymorphisms without requiring statistical analysis of intensity fluctuations.
Solution Approach 2:
The patent extracts the essential information (presence and type of hybrid molecules) directly from individual molecular detection events. By focusing on counting individual hybrids rather than analyzing overall fluorescence intensity patterns, the method extracts polymorphism information in a straightforward manner that eliminates complex statistical processing.
3Measurement precision
If specific nucleic acid probes are used to identify polymorphisms, then detection specificity can be improved, but non-specific hybridization occurs reducing accuracy
Solution Approach 1:
The patent changes the detection parameter from bulk fluorescence intensity to individual molecular counting. By detecting and counting individual hybrid molecules that pass through the confocal observation volume, the method can distinguish specific from non-specific hybridization based on the characteristic fluorescence signals of correctly matched hybrids versus mismatches, thereby improving specificity despite the presence of non-specific binding.
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 enables accurate identification of nucleic acid polymorphisms even at low concentrations by effectively suppressing non-specific hybridization and allowing for precise counting of hybrid molecules, thereby improving sensitivity and specificity in nucleic acid detection.
Implementation Method 1
a nucleic acid probe that specifically hybridizes with a single-stranded nucleic acid molecule having a first type of base sequence in a polymorphic sequence
Implementation Method 2
labeled with a fluorescent substance and a quenching substance, respectively... resulting in the emission of fluorescent light
Implementation Method 3
optics capable of detecting light from a microregion in a solution such as the optics of a confocal microscope or multiphoton microscope
Data Source
AI summary
The present invention provides a method for identifying polymorphism of nucleic acids in a sample solution in which the concentration or number density of the observed nucleic acids is lower than that of conventional photometric analysis technologies. It includes: preparing a sample solution comprising a first nucleic acid probe, which specifically hybridizes with a single-stranded nucleic acid molecule including a first type of base sequence, and a target nucleic acid molecule; forming a hybrid of the nucleic acid molecules in the sample solution; calculating a number of molecules of the hybrid including the first nucleic acid probe in the sample solution by the scanning molecule counting method; and identifying polymorphism of the target nucleic acid molecule based on the calculating result. The sample solution includes an oligonucleotide having a base sequence complementary to a base sequence different from the first type of base sequence in the polymorphic sequence.