Nucleic Acid Enumeration via Single-Molecule Segmentation
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Solution Overview
Problem
Current methods for detecting rare nucleic acid events, such as mutations and genomic imbalances, are not sufficiently accurate or efficient, particularly in early disease diagnosis and prenatal screening, leading to potential misdiagnosis and delayed detection.
Innovation Solution
A method involving single molecule amplification and hybridization-based probing, where polynucleotides from a biological sample are arranged to form reaction sites, amplified, and then hybridized with labeled probes specific to target and reference nucleic acids, allowing for precise enumeration and relative quantification of target nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If massively parallel sequencing is used to detect rare nucleic acid events, then measurement precision is improved, but productivity is reduced and cost increases
Solution Approach 1:
The method segments the detection process into discrete single-molecule reactions occurring in individually addressed microreaction sites (wells), where each site contains a single template molecule. This segmentation allows parallel processing of many samples while maintaining simplicity in each individual reaction, resolving the contradiction between precision and productivity.
Solution Approach 2:
The invention introduces solid support particles (beads) as intermediaries that carry multiple reaction components and enable single-molecule amplification. These beads serve as mediators between the template DNA and the detection system, allowing efficient signal generation from single molecules without requiring complex sequencing infrastructure.
2Measurement precision
If massively parallel sequencing is used to detect rare nucleic acid events, then measurement precision is improved, but device complexity increases
Solution Approach 1:
The invention extracts and isolates the essential function of DNA detection to single-molecule amplification and hybridization events, removing the complex sequencing and data processing requirements of massively parallel sequencing. By taking out only the necessary detection function and simplifying the reaction system, the method achieves high precision with reduced complexity.
3Reliability
If conventional amplification methods are used, then detection sensitivity is improved, but measurement precision deteriorates due to inability to detect rare events
Solution Approach 1:
The method performs preliminary single-molecule amplification in isolated microreaction sites before detection, ensuring that each site contains a known number of template molecules (ideally one). This preliminary action establishes a precise baseline for quantification that enables both high sensitivity for rare events and accurate measurement of relative amounts.
Solution Approach 2:
The invention creates locally optimized reaction conditions in each microreaction site, with individual amplification and detection parameters tailored to detect single molecules. This local quality control in each well enables simultaneous achievement of high sensitivity for rare event detection and precise quantification across the population of sites.
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 provides a faster, more sensitive, and cost-effective method for detecting rare nucleic acid events, achieving high sensitivity with low false positives and enabling early diagnosis of diseases and conditions, including fetal aneuploidies, with potential for use as early as 4-6 weeks gestation.
Implementation Method 1
amplifying the polynucleotides in the plurality of reaction sites
Implementation Method 2
determining by nucleic acid hybridization (i) a first number of first reaction sites containing a target nucleic acid sequence, or a portion thereof
Data Source
AI summary
Disclosed are methods and systems for enumeration of nucleic acids, including for the detection of rare events in a biological sample. In certain embodiments, the method may comprise arranging polynucleotides obtained from a biological sample to form a plurality of reaction sites, wherein each reaction site contains on average one polynucleotide; amplifying the polynucleotides in the plurality of reaction sites; determining by nucleic acid hybridization (i) a first number of first reaction sites containing a target nucleic acid sequence, or a portion thereof, and (ii) a second number of second reaction sites containing a reference nucleic acid sequence, or a portion thereof; comparing the first number of the first reaction sites to the second number of the second reaction sites to determine the relative amount of the target nucleic acid in the biological sample.