Electric-Field Concentration for Single-Molecule Sequencing
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Solution Overview
Problem
Existing single-molecule sequencing technologies struggle to accurately detect minority sequence variants due to their reliance on consensus determination methods, which treat sequence differences as noise, leading to inaccurate results when samples contain mixed DNA sequences.
Innovation Solution
A process involving a support with electrically conductive sample spots and an applied electrical field to concentrate single molecules, combined with optical analysis using multipoint illumination and detection, allows for the individual sequencing of nucleic acid molecules by detecting time-dependent fluorescence changes during enzymatic reactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If consensus determination methods are used for sequencing, then sequencing speed and throughput are improved, but detection accuracy of minority sequence variants deteriorates
Solution Approach 1:
The invention segments the sequencing process by immobilizing individual nucleic acid molecules at distinct positions on a solid support, allowing each molecule to be sequenced independently rather than through consensus determination of bulk populations. This enables detection of minority variants while maintaining high throughput through parallel processing of multiple individual molecules.
Solution Approach 2:
The invention creates multiple copies of the sequencing system by immobilizing many individual nucleic acid molecules simultaneously on a solid support array. Each molecule serves as an independent sequencing reaction unit, enabling parallel processing that maintains both speed and accuracy for detecting rare variants in the population.
2Reliability
If single molecule analysis is performed without concentration, then molecule individuality is preserved, but detection sensitivity deteriorates due to low signal intensity
Solution Approach 1:
The invention applies local quality enhancement by concentrating molecules into discrete focal spots on the solid support using electric fields. Each focal spot creates a localized region of high molecular concentration that generates sufficient signal intensity for sensitive detection while maintaining the individuality of each molecule through spatial separation from other molecules.
Solution Approach 2:
The invention introduces electric fields as an intermediary mechanism to concentrate individual molecules into focal spots on the solid support. This intermediary field enables the transition from dilute single-molecule solutions to concentrated localized configurations, enhancing detection sensitivity without compromising molecule individuality.
3Measurement precision
If electric field concentration is applied, then detection sensitivity is improved, but device complexity increases due to additional electrical components
Solution Approach 1:
The solid support structure serves multiple functions: it provides the physical platform for molecule immobilization, creates the focal spots for concentration, and acts as an electrode for applying electric fields. This multi-functionality reduces overall device complexity by combining several necessary components into a single integrated element.
Solution Approach 2:
The invention merges the support structure and electrical field generation into a single integrated system where the solid support itself functions as the electrode. This consolidation eliminates the need for separate electrical components and simplifies the overall device architecture while maintaining the ability to concentrate molecules for sensitive 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 method significantly enhances the concentration of single molecules, enabling accurate sequencing of multiple nucleic acid molecules in parallel, with improved detection sensitivity and accuracy, particularly for minority sequence variants.
Implementation Method 1
applying an electrical field across said reaction space, whereby a concentration of said at least one single molecule to be analysed at said sample spot is effected
Implementation Method 2
irradiating light into the support and generating an evanescent excitation field by way of internal reflection on the support surface
Implementation Method 3
generating an evanescent excitation field by way of internal reflection on the support surface in the region of the immobilized nucleic acid molecules
Implementation Method 4
determining simultaneously the base sequence of a plurality of nucleic acid molecules on the basis of the time-dependent change, caused when nucleotide building blocks are cleaved off, in the fluorescence of said nucleic acid molecules
Data Source
AI summary
The invention relates to a process and a device for analyzing single molecules, particularly to the parallel analysis of a plurality of single molecules. It is suitable for detecting interactions, e.g. binding between single molecules and/or reactions, e.g. elongation or degradation of single molecules. Particularly, the process of the invention relates to the sequencing of single nucleic acid molecules. The single molecule to be analyzed is present in free form, i.e. dissolved or suspended in a liquid medium, within a reaction space formed around the sample spot. According to the present invention, an electrical field is applied across the reaction space, whereby a concentration of single molecules, at the sample spots is effected.