Intermittent Detection for Nucleic Acid Sequencing
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Solution Overview
Problem
Current analytical techniques face challenges in maintaining reaction performance due to photo-induced damage from light sources, especially in low reactant volumes used in microfluidic or nanofluidic systems, and difficulties in sequencing noncontiguous portions of long nucleic acid templates, particularly those with repetitive sequences.
Innovation Solution
Implementing intermittent detection methods for analytical reactions, which involve alternating between detection and non-detection periods to minimize photo-induced damage and enhance reaction performance, allowing for the collection of reliable data from previously inaccessible regions of the reaction, and using nucleotides with detectable properties to generate noncontiguous sequence reads from single nucleic acid templates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescent labeling groups are used with excitation radiation source for detection, then signal detectability is improved, but photo-induced damage to reactants increases
Solution Approach 1:
The patent applies periodic action by using pulsed excitation radiation instead of continuous illumination. The excitation source is activated in periodic pulses during the sequencing reaction, allowing detection of fluorescent signals at specific intervals while providing dark periods that minimize cumulative photo-induced damage to the DNA template and other reactants. This periodic illumination strategy maintains sufficient signal detectability while reducing harmful effects.
2Productivity
If smaller reactant volumes are used in microfluidic or nanofluidic systems, then high throughput application efficiency is improved, but photo-induced damage impact increases
Solution Approach 1:
In microfluidic and nanofluidic systems where reactant volumes are minimized for high throughput applications, the patent implements periodic excitation to reduce the cumulative photo-induced damage impact. The pulsed illumination approach ensures that detection occurs at discrete time points while minimizing the total exposure time of the small reactant volumes to excitation radiation, thereby preserving reactant integrity in high-throughput sequencing applications.
Solution Approach 2:
The patent employs multiple identical sequencing reactions in parallel within microfluidic or nanofluidic arrays. Instead of attempting to sequence a single long template continuously, multiple copies of the same or different templates are sequenced simultaneously in separate reaction chambers. This copying approach increases throughput while each individual reaction experiences reduced photo-induced damage due to periodic excitation.
3Loss of information
If continuous detection is performed during sequencing reactions, then complete sequence data is obtained, but reaction performance degrades over time
Solution Approach 1:
The patent implements periodic detection during sequencing reactions by activating the excitation radiation source in pulses rather than continuously. Detection of fluorescently labeled nucleotides occurs at specific time points corresponding to pulsed excitation, providing sufficient sequence data while allowing the reaction to proceed during dark periods without cumulative photo-induced damage that would degrade reaction performance over time.
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 extends the processivity and fidelity of enzyme reactions, reduces photo-induced damage, and enables the collection of sequence data from discontiguous regions of nucleic acid templates, improving the accuracy and completeness of sequence data, especially for long and repetitive templates.
Implementation Method 1
intermittently monitoring the pattern of variations in current passing through the nanopore
Implementation Method 2
applying a voltage across the membrane; intermittently monitoring the pattern of variations in current passing through the nanopore as a nucleic acid template having a single-stranded linker connecting sense and antisense strands of a double-stranded polynucleotide is drawn through the nanopore
Data Source
Figure 1
Figure 2A~2B
Figure 3A~3C
AI summary
Methods, devices, and systems for performing intermittent detection during analytical reactions are provided. Such methods facilitate collection of reaction data from disparate reaction times. Further, such methods are useful for reducing photo-induced damage of one or more reactants in an illuminated analytical reaction at a given reaction time. In preferred embodiments, the reaction mixture is subjected to at least one illuminated and non-illuminated period and allowed to proceed such that the time in which the reaction mixture is illuminated is less than a photo-induced damage threshold period.