Length-Sensitive Reporter for Digital Assay Template Differentiation
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Solution Overview
Problem
Current digital assays face challenges in distinguishing and measuring multiple nucleic acid templates amplified by the same primers, as they require multiple optical channels and dyes, which are costly and impractical beyond a certain number, limiting the ability to detect variations such as insertions and deletions effectively.
Innovation Solution
A digital assay method using a length-sensitive reporter that generates luminescence varying according to amplicon length, allowing for the differentiation and quantification of templates of different lengths amplified by the same pair of primers, enabling the detection of insertions, deletions, and other variants without the need for multiple dyes or optical channels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple optical channels and dyes are used to detect different templates, then the ability to distinguish multiple templates is improved, but the cost and device complexity increase
Solution Approach 1:
The patent changes the parameter being measured from fluorescence wavelength (requiring multiple optical channels) to fluorescence intensity/amplitude (measurable by a single detector). Different templates produce different fluorescence amplitudes based on their amplicon lengths, allowing distinction without multiple optical channels or dyes
Solution Approach 2:
A single optical channel and single dye are used to detect multiple different templates simultaneously. The system achieves multiplexing capability through amplitude discrimination rather than requiring separate detection channels for each template, making the detection system universal for detecting various template types
2Adaptability or versatility
If multiple dyes are used to label template-specific probes, then detection of multiple templates is improved, but the cost increases
Solution Approach 1:
The invention shifts from using multiple dyes with different emission wavelengths to using a single dye where the fluorescence amplitude varies with amplicon length. This parameter change eliminates the need to purchase and use multiple expensive dyes while maintaining the ability to distinguish multiple templates
Solution Approach 2:
Instead of using different dyes to create distinct signals for each template, the system uses a single dye that produces amplitude variations as copies of the template are amplified. The amplitude of fluorescence serves as a copy-dependent signal that distinguishes different template types without requiring multiple dye molecules
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 the efficient and cost-effective detection of template variations, including those associated with cancer, by using conserved primers and fluorescence amplitude differences, providing quick and quantitative results in small volumes, such as droplets, while minimizing competitive effects among targets.
Implementation Method 1
The method may utilize a length-sensitive reporter generating luminescence that varies according to amplicon length
Implementation Method 2
The sequence-specific probe can be labeled with a dye that provides a fluorescence signal indicating whether or not the template has been amplified in each partition
Data Source
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AI summary
Digital assay system, includes methods, apparatus, and compositions, for distinguishing and measuring different types of templates according to the different lengths of corresponding amplicons, which may be amplified by the same pair of primers. The system may include a length-sensitive reporter generating luminescence that varies according to amplicon length. The system may, for example, be utilized to identify, distinguish, and/or quantify wild-type and mutant/variant templates, processed and unprocessed template, a target template and a primer dimer, or the like.