Orthogonal Nucleotide Deblocking for Lower-Cost DNA Sequencing
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Solution Overview
Problem
Current nucleic acid sequencing technologies are costly, complex, and require expensive hardware and reagents, limiting their use in clinical and research contexts where cost-effectiveness and convenience are crucial.
Innovation Solution
A method involving an array of sites with mixed nucleic acid templates, using primers with reversible blocking moieties and selective deblocking treatments to distinguish and sequence nucleotide analogs, allowing for orthogonal detection and super-resolution imaging of nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If sequencing-by-synthesis platforms use specially engineered biochemical components and expensive hardware, then detection precision is improved, but cost increases significantly
Solution Approach 1:
The patent uses inexpensive, disposable microbead particles as reaction substrates instead of expensive reusable hardware components. Each microbead carries a template strand and serves as a single-use reaction vessel, eliminating the need for costly, complex hardware systems while maintaining detection capability through optical imaging of the disposable particles.
Solution Approach 2:
The patent replaces complex mechanical and optical hardware systems (lasers, detection optics, fluid delivery systems) with a simpler chemical-biological system based on fluorescently labeled nucleotides and digital imaging. The detection is achieved through chemical fluorescence signals rather than complex mechanical scanning systems.
2Power
If DNA templates are amplified before sequencing-by-synthesis, then signal strength is improved, but device complexity and cost increase
Solution Approach 1:
The patent performs template amplification in advance by attaching multiple copies of the same template strand to each microbead particle before the sequencing reaction. This preliminary amplification step ensures sufficient signal strength during sequencing without requiring complex real-time amplification hardware during the actual sequencing-by-synthesis process.
3Manufacturing precision
If orthogonal blocking moieties are used for selective deblocking, then manufacturing precision is improved, but loss of substance increases due to selective removal steps
Solution Approach 1:
The patent uses nucleotide analogs with orthogonal blocking moieties that can be selectively removed by changing specific reaction parameters (different chemical or enzymatic treatments). Each blocking moiety responds to a unique deblocking condition, allowing precise selective removal without affecting other blocked positions, thereby maintaining manufacturing precision while minimizing substance loss through targeted rather than blanket removal.
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
Enhances information density and reduces costs by enabling efficient sequencing with fewer labels and hardware, facilitating use in cost-sensitive applications.
Implementation Method 1
a first primer is bound to the first nucleic acid template, and wherein a second primer is bound to the second nucleic acid template
Implementation Method 2
extending the first primer by addition of a first nucleotide analog that is attached to a reversible blocking moiety
Implementation Method 3
different nucleotide analogs having different reversible blocking moieties, respectively, thereby producing different primer extension products at each site
Implementation Method 4
detecting the different primer extension products to distinguish the different nucleotide analogs at each site
Data Source
AI summary
A method including steps of (a) providing an array of sites, wherein each site comprises a mixture of different nucleic acid templates; (b) extending primers hybridized to the different nucleic acid templates at each of the sites with different nucleotide analogs having different reversible blocking moieties, respectively, thereby producing different primer extension products at each site; (c) detecting the different primer extension products to distinguish the different nucleotide analogs at each site; and (d) removing the different reversible blocking moieties from the primer extension products at each of the sites using a first treatment that is selective for a first of the different reversible blocking moieties and a second treatment that is selective for a second of the different reversible blocking moieties.