Ethylene Carbonate Denaturation for Isothermal Nucleic Acid Sequencing
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Solution Overview
Problem
Next-generation sequencing methods require efficient preparation of nucleic acid molecules for sequencing, which is often time-consuming due to the need for thermal denaturation and separate steps for hybridization of sequencing primers, leading to longer workflow periods.
Innovation Solution
The use of ethylene carbonate to denature double-stranded nucleic acid molecules attached to a surface, allowing for simultaneous denaturation and hybridization with sequencing primers, thereby streamlining the workflow and enabling isothermal processing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If thermal denaturation and separate hybridization steps are used, then sequencing preparation can be performed, but workflow duration increases
Solution Approach 1:
The patent combines thermal denaturation and primer hybridization into a single simultaneous step. The sequencing primer is designed to hybridize to the template during the denaturation process, eliminating the need for separate steps and reducing workflow duration while maintaining preparation completeness
Solution Approach 2:
The sequencing primer is pre-designed with specific characteristics (such as including a binding domain and extension domain) that enable it to perform both denaturation and hybridization functions in advance, allowing the workflow to proceed more efficiently without compromising reliability
2Ease of operation
If multiple separate steps are used for denaturation and hybridization, then process control is easier, but productivity decreases
Solution Approach 1:
By merging denaturation and hybridization into one step, the patent increases productivity and workflow efficiency. The primer design ensures that both functions are achieved simultaneously, making the process more efficient without significantly complicating control mechanisms
3Reliability
If conventional denaturation methods are used, then nucleic acid molecules can be prepared, but workflow complexity increases
Solution Approach 1:
The patent reduces workflow complexity by combining multiple steps into one. The sequencing primer is designed to perform both denaturation and hybridization functions simultaneously, simplifying the overall workflow while maintaining reliable nucleic acid preparation
Solution Approach 2:
The sequencing primer is designed with multi-functionality, serving both as a binding element and an extension element while also facilitating denaturation. This universal design reduces the number of separate components and steps needed, thereby reducing workflow complexity
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 reduces the workflow duration by combining denaturation and hybridization into a single step, facilitating faster and more efficient preparation of nucleic acid molecules for sequencing while maintaining compatibility with upstream amplification and downstream sequencing analysis.
Implementation Method 1
contacting double-stranded nucleic acid molecules attached to a surface with ethylene carbonate to generate single-stranded nucleic acid molecules attached to the surface
Implementation Method 2
hybridizing sequencing primers to the single-stranded nucleic acid molecules, thereby generating sequencing hybrids
Data Source
AI summary
Provided herein are methods and are methods for preparing nucleic acid molecules for sequencing on a surface using ethylene carbonate. Further described are methods for sequencing said nucleic acid molecules on a surface, using flow sequencing methods.