Surface-Bound Splint Primer for Nucleic Acid Circularization
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Solution Overview
Problem
Current nucleic acid sequencing technologies face challenges in efficiently converting linear DNA templates to circular forms for next-generation sequencing (NGS) library preparation, which increases processing time and user burden due to the need for additional steps and purification processes.
Innovation Solution
A method involving the hybridization of a linear template polynucleotide to a splint primer immobilized on a surface, followed by circularization to form a continuous strand lacking free 5' and 3' ends, and subsequent amplification using a strand-displacing polymerase, allowing for direct sequencing of the circularized template.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If linear DNA is converted to circular form using conventional library prep methods, then sequencing compatibility is improved, but processing time and user burden increase due to additional steps and purification processes
Solution Approach 1:
The patent combines the linear-to-circular conversion step with the amplification step into a single integrated process. The splint primer design allows simultaneous circularization and rolling circle amplification, eliminating the need for separate purification steps and reducing overall processing time while maintaining NGS library compatibility
Solution Approach 2:
The splint primer is pre-designed with specific sequences that enable both circularization and serve as the starting point for amplification. By preparing the primer with dual functionality beforehand, the method eliminates intermediate purification steps and reduces user burden
2Quantity of substance
If conventional amplification methods are used, then DNA amplification is achieved, but product loss occurs and additional purification steps are required
Solution Approach 1:
The rolling circle amplification process is performed directly on the surface-bound circularized template, allowing the amplification to self-generate sufficient product without requiring extraction and re-amplification steps. This eliminates product loss associated with multiple purification cycles
3Reliability
If additional purification steps are performed, then amplification product quality is improved, but device complexity and user burden increase
Solution Approach 1:
The patent merges circularization, amplification, and sequencing preparation into a single continuous workflow. The surface-bound splint primer system allows all steps to occur in one location, eliminating the need for multiple purification and transfer steps, thereby reducing device complexity while maintaining product quality
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 number of steps required for nucleic acid template amplification, minimizes product loss, and eliminates the need for exonuclease treatment, thereby enhancing the signal intensity and accuracy of high-throughput sequencing methods.
Implementation Method 1
amplifying the circular template polynucleotide by extending the splint primer with a strand-displacing polymerase, wherein the extension generates a first extension product including one or more complements of the circular template polynucleotide
Implementation Method 2
hybridizing the linear template polynucleotide to a splint primer immobilized on a surface, wherein (i) the splint primer includes, in the 5′ to 3′ direction, a first sequence and a second sequence, (ii) the first sequence is complementary to a 5′ portion of the linear template polynucleotide, and (iii) the second sequence is complementary to a 3′ portion of the linear template polynucleotide
Data Source
AI summary
Disclosed herein, inter alia, are compositions and methods for amplification and sequencing of nucleic acids.