Rolling Circle Amplification with In-Situ DNA Circularization
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Solution Overview
Problem
The existing DNB-based sequencing technology requires additional steps of single-strand cyclization and purification, increasing complexity, time, and cost, and necessitates a large sample amount, limiting its application in rare samples and introducing errors with PCR amplification.
Innovation Solution
A method using double-stranded DNA as a template for rolling circle amplification, omitting single-strand cyclization and purification steps, simplifying the process and reducing sample requirements.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If single-stranded circular DNA is used as a template for rolling circle amplification, then sequencing accuracy is improved, but library preparation complexity and time are increased due to additional cyclization and purification steps
Solution Approach 1:
Instead of using single-stranded circular DNA as the template (conventional approach), the patent inverts the approach by using double-stranded DNA as the template. The mediating sequence anneals to the denatured single-stranded DNA, and ligase connects the two ends to form a circular structure during the amplification process itself, eliminating the need for separate cyclization and purification steps while maintaining sequencing accuracy
Solution Approach 2:
The patent merges the cyclization step with the rolling circle amplification process. The mediating sequence serves multiple functions: it anneals to the template DNA, provides a primer for amplification, and enables in-situ cyclization through ligase action. This consolidation of functions into a single integrated process eliminates separate preparation steps and reduces overall complexity
2Reliability
If single-strand cyclization and purification steps are added to prepare DNBs, then sequencing accuracy is improved, but preparation time and cost are increased
Solution Approach 1:
The cyclization and amplification processes are merged into a single reaction system. The mediating sequence enables both the formation of circular DNA structure and the initiation of rolling circle amplification simultaneously, eliminating the need for separate cyclization and purification steps that consume time and resources
3Manufacturing precision
If cyclization and purification steps are performed, then DNB quality is improved, but sample input requirements are increased due to low efficiency (10%-30%)
Solution Approach 1:
The system performs self-service cyclization where the mediating sequence and ligase work together within the reaction mixture to automatically form circular DNA structures from the denatured template. This in-situ cyclization eliminates the need for separate purification steps and reduces sample loss, allowing high-quality DNB formation from minimal input samples
Solution Approach 2:
The mediating sequence acts as an intermediary that facilitates the cyclization process by annealing to the template DNA ends and providing a platform for ligase action. This intermediary mechanism ensures efficient circularization without requiring excessive sample input or complex purification procedures
4Quantity of substance
If PCR amplification is used to compensate for insufficient sample volume, then sample amount is increased, but amplification errors are introduced
Solution Approach 1:
The rolling circle amplification system performs self-service amplification using the mediating sequence as a primer and the denatured single-stranded DNA as a template. The Phi29 polymerase continuously synthesizes DNA around the circular template, generating multiple copies without requiring PCR amplification, thus avoiding PCR-introduced errors while adequately increasing sample amount
Solution Approach 2:
The patent changes the amplification parameters by using isothermal rolling circle amplification instead of thermal cycling PCR. The Phi29 polymerase operates at a constant temperature (30°C) and uses a circular template with a primer binding site, enabling continuous DNA synthesis that amplifies sample quantity without the denaturation and annealing cycles that introduce PCR errors
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
Simplifies sample preparation, reduces costs, and avoids PCR errors, making it suitable for PCR-free and rare sample sequencing without compromising sequencing quality.
Implementation Method 1
simultaneously introducing a ligase and a polymerase into the system to connect the two ends of the single-stranded DNA under action of the ligase
Implementation Method 2
performing a rolling circle amplification reaction under action of the polymerase by using the mediating sequence as a primer and the single-stranded DNA as a template
Implementation Method 3
sequentially denaturing and annealing a double-stranded DNA and a mediating sequence (Splint oligo) in a same system, to complementarily pair the mediating sequence with two ends of a denatured single-stranded DNA
Data Source
AI summary
Provided are a rolling circle amplification method, a method for preparing a sequencing library, and a DNA nanoball prepared therefrom. The rolling circle amplification method includes: sequentially denaturing and annealing a double-stranded DNA and a mediating sequence in a same system, to complementarily pair the mediating sequence with two ends of a denatured single-stranded DNA; simultaneously introducing a ligase and a polymerase into the system to connect the two ends of the single-stranded DNA under action of the ligase; and performing a rolling circle amplification reaction under action of the polymerase by using the mediating sequence as a primer and the single-stranded DNA as a template, to obtain DNA nanoball.