DNA Nanoball Library Preparation Without Single-Strand Cyclization
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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, thereby simplifying the preparation process and reducing sample requirements.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If single-stranded circular DNA is used as a template for rolling circle amplification, then sequencing accuracy is improved, but library preparation complexity increases 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 linear DNA as the template. The mediating sequence facilitates rolling circle amplification directly from the linear double-stranded template, eliminating the need for prior cyclization steps while maintaining amplification efficiency and sequencing accuracy
Solution Approach 2:
The patent extracts and removes the mediating sequence after it has fulfilled its function of enabling rolling circle amplification from double-stranded linear DNA. This extraction of the mediating sequence eliminates the need for subsequent purification steps to remove cyclization reagents, simplifying the library preparation process
2Reliability
If single-strand cyclization and purification steps are added, then DNB preparation is achieved, but preparation time increases
Solution Approach 1:
The patent merges the rolling circle amplification step with the template preparation step by enabling RCA to proceed directly from double-stranded linear DNA using a mediating sequence. This consolidation eliminates the need for separate cyclization and purification steps, reducing preparation time while ensuring reliable DNB formation
Solution Approach 2:
The mediating sequence is designed to preliminarily bind to the double-stranded linear DNA template and facilitate the initiation of rolling circle amplification before the actual amplification begins. This preliminary action enables the process to skip subsequent cyclization steps, saving time
3Reliability
If single-strand cyclization and purification steps are added, then DNB preparation is achieved, but preparation cost increases
Solution Approach 1:
The patent merges multiple steps (template preparation and rolling circle amplification) into a single integrated process using double-stranded linear DNA as the template. This reduces the number of reagents and purification materials needed, thereby lowering preparation costs while maintaining reliable DNB preparation
Solution Approach 2:
The mediating sequence is extracted after serving its purpose of enabling amplification from double-stranded DNA, eliminating the need for expensive purification steps to remove cyclization reagents, thus reducing overall preparation costs
4Productivity
If cyclization efficiency and purification efficiency are considered, then sample input amount must be increased
Solution Approach 1:
The patent merges the rolling circle amplification with the template preparation, eliminating separate cyclization and purification steps. This integration ensures that nearly all input DNA molecules are converted to DNBs without loss during intermediate purification steps, maximizing productivity with minimal sample input
Solution Approach 2:
The mediating sequence is removed after facilitating amplification, and the process is designed so that this extraction does not require loss-prone purification steps. This ensures high recovery efficiency of the amplified products, maintaining productivity with low sample input
5Quantity 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 patent changes the fundamental parameter of the amplification template from single-stranded circular DNA to double-stranded linear DNA. This parameter change enables the use of rolling circle amplification directly on native genomic DNA without prior PCR, preserving sample integrity and avoiding PCR-introduced errors while still achieving sufficient amplification for sequencing
Solution Approach 2:
The rolling circle amplification process creates multiple copies of the DNA template through isothermal amplification using a mediating sequence and polymerase. This copying mechanism achieves the necessary sample volume increase without using PCR, thereby maintaining amplification accuracy and avoiding PCR-related 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
This approach reduces preparation time and cost, minimizes sample input, and avoids PCR-related errors, facilitating PCR-free sample preparation, especially for rare samples, without compromising sequencing quality.
Implementation Method 1
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 2
connect the two ends of the single-stranded DNA under action of the ligase
Implementation Method 3
denaturing and annealing a double-stranded DNA and a mediating sequence
Implementation Method 4
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
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.