Rolling Circle Amplification Double-Strand Conversion
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Solution Overview
Problem
Existing methods for converting single-stranded products of rolling circle amplification (RCA) into double-stranded DNA face challenges such as incomplete secondary strand synthesis, complex secondary structures, and the generation of numerous by-products, which affect the stability and utility of the resulting DNA molecules.
Innovation Solution
A method involving subjecting a single-stranded circular DNA to rolling circle amplification with a first primer, opening the DNA through a disconnection mechanism to form a single-stranded linear DNA, and then performing amplification in the opposite direction using the linear DNA as a second primer to obtain a double-stranded sequence, which reduces by-products and simplifies the secondary structure.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If double-primed RCA is used to convert single-stranded products into double-stranded DNA, then amplification efficiency is improved, but the structure becomes extremely complicated with single and double strands coexisting
Solution Approach 1:
The patent segments the amplification process into two distinct phases: first generating single-stranded RCA products, then using a separate second primer to synthesize the complementary strand. This segmentation prevents the complex mesh structure formation that occurs when both primers act simultaneously, while still achieving complete double-stranded conversion.
Solution Approach 2:
The patent performs the first RCA amplification to generate single-stranded products as a preliminary step before introducing the second primer for complementary strand synthesis. This preliminary action allows the first strand to serve as a clean template, preventing the spatial structure limitations and incomplete synthesis issues of double-primed RCA.
2Manufacturing precision
If random primers are used for RCA to generate double-stranded products, then secondary strand synthesis is more complete, but numerous by-products are generated
Solution Approach 1:
The patent uses a second primer with specific local complementarity to the 3' end of the single-stranded RCA product, rather than random primers that bind throughout the sequence. This localized priming ensures complete secondary strand synthesis at the critical region while minimizing non-specific binding and by-product formation.
3Productivity
If adapters and label sequences are added for LAMP method, then double-stranded DNA with multiple copies is obtained, but redundant information increases
Solution Approach 1:
The patent extracts and removes the unnecessary adapters and label sequences that accumulate during the amplification process, keeping only the essential complementary strand synthesis. This extraction maintains the high amplification efficiency of LAMP-like methods while eliminating the redundant information that complicates downstream applications.
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 generates double-stranded RCA products with fewer by-products, simpler structures, and improved biochemical performance, making them more similar to conventional DNA molecules and suitable for single-molecule sequencing applications.
Implementation Method 1
Rolling circle amplification (RCA) or rolling circle replication (RCR) generally refers to subjecting a single-stranded circular DNA molecule as a template to polymerase chain reaction (PCR) with use of a DNA polymerase possessing strand displacement activity
Implementation Method 2
the DNA polymerases possessing strand displacement activity will take the added DNA or RNA fragment as a primer, and take the single-stranded circular DNA molecule as a template, to amplify the DNA template
Implementation Method 3
the generated single stranded DNA will fold and intertwine therebetween, forming a complexly spatial secondary structure
Implementation Method 4
which coordinates with metal ions to form a structurally dense DNA nanoball (DNB)
Implementation Method 5
opening the single-stranded circular DNA through the disconnection mechanism, to obtain a single-stranded linear DNA
Implementation Method 6
performing amplification in the opposite direction of the initial direction of the RCA, by taking the opened single-stranded linear DNA as a primer and the amplified sequence as a template
Data Source
AI summary
Provided is a method for obtaining a double-stranded sequence by single-stranded rolling circle amplification, comprising: 1) performing rolling circle amplification reaction on single-stranded circular DNA by means of a first primer to obtain an amplified sequence, the first primer being complementary to a partial region of the single-stranded circular DNA, and the single-stranded circular DNA having a break mechanism that can cause the single-stranded circular DNA to ring-open; 2) ring-opening the single-stranded circular DNA by means of the break mechanism to obtain single-stranded linear DNA; and 3) using the single-stranded linear DNA as a second primer and using the amplified sequence obtained in step 1) as a template to perform amplification reaction to obtain an amplified double-stranded sequence.