Rolling Circle Amplification Specificity for Circular Genomes
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Solution Overview
Problem
Current nucleic acid amplification methods struggle to differentially amplify circular genomes from mixed samples, often resulting in significant amplification of non-target nucleic acids, which complicates the isolation of specific circular genomes such as mitochondrial or viral genomes.
Innovation Solution
A method involving rolling circle amplification with a set of primers complementary to the circular genome of interest, using DNA polymerase under isothermal conditions, which selectively amplifies the circular genome of interest relative to non-target nucleic acids by strand displacement replication, allowing for high-fold differential amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional nucleic acid amplification methods are used, then amplification of nucleic acids can be achieved, but non-target nucleic acids are also significantly amplified, reducing the specificity for circular genomes
Solution Approach 1:
The invention uses a set of multiple specific primers instead of a single primer or random primers. Each primer in the set is designed to be complementary to a different region of the circular genome, allowing selective amplification of the circular genome while excluding non-target nucleic acids. This segmentation of the amplification process into multiple specific binding events enhances specificity.
Solution Approach 2:
The primers are designed with specific local complementarity to regions unique to the circular genome. By optimizing the primer sequences to match specific local regions of the circular genome and not bind to non-target sequences, the method achieves high specificity amplification of the desired target while leaving non-target nucleic acids unaffected.
2Productivity
If rolling circle amplification with a single primer is used, then linear amplification can be achieved, but the amplification fold is limited compared to exponential amplification methods
Solution Approach 1:
The invention merges the advantages of rolling circle amplification (specificity for circular templates, isothermal conditions) with the high amplification efficiency of exponential methods by using multiple primers. The combination of multiple specific primers with rolling circle mechanics enables multiply-primed exponential amplification of circular genomes, achieving amplification folds of 10^6 to 10^9 while maintaining the circular template specificity.
Solution Approach 2:
The method maintains continuous rolling circle replication with multiple primers simultaneously active on the circular template. This continuous action with multiple primers working in parallel enables exponential amplification without the need for thermal cycling, achieving high productivity under isothermal conditions.
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 method enables efficient and consistent amplification of circular genomes, achieving up to several thousand-fold amplification of the genome of interest compared to non-target nucleic acids, facilitating the isolation of specific circular genomes from complex samples without substantial amplification of non-target sequences.
Implementation Method 1
The invention is based on rolling circle amplification of the circular genomes which involves strand displacement replication by primers
Implementation Method 2
which involves strand displacement replication by primers
Data Source
Figure 1~2
AI summary
Disclosed are compositions and a method for amplification of circular genomes. The method is based on rolling circle amplification of the circular genomes which involves strand displacement replication by primers. The disclosed method allows differential amplification of circular genomes of interest. In genomic nucleic acid samples containing both a circular genome of interest and non-target nucleic acids, such as non-target genomes, the disclose methods and compositions can result in many-fold differential amplification of the circular genome of interest over non-target nucleic acids. It has been discovered that selection of a set of primers complementary to a circular genome of interest can result in much greater amplification of the circular genome of interest relative to non-target nucleic acids present. Such differential amplification of circular genomes is very useful for obtaining useful amounts of genomes of interest from a mixed nucleic acid sample. For example, mitochondrial genomes, which, absent complicated and time consuming purification, are in the presence of non-target nucleic acids (such as the host cell genome), can be differentially amplified relative to the host cell genome and other non-target nucleic acids using the disclosed methods and composition.