Rolling Linear Amplification Hairpin Template
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Solution Overview
Problem
Existing rolling circle amplification (RCA) methods face challenges in circularizing DNA, requiring knowledge of the single strand sequence and excluding the template from the amplified product, limiting their applicability and information retrieval.
Innovation Solution
A method of rolling linear amplification using a polymerase to copy a 5′ hairpin with non-canonical bases, allowing for the formation of a complementary 3′ hairpin, enabling the inclusion of the original target sequence in the amplified product and allowing for target-specific or non-specific amplification without prior sequence knowledge.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If rolling circle amplification is used to amplify DNA, then amplification can be achieved, but the template DNA cannot be included in the amplified product and sequence knowledge is required
Solution Approach 1:
The patent inverts the conventional RCA approach by using a linear template with hairpin structures instead of circular templates. The 5' hairpin contains the template sequence and is copied by the polymerase to generate a complementary 3' hairpin, allowing the original template to be included in the amplified product while eliminating the need for pre-existing sequence knowledge.
Solution Approach 2:
The patent segments the template structure into distinct functional elements: a 5' hairpin containing the template sequence, a target region, and a 3' hairpin formation site. This segmentation allows the template sequence to be preserved in the 5' hairpin while the polymerase amplifies the target region, resolving the contradiction between template inclusion and amplification efficiency.
2Productivity
If conventional RCA methods are used, then amplification occurs, but the process is complex and requires multiple steps including circularization
Solution Approach 1:
The patent extracts the circularization step from the amplification process entirely. By using a linear template with pre-formed 5' hairpin structures that directly serve as primers, the method eliminates the complex circularization steps required in conventional RCA, simplifying the overall process while maintaining high amplification efficiency.
Solution Approach 2:
The patent performs preliminary action by pre-forming the 5' hairpin structure on the template before amplification begins. This pre-formed structure serves as both the template and primer, eliminating the need for separate circularization or primer annealing steps, thus reducing process complexity while maintaining productivity.
3Measurement precision
If RCA is used for biological detection, then signal amplification is achieved, but the template is excluded from the product limiting information retrieval
Solution Approach 1:
The patent uses copying by having the polymerase copy the 5' hairpin sequence to generate a complementary 3' hairpin. This copying process creates a new template that includes the original target sequence information, allowing both signal amplification and information retrieval while maintaining measurement precision for detection 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
Enables efficient and specific amplification of polynucleotides, including the original target sequence, with the ability to repeat the amplification cycle multiple times, facilitating the detection of modifications such as DNA methylation and providing longer amplification products.
Implementation Method 1
the polymerase extends, using the canonical nucleotides, the template polynucleotide from its 3′ end to form a first extended polynucleotide
Implementation Method 2
the complement of the 5′ hairpin forms a 3′ hairpin
Data Source
AI summary
A method of amplifying a target polynucleotide, comprising: providing a template polynucleotide comprising a 5′ hairpin, a target polynucleotide and a 3′ hairpin, wherein the 5′ hairpin comprises one or more non-canonical nucleotides and contacting the template polynucleotide with a polymerase and canonical nucleotides, wherein the polymerase extends, using the canonical nucleotides, the target polynucleotide from its 3′ end to form a first extended polynucleotide comprising the 5′ hairpin at its 5′ end and the complement of the 5′ hairpin at its 3′ end, wherein the complement of the 5′ hairpin forms a 3′ hairpin; and the polymerase extends the first extended polynucleotide from its 3′ end to form a second extended polynucleotide comprising the 5′ hairpin at its 5′ end and the complement of the 5′ hairpin at its 3′ end, wherein the complement of the 5′ hairpin forms a 3′ hairpin.