Circularized Nucleic Acid Template Rolling Circle Amplification
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Solution Overview
Problem
Current nucleic acid amplification methods lack sensitivity, precision, reproducibility, and multiplexing capability, are time-consuming, and require complex equipment, making them unsuitable for rapid and cost-effective applications such as sequencing and in vitro diagnostics.
Innovation Solution
The method involves using circularized nucleic acid templates with specific primers and bridging oligonucleotides to achieve exponential rolling circle amplification, utilizing non-natural nucleotides and polymerase enzymes to produce concatemeric amplicons, allowing for repeated cycles of amplification and multiplexing under isothermal conditions with simpler equipment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If linear amplification methods are used, then the process is simple, but sensitivity and amplification yield are insufficient
Solution Approach 1:
The patent employs circularized nucleic acid templates instead of linear templates. The circular topology enables rolling circle amplification where the polymerase continuously synthesizes DNA around the circle, generating long concatemeric amplicons. This curved/circular structure fundamentally changes the amplification mechanism from linear to exponential, dramatically increasing amplification yield while maintaining procedural simplicity.
Solution Approach 2:
The rolling circle amplification process enables continuous synthesis by the polymerase enzyme as it travels around the circular template repeatedly. This continuous action without termination allows for exponential accumulation of amplicons, transforming the discrete linear amplification steps into a continuous exponential process that dramatically increases yield.
2Quantity of substance
If exponential amplification methods are used, then amplification yield increases, but the process becomes time-consuming and tedious
Solution Approach 1:
The patent incorporates a bridging oligonucleotide with a 5' overhang that is pre-designed to anneal to the 3' end of the amplified DNA. This preliminary structural preparation enables the bridging oligo to automatically prime the next round of synthesis without requiring additional enzymes or steps, streamlining the exponential amplification process and reducing time.
Solution Approach 2:
The bridging oligonucleotide serves multiple functions: it anneals to the amplified product, provides a primer for the next synthesis round, and contains the 5' overhang sequence that becomes part of the final amplicon. This multi-functionality eliminates the need for separate priming and extension steps, making the exponential amplification process more efficient and less time-consuming.
3Measurement precision
If conventional amplification methods are used, then the equipment requirements are standard, but sensitivity, precision, and reproducibility are insufficient
Solution Approach 1:
The patent changes the topological parameter of the nucleic acid template from linear to circular, and modifies the primer structure to include 5' overhangs with specific sequences. These parameter changes enable the rolling circle amplification mechanism that produces exponential amplification with high precision and reproducibility, while the isothermal condition parameter eliminates the need for complex thermal cycling equipment.
Solution Approach 2:
The patent replaces the mechanical thermal cycling system (heating and cooling cycles required by PCR) with an isothermal chemical-biological system. The rolling circle amplification proceeds at a constant temperature using the bridging oligonucleotide priming mechanism, substituting complex mechanical temperature control with a simpler isothermal biochemical process that achieves equivalent or superior precision.
4Adaptability or versatility
If standard amplification protocols are used, then the procedure is established, but multiplexing capability is limited
Solution Approach 1:
The patent enables multiplexing by designing different target-specific primers and bridging oligonucleotides with unique 5' overhang sequences. Each target can be amplified with its specific primer-bridging pair, and the segmented nature of the bridging oligos allows multiple reactions to occur simultaneously in the same tube without cross-interference, dramatically increasing multiplexing capability.
Solution Approach 2:
The bridging oligonucleotide acts as an intermediary that connects the target-specific primer to the universal amplification mechanism. Different bridging oligos with distinct 5' overhangs serve as mediators for different targets, allowing multiple specific reactions to be coordinated simultaneously through the same rolling circle mechanism, enabling high-level multiplexing while maintaining ease of execution.
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 enables rapid, sensitive, precise, and reproducible amplification of nucleic acids, reducing assay costs and time, and facilitating the detection of multiple targets in a single assay, thereby improving sequencing and diagnostic processes.
Implementation Method 1
The first primer is annealed to the target nucleic acid and is extended with a polymerase enzyme
Implementation Method 2
extended with a polymerase enzyme to form a first duplex nucleic acid
Implementation Method 3
The second primer is annealed to the first nucleic acid and extended with a polymerase enzyme
Implementation Method 4
extended with a polymerase enzyme to produce a second duplex nucleic acid
Implementation Method 5
The bridging oligonucleotide anneals to both the 3′- and 5′-termini of the second nucleic acid bringing them together
Implementation Method 6
The blunt ends are optionally ligated with ligase to produce a circular nucleic acid
Implementation Method 7
The bridging oligonucleotide is extended in the presence of polymerase and non-natural nucleotide triphosphates complementary to the non-natural nucleotides of the second primer producing a concatemeric amplicon
Implementation Method 8
The second primer is annealed to the third nucleic acid at one or more locations along the amplicon and extended by polymerase
Implementation Method 9
extended by polymerase in the absence of the non-natural nucleotide triphosphates contained in the second primer to produce additional copies
Implementation Method 10
The bridging oligonucleotide is annealed to the additional second nucleic acids to produce additional circular nucleic acids
Implementation Method 11
The 3′- and 5′-termini of the additional circularized nucleic acids may be optionally joined by ligase
Data Source
AI summary
The present invention provides methods of amplifying a target nucleic acid utilizing a circularized template. Circularization may be achieved utilizing a bridging oligonucleotide or an inverter primer. The bridging oligonucleotide or inverted primer is extended forming a concatemeric amplicon that can then be used as a template to provide exponential amplification of the target nucleic acid.