Hairpin Internal Control for Isothermal Amplification Monitoring
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Solution Overview
Problem
Existing nucleic acid amplification methods, such as PCR, require thermocycling and can be time-consuming, and competitive internal controls (ICs) can lower target amplification efficiency, while noncompetitive ICs require careful optimization to avoid primer competition.
Innovation Solution
A method using a quality control template with a 5′ subdomain, 3′ subdomain, and loop domain that forms a paired stem, and a quality control primer for isothermal amplification, generating a hairpin structure to monitor amplification without interfering with target amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If competitive internal control strategy is used, then internal control can be implemented in the amplification reaction, but target amplification efficiency is lowered due to competition for primers
Solution Approach 1:
The internal control template is segmented into two separate domains: a first domain that is amplified by the internal control primer, and a second domain that is amplified by the target primers. This segmentation ensures that the internal control amplification and target amplification occur independently without competing for the same primers, thereby maintaining both reliable internal control monitoring and high target amplification efficiency.
2Productivity
If noncompetitive internal control strategy is used, then target amplification efficiency is maintained, but primer and polymerase competition still occurs requiring careful optimization
Solution Approach 1:
The internal control template is divided into distinct domains with specific primer binding sites. The first domain contains a binding site for the internal control primer that is unique and does not overlap with target primer binding sites. This structural segmentation eliminates the need for careful optimization of primer concentrations and copy numbers, as the segmented design inherently prevents competition for primers and polymerase.
3Productivity
If PCR thermocycling is used, then nucleic acid amplification can be achieved, but the process is time-consuming and requires specialized machinery
Solution Approach 1:
The patent replaces the mechanical thermocycling system with an isothermal amplification system using Bst DNA polymerase. This enzyme enables amplification at a constant temperature (e.g., 60-65°C) without requiring temperature cycling, thereby eliminating the need for specialized thermocycling machinery and significantly reducing amplification time while maintaining amplification capability.
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
The method allows for rapid, efficient nucleic acid amplification with reduced assay complexity and improved target amplification efficiency, even at low copy numbers, by using a hairpin IC that minimizes primer competition and interference.
Implementation Method 1
a quality control template comprising: a 5′ subdomain; a 3′ subdomain; and a loop domain situated between the 5′ subdomain and the 3′ subdomain, wherein intramolecular nucleotide base pairing between the 5′ subdomain and the 3′ subdomain are capable of forming a paired stem domain
Data Source
AI summary
Disclosed herein include methods, compositions, and kits for monitoring an amplification reaction. Some embodiments include a quality control template comprising a 5′ subdomain, a 3′ subdomain, and a loop domain situated between the 5′ subdomain and the 3′ subdomain. Intramolecular nucleotide base pairing between the 5′ subdomain and the 3′ subdomain are capable of forming a paired stem domain. A quality control primer can be capable of hybridizing to at least a portion of the 3′ subdomain. Subjecting the quality control template and the quality control primer to an amplification reaction can generate a first quality control product. The method can comprise detecting the first quality control product.