Amplicon Species Generation for Wide Dynamic Range Nucleic Acid Quantitation
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Solution Overview
Problem
Current nucleic acid amplification and detection methods struggle to accurately quantify target nucleic acids over a wide dynamic range, often requiring additional steps and increasing complexity, which can lead to errors and false results due to limitations in sensitivity and dynamic range.
Innovation Solution
The method generates multiple differentiable amplicon species at unequal ratios using a single amplification system in a single vessel, allowing for detection across an extended dynamic range by using unequal amounts of amplification reaction components, such as primers or NTPs, to span the required linear dynamic range and accommodate detection system limitations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional nucleic acid amplification and detection methods are used, then detection sensitivity is achieved, but the dynamic range is limited and additional steps are required
Solution Approach 1:
The patent segments the amplification process by introducing multiple primers with different concentrations, each targeting the same nucleic acid sequence but producing amplicons at different amplification rates. This segmentation allows simultaneous detection across multiple concentration ranges within a single reaction, effectively extending the dynamic range while maintaining detection sensitivity.
Solution Approach 2:
The patent changes the concentration parameter of primers to create a gradient of amplification efficiencies. By using primers at varying concentrations (e.g., 100 nM, 10 nM, 1 nM), the system generates amplicons that accumulate at different rates, allowing the detection system to accurately quantify target nucleic acids across a wide dynamic range without requiring additional detection steps.
2Adaptability or versatility
If multiple amplification steps are used to extend dynamic range, then detection range is improved, but process complexity increases
Solution Approach 1:
The patent merges multiple amplification reactions into a single reaction vessel by combining multiple primers with different concentrations. This merging eliminates the need for separate amplification steps, reducing process complexity while maintaining the extended dynamic range capability. The detection system simply measures the combined amplicon signals to determine the target concentration.
Solution Approach 2:
The patent creates a universal amplification system where multiple primers serve different functions simultaneously - each primer contributes to amplifying the target sequence at a different rate, and collectively they provide coverage across the entire dynamic range. This multi-functionality allows a single reaction mixture to perform what would traditionally require multiple separate reactions.
3Measurement precision
If additional detection steps are implemented, then quantitation accuracy over wide range is improved, but time consumption and error potential increase
Solution Approach 1:
The patent performs preliminary action by pre-calculating the expected amplicon concentrations based on primer concentrations and target concentration. The detection system uses these pre-established relationships to directly calculate the target nucleic acid concentration from the measured amplicon signals, eliminating the need for additional quantitation steps and reducing both time and error potential.
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 accurate and reproducible quantitation of nucleic acid sequences across a wide range, reducing the need for additional steps and minimizing errors, by generating amplicons that are detectable within overlapping linear ranges, thus extending the dynamic range of detection.
Implementation Method 1
Methods for amplifying a target nucleic acid sequence that may be present in a test sample are known and include methods such as the polymerase chain reaction (PCR)
Implementation Method 2
Methods for detecting non-amplified or amplified target nucleic acid sequences that may be present in a test sample are also known
Data Source
AI summary
Disclosed are compositions and methods for making differentiable amplicon species at unequal ratios using a single amplification system in a single vessel. The number of differentiable amplicons and their ratios to one another are chosen to span the required linear dynamic range for the amplification reaction and to accommodate limitations of the measuring system used to determine the amount of amplicon generated. Unequal amounts of distinguishable amplicon species are generated by providing unequal amounts of one or more amplification reaction components (e.g., distinguishable amplification oligomers, natural and unnatural NTP in an NTP mix, or the like). The amount of target nucleic acid present in a test sample is determined using the linear detection range generated from detection of one or more amplicon species having an amount within the dynamic range of detection.