qPCR Normalization Using Quenching Primers for Multiplex Detection
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Solution Overview
Problem
Existing qPCR methods face challenges in accurately quantifying nucleic acids due to skewed reaction efficiency and the presence of inhibitors, leading to over- or under-quantitation, and are limited in simultaneously amplifying multiple targets using intercalating dyes.
Innovation Solution
The use of fluorescence quenching primers with multiple quenchers positioned within the primer to quench the fluorescence of control nucleic acid amplicons, combined with a detectable probe that emits a distinguishable signal, allows for simultaneous amplification and detection of multiple targets in the same reaction well.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescent DNA intercalators are used to detect amplification products, then fluorescence signal is generated for quantitation, but skewed reaction efficiency and inhibitors cause over- or under-quantitation
Solution Approach 1:
A control nucleic acid with a distinguishable sequence is introduced as an intermediary standard. This control serves as a reference that is amplified alongside the target nucleic acid, allowing normalization of fluorescence signals to account for variations in reaction efficiency and inhibitor effects, thereby improving quantitation accuracy
Solution Approach 2:
The invention changes the detection parameter by using differential fluorescence detection. The control nucleic acid is detected at a first wavelength and the target nucleic acid at a second wavelength, allowing separate quantitation that can be normalized to correct for reaction variability
2Productivity
If multiple targets are amplified simultaneously using intercalating dyes, then productivity increases, but fluorescence signals from multiple targets cannot be distinguished
Solution Approach 1:
The invention applies local quality by assigning different detection wavelengths to different nucleic acid targets. The control nucleic acid is detected at a first wavelength while the target nucleic acid is detected at a second wavelength, allowing simultaneous amplification and distinct detection of multiple targets in the same reaction well
Solution Approach 2:
The fluorescent DNA intercalator serves multiple functions by binding to and enabling detection of both control and target nucleic acids simultaneously. A single intercalating dye performs the function of detecting multiple different nucleic acid sequences through wavelength differentiation, eliminating the need for multiple different dyes
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 normalization of qPCR results by quenching excess fluorescence, allowing for concurrent amplification and detection of multiple targets with improved quantitation accuracy and efficiency.
Implementation Method 1
each of the forward quenching primer and the reverse quenching primer comprises two or more quenchers and wherein the two or more quenchers are positioned within the primer such that the quenchers quench all or substantially all of the fluorescence from an amplification product of the control nucleic acid
Implementation Method 2
fluorescence emitted by a fluorescent DNA intercalator bound to the amplification product of the control nucleic acid
Data Source
Figure 1
Figure 2A~2B
Figure 3A
AI summary
Compositions, kits, and methods are provided for the normalization of a quantitative polymerase chain reaction (PCR) amplification. Also provided are compositions, kits, and methods for multiplexing qPCR amplification of two or more target nucleic acids in the same well.