Multiplex Dye Probe Structure With Internal Quencher Placement
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Solution Overview
Problem
Current qPCR probe designs with a reporter dye at the 5′ end and a quencher molecule at the 3′ end have limitations due to the distance between the fluorophore and quencher, leading to suboptimal quenching and background noise, which affects assay accuracy and robustness.
Innovation Solution
Development of a compound with a divalent nucleobase, linker, and quencher moiety configuration that allows for improved quenching by placing the quencher closer to the fluorophore, enhancing the accuracy and robustness of nucleic acid detection assays.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the quencher molecule is placed at the 3′ end with 20-30 bases between the fluorophore and quencher, then the probe structure is stable and easy to manufacture, but the quenching efficiency is insufficient and background noise increases
Solution Approach 1:
The patent introduces an internal quencher placed at a specific location within the probe (6-12 bases from the fluorophore) rather than relying solely on the terminal quencher. This creates a local high-concentration quenching zone that enhances quenching efficiency and reduces background noise without compromising overall probe stability or manufacturability.
2Measurement precision
If an internal quencher is placed closer to the fluorophore within 6-12 bases, then quenching efficiency improves and background noise decreases, but the probe structure becomes more complex
Solution Approach 1:
The internal quencher moiety serves multiple functions simultaneously: it enhances quenching efficiency, reduces background noise, and maintains probe stability. By integrating this single component with multifunctional properties, the patent improves measurement precision without proportionally increasing structural complexity.
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 new compound design improves quenching efficiency, reducing background noise and increasing the accuracy and reliability of nucleic acid detection assays.
Implementation Method 1
There are typically 20 to 30 bases between the fluorophore and the quencher. An internal quencher can be placed closer to the fluorophore dye within 6-12 bases between the reporter dye and quencher.
Data Source
AI summary
Described herein, inter alia, are multiplex dye compounds and methods of use thereof.