Direct qPCR Without Minor Groove Binders
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Solution Overview
Problem
Current qrtPCR methods require DNA extraction and removal from filter paper, leading to sample loss and laborious processes, and the presence of filter paper in reaction tubes increases background fluorescence, making direct quantification challenging.
Innovation Solution
A method for direct nucleic acid quantification without prior extraction, involving the use of a solid support like filter paper in the qrtPCR reaction vessel without a Minor Groove Binder (MGB), which reduces background fluorescence by employing a probe lacking an MGB and using a kit with a 5'-exonuclease probe and primer pair that hybridizes to a target sequence without forming a stable stem-loop structure.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If filter paper is present in the reaction tube during qrtPCR, then direct quantification without extraction is achieved, but background fluorescence increases
Solution Approach 1:
The patent removes the Minor Groove Binder (MGB) component from the probe structure to eliminate the source of background fluorescence interference. This extraction of the problematic element allows filter paper to remain in the reaction tube without compromising signal quality, enabling direct quantification.
Solution Approach 2:
The patent changes the chemical composition parameter of the probe by eliminating the MGB moiety. This parameter change fundamentally alters the fluorescence characteristics of the system, reducing background signal while maintaining target detection capability.
2Object-affected harmful factors
If DNA extraction and removal from filter paper is performed, then background fluorescence is reduced, but sample loss and labor increase
Solution Approach 1:
The patent extracts/removes the MGB component from the probe design to eliminate background fluorescence interference. This removal eliminates the need for DNA extraction from filter paper, as the background issue is solved at the probe level rather than requiring sample preparation steps.
Solution Approach 2:
The probe design itself provides the solution to background fluorescence by excluding MGB. The system becomes self-sufficient, requiring no additional extraction or purification steps to achieve low background signals.
3Stability of the object's composition
If MGB is present in the probe, then probe stability is improved, but background fluorescence increases
Solution Approach 1:
The patent removes the MGB component from the probe structure to eliminate background fluorescence. This extraction accepts a trade-off in probe stability but achieves the primary goal of reducing background interference in direct qrtPCR with filter paper.
Solution Approach 2:
The patent changes the chemical composition of the probe by eliminating the MGB moiety, fundamentally altering the fluorescence properties of the system to reduce background signal while maintaining target detection 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
This approach allows for accurate DNA quantification without extraction, minimizing sample loss and labor, and significantly reduces background fluorescence by eliminating the interference caused by MGBs, enabling reliable qrtPCR assays even with filter paper present.
Implementation Method 1
employing a probe without a MGB... wherein the probe hybridizes to a target sequence
Implementation Method 2
employing a 5'-exonuclease probe... detecting the level of fluorescence emitted from the vessel
Implementation Method 3
fluorescent dyes are generally used to detect PCR products... fluorescence emanating from a reaction tube is collected by the fluorimeter
Data Source
AI summary
Disclosed herein are methods, compositions and kits for the quantification of a nucleic acid target present on a solid support. This entails quantitative real-time polymerase chain reaction wherein minor groove binders are excluded.