Real-Time Flap Cleavage PCR for Low-Ratio Mutation Detection
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Solution Overview
Problem
Current methods for detecting point mutations in the human genome are inefficient in distinguishing between low ratios of mutant to wild-type sequences, particularly in real-time PCR assays.
Innovation Solution
A cleavage-based real-time PCR assay method involving two sets of thermocycling conditions, including high and low temperatures, is used to amplify and detect nucleic acid targets, with real-time measurement of flap probe cleavage during the second set of cycles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional real-time PCR assays are used for detecting point mutations, then the detection process is simplified, but the ability to distinguish low ratios of mutant to wild-type sequences is poor
Solution Approach 1:
The assay is divided into two distinct sets of thermocycling conditions: a first set for initial amplification and a second set for detection. This segmentation allows each phase to be optimized independently, with the first set focusing on amplification efficiency and the second set on detection precision, thereby resolving the contradiction between simplified detection and improved mutant ratio discrimination
Solution Approach 2:
The patent changes thermocycling parameters between the two sets of cycles. The first set uses standard amplification temperatures, while the second set uses modified temperatures (including a holding temperature of 60°C) that optimize flap probe cleavage and detection. This parameter change enables high precision detection of low mutant ratios without requiring entirely separate assay systems
2Measurement precision
If a single set of thermocycling conditions is used, then the assay procedure is simplified, but the resolution of mutant to wild-type ratios is insufficient
Solution Approach 1:
The patent merges amplification and detection into a single continuous reaction without removing or adding reagents between the two sets of thermocycling conditions. This merging allows the assay to achieve high ratio resolution (1:10,000) while maintaining efficiency, as the same reaction mixture serves both amplification and detection functions throughout the extended cycling protocol
3Measurement precision
If additional reagents are added between thermocycling sets, then detection sensitivity is improved, but the assay procedure becomes more complex
Solution Approach 1:
The flap probe serves multiple functions: it acts as a primer during amplification, becomes a substrate for cleavage during detection, and provides the fluorescent signal for measurement. This multi-functionality eliminates the need for additional reagents between thermocycling sets, maintaining operational simplicity while achieving high detection sensitivity for point mutations
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 achieves improved resolution of mutant to wild-type ratios from 1:10 to 1:10,000, enabling accurate and efficient detection of point mutations in nucleic acid samples.
Implementation Method 1
a flap endonuclease, and a fluorophore-coupled FRET cassette... the flap endonuclease is capable of cleaving the flap probe at a junction between a single-stranded region and a double-stranded region
Implementation Method 2
subjecting a reaction mixture comprising a) PCR reagents for amplifying a nucleic acid target... to two sets of thermocycling conditions... i. a first temperature of at least 90° C.; ii. a second temperature in the range of 60° C. to 75° C.
Implementation Method 3
a fluorescently-labeled flap probe... that hybridizes to the amplified nucleic acid target
Data Source
AI summary
A cleavage-based real-time PCR assay method is provided. In general terms, the assay method includes subjecting a reaction mixture comprising a) PCR reagents for amplifying a nucleic acid target, and b) flap cleavage reagents for performing a flap cleavage assay on the amplified nucleic acid target to two sets of thermocycling conditions. No additional reagents are added to the reaction between said first and second sets of cycles and, in each cycle of the second set of cycles, cleavage of a flap probe is measured.