MLPA Point Mutation Quantification Beyond Signal Saturation
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Solution Overview
Problem
Conventional MLPA methods can only perform qualitative testing for point mutations, unable to measure the rate of mutant-type (MT) to wild-type (WT) cells due to the narrow dynamic range of capillary sequencers, which saturate at high WT signal intensities.
Innovation Solution
A point mutation rate detection method using multiplex ligation-dependent probe amplification (MLPA) that measures fluorescence signals from mutation and wild-type sites, expanding the dynamic range to calculate the MT/WT ratio by setting a reference value above saturation, allowing for quantitative testing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional capillary sequencers are used for MLPA point mutation detection, then qualitative testing is possible, but quantitative measurement of MT/WT ratio is not possible due to signal saturation at high WT intensities
Solution Approach 1:
The patent changes the reference value parameter from a saturated control binning DNA signal to an unsaturated reference signal with higher intensity. By setting the reference value above the saturation limit of the detection system, the system can accurately measure MT/WT ratios across a wide dynamic range without signal saturation interfering with quantitative accuracy.
2Measurement precision
If the reference value is set at the saturation limit of capillary sequencers, then qualitative detection is sufficient, but quantitative ratio calculation becomes inaccurate
Solution Approach 1:
The patent modifies the reference value parameter to be higher than the saturation limit, enabling accurate quantitative measurements without requiring complex signal normalization or multiple measurement systems. This single parameter change allows the existing capillary sequencer to perform both qualitative and quantitative analysis accurately.
3Adaptability or versatility
If next-generation sequencers are used to measure multiple genes, then comprehensive cancer gene detection is possible, but cost becomes significantly high
Solution Approach 1:
The patent makes the capillary sequencer system universal by enabling it to perform both qualitative and quantitative point mutation detection across multiple genes using a single system. The MLPA method with the modified reference value approach allows one capillary sequencer to replace expensive next-generation sequencers for detecting multiple cancer-related genes, significantly reducing costs while maintaining comprehensive 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
Enables accurate quantitative measurement of MT/WT ratios, overcoming the limitations of conventional capillary sequencers by expanding the dynamic range and compensating for variations in sample loading, facilitating cost-effective and precise cancer diagnosis.
Implementation Method 1
a capillary sequencer makes it possible to separate and detect PCR products according to their molecular weights by performing electrophoresis after the PCR
Implementation Method 2
measuring at least an intensity SMT among the intensity SMT of a mutation-derived signal as a fluorescence signal emitted from a mutation site of a sample and an intensity SWT of a wild-type derived signal as a fluorescence signal emitted from a site of the sample other than the mutation site
Data Source
AI summary
The point mutation rate detection method according to the present invention is used in a multiplex ligation-dependent probe amplification (MLPA) measurement, and includes: a measurement step of measuring at least an intensity SMT among the intensity SMT of a mutation-derived signal as a fluorescence signal emitted from a mutation site of a sample and an intensity SWT of a wild-type derived signal as a fluorescence signal emitted from a site of the sample other than the mutation site, using an electrophoresis device; and a rate calculation step of calculating a rate of the intensity SMT to a reference value having a higher intensity than the SMT. In the point mutation rate detection method, an upper limit of a dynamic range of measurement for the fluorescence signal of the electrophoresis device is equal to or more than a predetermined value.