Multiplex Allele-Specific PCR for ESR1 Mutation Detection

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Solution Overview

Problem

Current methods for detecting ESR1 mutations associated with hormone resistance in breast cancer, such as next-generation sequencing and digital PCR, are labor-intensive, costly, and limited by equipment constraints, making them inefficient for widespread use in clinical settings.

Innovation Solution

Development of multiplex allele-specific PCR kits and assays that include specific primer pairs and probes for various ESR1 mutations, allowing for simultaneous detection of multiple mutations in a single reaction using non-invasive samples, reducing the need for specialized equipment and improving efficiency.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If next generation sequencing is used for ESR1 mutation detection, then multiple mutations can be detected simultaneously, but the process becomes labor-intensive, lengthy and expensive

Engineering Contradiction:

Improvedetection capabilityVSAvoidtesting efficiency

Core Design Contradiction:

Adaptability or versatilityVSProductivity

Solution Approach 1:

The patent combines multiple allele-specific PCR assays into a single multiplex reaction system that detects multiple ESR1 mutations simultaneously in one tube, merging the functions of separate tests into one integrated assay that maintains high detection capability while improving efficiency

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The invention creates a universal PCR assay platform using common primers and mutation-specific probes that can detect various ESR1 mutations (K303R, E380Q, L536Q, Y537S, D538G, etc.) through a single standardized protocol, making the system multi-functional for different mutation types without requiring separate specialized procedures

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Measurement precision

If digital PCR is used for ESR1 mutation detection, then high sensitivity is achieved, but the workflow becomes lengthy and requires special equipment with limited multiplexing capability

Engineering Contradiction:

Improvedetection sensitivityVSAvoidequipment requirement

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent replaces complex digital PCR instrumentation with conventional PCR equipment by using allele-specific probes with fluorophore-quencher pairs that generate detectable signals through standard real-time PCR amplification, substituting specialized mechanical/digital systems with accessible laboratory infrastructure while maintaining detection sensitivity

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The invention changes the detection parameter from digital counting of individual molecules to fluorescent signal intensity measurement during amplification, using the ratio of mutant to wild-type signal intensities to determine mutation presence, thereby achieving high sensitivity through parameter optimization rather than complex equipment

Inventive Principle:

Principle #35Parameter changes

3Measurement precision

If multiple separate PCR assays are performed for different ESR1 mutations, then each mutation is detected accurately, but the overall process becomes time-consuming and resource-intensive

Engineering Contradiction:

Improvemutation detection accuracyVSAvoidtesting duration

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The patent merges multiple separate allele-specific PCR assays into a single multiplex reaction by combining common primers with multiple mutation-specific probes in one tube, allowing simultaneous detection of multiple ESR1 mutations (K303R, E380Q, L536Q, Y537S, D538G, etc.) in a single amplification cycle, thereby reducing testing duration while preserving detection accuracy through probe-specific fluorescent signals

Inventive Principle:

Principle #5Merging (Combining)

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 multiplex PCR approach enables rapid, accurate, and cost-effective detection of ESR1 mutations, facilitating personalized treatment decisions for hormone-resistant breast cancer patients by identifying specific mutations that confer therapy resistance.

Implementation Method 1

multiplex allele-specific PCR assays for detecting mutations in the ESR1 gene

Methodology Applied

Scientific EffectPCR amplification:

Implementation Method 2

The reactions are performed in triplicate in a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA)

Methodology Applied

Scientific EffectThermal cycling:

Implementation Method 3

Each probe was labeled with a fluorophore and quencher

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentEP3500681B1Multiplex allele specific PCR assays for detection of estrogen receptor ESR1 mutations

Publication Date: 2021.01.20 ROCHE DIAGNOSTICS GMBH

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AI summary

Provided herein are methods and compositions to detect mutations in estrogen receptor (ESR1).