EGFR Mutation Detection via DNA Cyclization and Tagging
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Solution Overview
Problem
Current methods for detecting EGFR gene mutations in Non Small Cell Lung Cancer (NSCLC) are inefficient, requiring invasive procedures, high costs, and lengthy sequencing times, with limited sensitivity and accuracy, especially when dealing with plasma DNA fragments.
Innovation Solution
A method involving the design of specific primers for EGFR gene exons, extraction of plasma DNA, connection with tagging linkers, PCR pre-amplification, cyclization of DNA fragments, and high-throughput sequencing to non-invasively detect EGFR gene mutations using second-generation sequencing technology.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional sequencing is used to detect EGFR gene mutations, then high accuracy is achieved, but the method requires invasive procedures, has long sequencing time, and high cost
Solution Approach 1:
The patent extracts and amplifies only the specific EGFR gene regions containing mutation hotspots (exons 18-21) using targeted PCR primers, rather than sequencing the entire genome or gene. This extraction of relevant genetic material enables rapid, accurate detection of EGFR mutations in plasma DNA without the time and resource burden of comprehensive sequencing
Solution Approach 2:
The patent divides the EGFR gene into specific segments (exons 18-21) that contain the majority of clinically relevant mutations. By designing primers that target these specific segments and using cycle sequencing to read only these portions, the method achieves high detection accuracy for clinically important mutations while dramatically reducing sequencing time and cost compared to full gene or genome sequencing
2Ease of operation
If PCR-SSCP is used to detect EGFR gene mutations, then simple operation and high sensitivity are achieved, but the method has high false positive rate and requires parallel standard control
Solution Approach 1:
The patent replaces the complex PCR-SSCP mechanical process (which requires gel electrophoresis, heat denaturation, and visual comparison of conformational changes) with direct cycle sequencing and automated sequence analysis. This substitution eliminates the subjective interpretation and conformational variability that cause false positives in PCR-SSCP, while maintaining operational simplicity through automated sequencing machines
Solution Approach 2:
The patent uses cycle sequencing to create multiple copies of the DNA fragments with fluorescently labeled terminators, generating sufficient signal for direct detection without requiring the parallel standard controls needed in PCR-SSCP. The sequencing reaction produces abundant copies of the target regions that can be directly analyzed, eliminating the need for comparative conformational analysis
3Measurement precision
If mutation enriched PCR is used to detect EGFR gene mutations, then high sensitivity is achieved, but the method needs twice PCR and enzyme digestion making it complex and time-consuming
Solution Approach 1:
The patent merges the enrichment and detection steps into a single streamlined process. By designing primers that directly amplify the mutation-containing regions and using cycle sequencing for detection, the method combines the enrichment function (targeted amplification) with the detection function (sequence reading) in one integrated workflow, eliminating the separate enrichment PCR, enzyme digestion, and detection steps required by mutation enriched PCR
4Measurement precision
If AMRS or micro digital PCR is used to detect EGFR gene mutations, then detection sensitivity is improved, but their application in clinic still needs time
Solution Approach 1:
The patent uses standard, widely available reagents and equipment (PCR reagents, cycle sequencing kits, automated sequencers) that are already established in clinical laboratories, rather than requiring specialized long-development technologies like AMRS or micro digital PCR. This approach achieves high detection sensitivity using accessible, cost-effective materials that can be immediately implemented in clinical settings without extensive validation or infrastructure changes
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 enables rapid, accurate, and sensitive detection of EGFR gene mutations in plasma DNA, improving diagnostic efficiency and reducing the need for invasive procedures, with high throughput and cost-effectiveness.
Implementation Method 1
PCR pre-amplifying the tagging linkers connected plasma DNAs
Implementation Method 2
cyclizing the amplified DNAs to obtain cyclised DNAs
Data Source
AI summary
The present invention discloses a method for non-invasively detecting EGFR gene mutations in subjects, comprising the following steps: designing primers according to EGFR gene exons; extracting plasma DNAs in subjects; connecting the extracted plasma DNAs with tagging linkers; PCR pre-amplifying the tagging linkers connected plasma DNAs; cyclising the pre-amplified DNAs to obtain cyclised DNAs; PCR amplifying the cyclised DNAs using the designed primers; and high throughput sequencing the PCR amplified product and analyzing the EGFR gene mutations. The present invention also discloses a corresponding kit.