UID-PCR Mutation Detection Kit for KIT Gene Analysis
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Solution Overview
Problem
Conventional next-generation sequencing (NGS) methods struggle to distinguish between sequence alterations and errors introduced by polymerases, leading to inaccurate detection of mutations in the KIT gene, which is crucial for cancer diagnosis and therapy monitoring.
Innovation Solution
A reagent kit and method utilizing UID-PCR with barcoded primers for multiplex sequencing, where each amplicon is labeled with a unique identifier, allowing for the detection of mutations present in a threshold proportion of amplicons to differentiate between true mutations and polymerase errors, thereby enhancing the accuracy and sensitivity of mutation detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional NGS methods are used for mutation detection, then sequencing capability is provided, but polymerase errors cannot be distinguished from true sequence alterations
Solution Approach 1:
The method segments the mutation detection process into multiple independent PCR amplifications, each with its own unique molecular identifier (UMI). By dividing the sample into multiple aliquots and performing separate amplifications, the patent creates distinct molecular families that can be independently tracked and analyzed to distinguish true mutations from polymerase errors.
Solution Approach 2:
The patent introduces unique molecular identifiers (UMIs) as intermediary tags that are incorporated into each DNA molecule during initial amplification. These UMI tags serve as mediators to track and group descendant molecules, allowing the system to identify and filter out polymerase errors while preserving true mutations through consensus sequencing within each UMI family.
2Measurement precision
If multiplex sequencing with barcoded primers is used, then sensitivity and accuracy of mutation detection are improved, but false positive results are reduced
Solution Approach 1:
The patent implements a feedback mechanism through consensus sequencing analysis. By sequencing multiple descendant molecules from each UMI family and requiring a threshold proportion (e.g., ≥50%) to share the same mutation, the system provides feedback that filters out false positives while maintaining true mutation detection. This consensus approach creates a self-correcting system that reduces false positive results.
3Reliability
If UID-PCR with unique identifiers is implemented, then ability to differentiate true mutations from polymerase errors is enhanced, but detection reliability is improved
Solution Approach 1:
The patent uses the copying principle by creating multiple identical copies of each original DNA molecule through PCR amplification, with each copy carrying the same unique molecular identifier (UMI). These replicated molecules form families that can be sequenced and compared, allowing the system to identify true mutations that appear consistently across copies while filtering out random polymerase errors that do not recur in the same UMI family.
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 significantly reduces false positive results and provides reliable detection of mutations, enabling precise cancer diagnosis and monitoring, as well as assessing susceptibility or resistance to KIT-targeting therapies.
Implementation Method 1
the primers comprise, or consist of, a universal sequence (e.g., a WBC-PCR primer binding site), the digital unique identifier (UID), and a gene-specific binding site (which is hereinafter also called "complementary sequence portion")
Data Source
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AI summary
Provided herein are methods for the detection of genetic mutations associated with cancer, particularly in the KIT gene.