Fusion Probe Gene Fusion Detection via Nuclease Treatment
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Solution Overview
Problem
Current methods for detecting gene fusions, particularly oncogenic gene fusions, are limited in sensitivity and specificity, and often require extensive sample handling and nucleic acid purification, which can lead to degradation and interassay variability.
Innovation Solution
The use of fusion probes that span the fusion point between two nucleic acids, combined with nuclease treatment and microarray detection, allows for sensitive and specific detection of gene fusions in DNA or RNA samples, including previously unknown fusions, without the need for extensive sample preparation or nucleic acid purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current methods for detecting gene fusions are used, then detection can be performed, but sensitivity and specificity are limited and extensive sample handling and nucleic acid purification are required
Solution Approach 1:
The detection method is segmented into distinct functional components: fusion probes that specifically target fusion transcripts, nuclease treatment steps that selectively degrade non-specific nucleic acids, and microarray detection components. This segmentation allows each component to be optimized independently, improving sensitivity and specificity while reducing the complexity of overall sample handling.
Solution Approach 2:
Fusion probes serve as intermediaries that bridge the target fusion transcripts and the detection system. These probes hybridize specifically to fusion transcripts and are subsequently detected via microarray, eliminating the need for extensive nucleic acid purification while maintaining high detection specificity.
2Reliability
If extensive sample handling and nucleic acid purification are performed, then detection can be achieved, but sample degradation and interassay variability increase
Solution Approach 1:
The method performs preliminary nuclease treatment on the sample before detection to selectively remove non-specific nucleic acids. This preliminary action protects against sample degradation during subsequent handling steps and reduces interassay variability by standardizing the sample preparation process.
Solution Approach 2:
The method converts the potential harm of extensive sample handling into a benefit by using controlled nuclease treatment. The nuclease selectively degrades non-specific nucleic acids that would otherwise interfere with detection, while the specific fusion probe-nucleic acid hybrids are protected from degradation, thereby improving detection reliability.
3Measurement precision
If fusion probes with nuclease treatment and microarray detection are used, then sensitivity and specificity are highly improved, but the method complexity increases
Solution Approach 1:
The method merges multiple functions into integrated components: fusion probes combine specific hybridization capability with nuclease resistance, and the microarray system integrates detection of multiple fusion transcripts simultaneously. This merging reduces overall method complexity while maintaining high sensitivity and specificity.
Solution Approach 2:
The fusion probe design and microarray detection system are universally applicable to detect various gene fusions. The same basic methodology can detect different fusion transcripts by simply changing the probe sequences, eliminating the need for separate complex procedures for each fusion type.
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 highly sensitive and specific detection of gene fusions, including single copy detection in a few cells, with reduced background noise and the ability to multiplex for simultaneous detection of multiple fusions, facilitating advanced diagnostic and therapeutic applications.
Implementation Method 1
contacting the sample with a fusion probe that has a 5' portion complementary to a first nucleic acid and a 3' portion complementary to a second nucleic acid, wherein the fusion probe spans a fusion point of the first nucleic acid and the second nucleic acid
Implementation Method 2
The sample is contacted with a nuclease specific for single-stranded nucleic acids
Data Source
AI summary
Disclosed herein are methods of detecting presence of a gene fusion in a sample from a subject. In some embodiments, the methods of detecting presence of a fusion gene in a sample from a subject utilize a fusion probe that spans the point of fusion between two nucleic acids or genes. In other embodiments, the methods of detecting presence of a fusion gene in a sample from a subject utilize two or more probes that flank the point of fusion between two nucleic acids or genes. In additional embodiments, the methods can include determining the percentage of gene fusion in the sample relative to the first nucleic acid or the second nucleic acid.