Circularized Nucleic Acid Fusion Gene Detection
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Solution Overview
Problem
Current methods for detecting gene fusions, such as multiplex PCR, anchored multiplex PCR, and hybrid capture, face challenges including inability to identify fusions involving novel breakpoints, high input requirements, complex workflows, reduced sensitivity, and robustness issues with degraded samples like FFPE preserved tissue and cfDNA.
Innovation Solution
A method involving differential amplification of fusion genes using circularized nucleic acid molecules, where linear molecules are converted into circular templates, and a blocking element is used to inhibit amplification of non-fusion templates, allowing for preferential amplification of fusion-containing templates using primers and a polymerase.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Device complexity
If multiplex PCR is used for targeted sequencing of gene fusions, then cost and analysis complexity are reduced, but the ability to identify fusions involving novel breakpoints and partners is lost
Solution Approach 1:
The method segments the gene fusion detection process into two distinct phases: (1) circularization of linear nucleic acid molecules to create circular templates, and (2) selective amplification using primers that preferentially amplify circular templates containing fusion genes. This segmentation allows the method to maintain simplicity while enabling detection of novel breakpoints, as the circularization step preserves all original sequence information including novel fusion junctions that would be missed by primer-based multiplex PCR.
2Measurement precision
If anchored multiplex PCR is used for targeted sequencing of gene fusions, then sensitivity is improved, but input requirements increase and workflow complexity increases
Solution Approach 1:
Instead of using anchored multiplex PCR to selectively amplify fusion genes from linear templates (which requires complex primer designs and high input material), the invention inverts the approach by first converting all templates to circular form, then using the circular structure itself as the selection mechanism. The circularized templates containing fusion genes are preferentially amplified because the circular structure provides a continuous template for primer extension, eliminating the need for complex anchored primer designs and reducing input requirements.
3Quantity of substance
If hybrid capture is used for targeted sequencing of gene fusions, then coverage is improved, but workflow complexity increases and sensitivity is reduced
Solution Approach 1:
The invention replaces the mechanical hybridization capture process (which requires complex probe designs, high input material, and multiple steps) with a biochemical approach based on circular DNA topology. By converting linear nucleic acid molecules to circular templates in vitro, the method creates a structural feature that can be selectively amplified using standard PCR primers. This substitution eliminates the need for complex hybrid capture workflows while maintaining comprehensive coverage of fusion genes, including those with novel breakpoints.
4Reliability
If standard PCR approaches are used for gene fusion detection, then robustness to degraded samples is improved, but ability to detect low-abundance fusion genes is reduced
Solution Approach 1:
The method applies preliminary anti-action by first circularizing all linear nucleic acid molecules in the sample, including degraded FFPE and cfDNA. This circularization step creates a protective structural feature that prevents degradation and enables selective amplification. By converting all templates to circular form before amplification, the method ensures that even low-abundance fusion genes are represented in the circularized pool and can be detected with high sensitivity using standard PCR, thereby maintaining both robustness to degraded samples and detection sensitivity.
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 high-sensitivity targeted analysis of gene fusions with minimal workflow complexity and input requirements, while maintaining robustness to degraded materials, effectively identifying fusion genes across different chromosomes.
Implementation Method 1
circularizing a plurality of linear nucleic acid molecules to form a plurality of circular template polynucleotides
Implementation Method 2
binding a blocking element to the one or more non-fusion circular template polynucleotides
Implementation Method 3
hybridizing a first primer and a second primer to the one or more non-fusion circular template polynucleotides and the one or more fusion circular template polynucleotides and extending with a polymerase to generate a first number of non-fusion polynucleotide amplification products and a second number of fusion polynucleotide amplification products
Data Source
AI summary
Disclosed herein, inter alia, are compositions and methods providing sequencing-efficient solutions for detecting genetic features and aberrations.