DNA Target Capture With Barcodes for Rare Alteration Detection
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Solution Overview
Problem
Conventional methods for detecting and quantifying rare genetic events, such as low-level microbial DNA, are hindered by high error rates and require extensive amplification, which compromises accuracy, and hybridization-based capture methods lack specificity and efficiency in capturing both defined and undefined target regions.
Innovation Solution
A method involving primers with barcode sequences and separation molecules allows simultaneous capture and identification of defined and undefined target regions in DNA samples, using polymerase-mediated elongation and bead binding to enhance specificity and sensitivity, enabling detection of various genetic alterations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If amplification methods are used to overcome limitations of sample input amount, then the quantity of detectable genetic material is improved, but the reliability and accuracy of detection deteriorates due to errors introduced during amplification
Solution Approach 1:
The patent applies preliminary action by performing target capture and enrichment of DNA fragments before amplification. The method uses hybridization-based capture with biotinylated probes to selectively enrich target sequences from the original sample, followed by streptavidin bead separation. This preliminary enrichment step increases the quantity of target material available for subsequent amplification and detection, while the selective capture process maintains reliability by focusing amplification resources on verified target sequences rather than random genomic DNA.
2Adaptability or versatility
If hybridization-based capture methods are used to capture target regions, then the ability to capture both defined and undefined target regions is improved, but the specificity deteriorates due to off-target region capture
Solution Approach 1:
The patent applies local quality by designing different probe types with distinct properties for different target regions. Captive probes are designed with high specificity for defined target regions (known sequences), while dispersive probes provide broader coverage for undefined target regions (potential novel sequences). The biotinylated capture probes use locally optimized hybridization conditions and probe designs to maximize on-target capture while minimizing off-target binding, thereby achieving both versatility and precision.
Solution Approach 2:
The patent implements feedback through a two-stage enrichment process with intermediate analysis. After initial hybridization-based capture, the enriched material undergoes sequencing or analysis to identify off-target captures. This information feeds back into probe design optimization and enrichment parameter adjustment, allowing iterative improvement of specificity while maintaining the ability to capture both defined and undefined targets.
3Ease of manufacture
If conventional PCR-based methods are used for target capture, then the simplicity of the method is improved, but the ability to detect structural variants with unknown breakpoints deteriorates
Solution Approach 1:
The patent applies segmentation by dividing the target capture process into multiple functional components: hybridization-based enrichment using biotinylated probes, streptavidin bead-based separation, and subsequent amplification. This segmented approach replaces the single-step PCR method, enabling detection of structural variants with unknown breakpoints through the hybridization mechanism that does not require predefined primer binding sites, while maintaining operational simplicity through standardized laboratory procedures.
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 method achieves high sensitivity and specificity in detecting and quantifying genetic alterations, including viral DNA, microsatellite instability, and structural rearrangements, without requiring initial sample splitting, thus improving detection efficiency and accuracy.
Implementation Method 1
allowing the polymerase to elongate the primer A and the primer B thereby obtaining a double stranded product A and a double stranded product B
Implementation Method 2
adding a bead that binds the separation molecule to the main mixture and allowing the separation molecule in the double stranded product B to bind to the bead thereby forming a double stranded complex B
Data Source
AI summary
Disclosed is a method of simultaneously capturing and identifying distinct targets within a DNA sample, wherein the distinct targets comprise a defined target region and an undefined target region, wherein the undefined target region comprises a structural variation or rearrangement or fusion. Also disclosed is a kit comprising the reagents for use in the methods as described herein.