HTGTS Detection of Non-Specific DNA Double-Strand Breaks
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Solution Overview
Problem
Current methods for high-throughput genome-wide translocation sequencing (HTGTS) are limited in sensitivity, specificity, and efficiency, particularly in identifying off-target hotspots and non-specific DNA double-strand breaks (DSBs) caused by agents like nucleases used in gene therapy, and are costly and time-consuming.
Innovation Solution
An enhanced HTGTS approach involving Linear Amplification Mediated (LAM)-PCR with locus-specific primers, adapter ligation, and nested PCR amplification, followed by sequencing and alignment, to detect and analyze DNA double-strand breaks and translocations, allowing for the identification of on-target and off-target DSBs and collateral genomic damage with increased sensitivity and cost-effectiveness.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If existing HTGTS methods are used to detect genome-wide translocations, then translocation detection can be performed, but sensitivity is limited and off-target hotspots are not adequately identified
Solution Approach 1:
The patent segments the detection process into multiple specialized stages: initial DSB capture using biotinylated oligos, streptavidin-bead enrichment, and iterative PCR amplification. Each stage is optimized for specific functions, allowing high sensitivity detection while maintaining reliability through controlled enrichment of true signals over background noise.
Solution Approach 2:
The method performs preliminary enrichment of DSB fragments using biotin-streptavidin binding before sequencing. This preliminary action concentrates rare off-target events from complex genomic backgrounds, enabling sensitive detection without requiring excessive sequencing depth, thus improving both sensitivity and cost-effectiveness.
2Reliability
If existing HTGTS methods are used, then translocation detection is possible, but the process is time-consuming and costly
Solution Approach 1:
The patent employs iterative PCR amplification where only the necessary number of cycles are performed to achieve sufficient enrichment. This partial action approach avoids over-amplification artifacts while achieving reliable detection, reducing both time and cost compared to methods requiring excessive sequencing or multiple validation steps.
Solution Approach 2:
The patent introduces biotinylated oligonucleotides as intermediaries that specifically bind to DSB fragments. This intermediary mechanism enables selective enrichment of relevant DNA fragments from complex genomic material, streamlining the workflow and reducing the time required for reliable detection compared to non-specific methods.
3Measurement precision
If existing HTGTS methods are used, then genome-wide translocation sequencing can be performed, but specificity is limited and non-specific DSBs are not adequately distinguished
Solution Approach 1:
The detection workflow is segmented into distinct functional modules: DSB capture, enrichment, amplification, and sequencing. Each module is optimized to preserve specific information while filtering noise, enabling accurate characterization of non-specific DSBs by maintaining their sequence context through controlled enrichment and amplification.
Solution Approach 2:
The iterative PCR amplification process incorporates feedback control where amplification cycles are monitored and adjusted to achieve optimal enrichment without over-amplification. This feedback mechanism preserves specificity by preventing the amplification of non-specific products while ensuring sufficient signal from true DSBs for accurate characterization.
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 provides robust and efficient detection of DNA double-strand breaks and translocations, enhancing sensitivity and specificity, reducing costs and time, and enabling the characterization of DSB-generating agents' activity across the genome.
Implementation Method 1
performing a nested-PCR with an adapter- and a locus-specific primer
Implementation Method 2
producing a ligated DNA product by ligating the single-stranded PCR product produced in step (e) to an adapter
Data Source
AI summary
Described herein are methods and systems relating to high throughput, genome-wide translocation sequencing (HTGTS) and/or detection of double-stranded DNA break (DSB) locations. The methods described herein can comprise generating DSBs in a nucleic acid sequence and performing nested PCR with the primers described herein. Described herein is an enhanced HTGTS approach. The assays and methods described herein permit the measurement of various DNA double-strand break (DSB) activities either intrinsic to the biological system or from outside agents.