Recombinase-Mediated Nucleic Acid Cleavage for Unbiased Pathogen Sequencing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing nucleic acid detection methods require prior knowledge of the pathogen and involve costly and time-consuming regulatory approval for new detection reagents, limiting the detection of previously unknown pathogens.
Innovation Solution
Recombinase-mediated selective cleavage of target nucleic acids using single-stranded probes and nucleases, such as nuclease S1, to enrich for non-target nucleic acids in a sample, and remove cleaved target nucleic acids, enabling unbiased sequencing without prior pathogen knowledge.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If antibody-based methods or PCR are used for pathogen detection, then detection sensitivity is improved, but prior knowledge of the pathogen is required and regulatory approval is needed for new reagents
Solution Approach 1:
Instead of directly detecting the pathogen using targeted reagents, the invention inverts the approach by detecting host DNA that is absent or depleted in the sample. By removing host DNA through nuclease treatment after probe hybridization, any remaining DNA must be of pathogenic origin, enabling detection of unknown pathogens without requiring prior pathogen-specific reagents
Solution Approach 2:
The invention uses host DNA as an intermediary marker for pathogen detection. Rather than targeting pathogen DNA directly, the method targets complementary host DNA sequences that should be present if no pathogen is detected. The absence or depletion of host DNA indirectly indicates pathogen presence, allowing detection without direct pathogen targeting
2Measurement precision
If targeted nucleic acid capture followed by sequencing is used, then detection accuracy is improved, but the method requires prior knowledge of the pathogen and involves costly regulatory approval
Solution Approach 1:
The invention uses inexpensive, easily synthesized nucleic acid probes targeting host DNA sequences. These probes can be rapidly designed and synthesized without requiring expensive, regulated pathogen-specific reagents. The probes are single-use consumables that can be quickly updated to target different host sequences, avoiding lengthy regulatory approval processes
Solution Approach 2:
The host DNA targeting approach provides universal applicability across multiple pathogen types. A single set of host-targeting probes can detect various pathogens by identifying the absence of expected host DNA, eliminating the need to develop and approve separate reagents for each pathogen
3Adaptability or versatility
If unbiased sequencing is performed without host DNA removal, then comprehensive pathogen detection is possible, but host DNA dominates the sample and reduces sensitivity
Solution Approach 1:
The invention extracts and removes host DNA from the sample through nuclease treatment following probe hybridization. By selectively depleting host DNA while leaving pathogen DNA intact, the method enriches the sample for pathogenic sequences, dramatically improving detection sensitivity without requiring prior knowledge of the pathogen
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
Enhances pathogen detection sensitivity and reduces costs by enriching for non-host nucleic acids, allowing detection of unknown pathogens without new reagents or regulatory approval.
Implementation Method 1
contacting the target nucleic acid with a single-stranded nucleic acid probe and a recombinase such that a D-loop in the target nucleic acid is formed
Implementation Method 2
contacting the D-loop with a nuclease thereby cleaving the target nucleic acid
Data Source
Figure 1
Figure 2
Figure 3
AI summary
Embodiments of the methods and compositions provided herein relate to the selective cleavage of target nucleic acids. Some embodiments include recombinase-mediated selective cleavage of target nucleic acids with single-stranded nucleic acid probes and a recombinase. Some embodiments also include the enrichment of non-target nucleic acids in a sample by selective cleavage of target nucleic acids in the sample, and removal of the cleaved target nucleic acids from the sample.