Cas Endonuclease Complexes for Nucleic Acid Detection
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Solution Overview
Problem
Current methods for detecting pathogens are hindered by the presence of irrelevant nucleic acids, which interfere with PCR and probe hybridization, and lack sensitivity to detect large DNA fragments or multiple pathogens simultaneously.
Innovation Solution
The method involves using Cas endonuclease complexes to protect the target nucleic acid of interest and degrade all other nucleic acids, allowing for the isolation and detection of the target nucleic acid through selective degradation, enabling the detection of pathogens in complex samples without the need for amplification or copying.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If PCR or probe hybridization is used to detect pathogen nucleic acids, then detection can be performed, but irrelevant nucleic acids from host or patient interfere with the detection process
Solution Approach 1:
The patent extracts and removes irrelevant nucleic acids from the sample through selective degradation using exonucleases. The method specifically degrades unprotected nucleic acids while leaving the target pathogen nucleic acids intact, thereby eliminating interference from host or patient nucleic acids and improving detection accuracy
Solution Approach 2:
The patent introduces binding proteins as intermediary agents that selectively bind to and protect target pathogen nucleic acids from degradation. These binding proteins act as mediators that distinguish between relevant and irrelevant nucleic acids, allowing selective preservation of target sequences while degrading background nucleic acids
2Measurement precision
If PCR amplification is used to detect target nucleic acids, then sensitivity is improved, but errors are introduced and epigenetic modifications such as methylation are lost
Solution Approach 1:
The patent avoids creating copies of the target nucleic acid through PCR amplification. Instead, it directly detects the original target sequences after selective removal of background nucleic acids, thereby preserving all original information including epigenetic modifications and avoiding amplification errors
Solution Approach 2:
The patent performs preliminary selective degradation of irrelevant nucleic acids before detection, enriching the target sequences in advance. This preliminary action improves detection sensitivity without requiring subsequent amplification steps that would cause information loss
3Reliability
If capture- or amplification-based methods are used to detect pathogens, then detection can be performed, but sensitivity is insufficient when target-to-background ratio is very low
Solution Approach 1:
The patent converts the harmful effect of abundant background nucleic acids into a beneficial selective degradation target. By using exonucleases that specifically degrade unprotected nucleic acids, the method exploits the presence of high background levels to achieve selective enrichment of protected target sequences, thereby improving detection sensitivity even when target-to-background ratio is very low
4Reliability
If traditional detection methods are used, then single pathogen detection is possible, but multiplexing capability is limited making it difficult to detect multiple pathogens simultaneously
Solution Approach 1:
The patent employs a universal detection platform using binding proteins and exonucleases that can recognize and protect various different target sequences. By using different binding proteins with specificities for different pathogens, the system achieves multi-functionality and can detect multiple pathogens simultaneously in a single assay without compromising detection accuracy
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 allows for the sensitive detection of nucleic acids of various sizes, including those over 10 kb, and multiple targets in a single assay, overcoming limitations of existing methods by isolating the target nucleic acid and reducing interference from irrelevant nucleic acids.
Implementation Method 1
protecting the ends of the nucleic acid of interest using binding proteins such as Cas endonuclease complexes
Implementation Method 2
degrading the unprotected nucleic acids, e.g., with an exonuclease
Data Source
AI summary
Provided herein are methods of detecting nucleic acids. The nucleic acid of interest may be detected by using Cas endonuclease to degrade substantially all nucleic acid in a sample except for the nucleic acid of interest, leaving the nucleic acid of interest isolated and amenable to detection. In related methods, Cas endonuclease complexes are used to protect the nucleic acid of interest while unprotected nucleic acid is digested, e.g., by exonuclease, after which the isolated nucleic acid of interest is detected.