Duplex-Specific Nuclease Nucleic Acid Detection
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Solution Overview
Problem
Current nucleic acid detection methods are not sufficiently sensitive, accurate, or rapid, often requiring purification and amplification steps, making them inefficient for direct detection from patient samples.
Innovation Solution
The use of duplex-specific nuclease (DSN), such as Kamchatka crab DSN, to digest nucleic acid duplexes formed by hybridizing target nucleic acids with probes, releasing detectable components for sensitive and accurate detection without the need for extensive sample preparation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If hybridization-based methods or PCR amplification are used to detect target nucleic acids, then detection sensitivity can be improved, but the detection time and process complexity increase significantly
Solution Approach 1:
The invention performs preliminary hybridization between the target nucleic acid and the detection probe before adding the DSN enzyme. This preliminary action creates a duplex structure that is specifically recognized and digested by DSN, allowing for rapid detection without requiring time-consuming amplification steps. The probe is designed with a detectable label that is released upon DSN digestion, enabling direct detection.
Solution Approach 2:
The invention extracts only the essential detection function by using a simple hybridization-DNA digestion-detection workflow. Instead of performing complete PCR amplification or complex purification steps, the method extracts the key step of target recognition through hybridization and uses DSN to specifically digest the hybridized duplex, releasing the detectable label for direct observation.
2Quantity of substance
If purification and amplification steps are performed prior to detection, then sufficient starting material is obtained for detection, but the overall process becomes more complex and time-consuming
Solution Approach 1:
The detection probe itself serves multiple functions: it acts as both the capture agent for target recognition and the source of the detectable label. Upon hybridization with the target nucleic acid, the probe-DSN complex automatically processes the detection without requiring external purification or amplification steps. The system is self-sufficient, eliminating the need for separate preparation steps.
Solution Approach 2:
The detection probe is designed as a multi-functional molecule that performs target recognition, signal generation, and detection functions simultaneously. The probe contains both the hybridization region for target binding and the detectable label for signal generation. This universal design eliminates the need for separate purification and amplification reagents, simplifying the overall process.
3Reliability
If existing nucleic acid detection methods are used, then detection can be performed, but accuracy and sensitivity are insufficient for direct detection from patient samples
Solution Approach 1:
The detection probe is designed with localized functional regions: a hybridization region for specific target binding and a detectable label region for signal generation. The DSN enzyme specifically recognizes and digests only the duplex structure formed by perfect or near-perfect hybridization, providing local quality control that enhances detection accuracy even in complex patient samples without extensive preparation.
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 method enables rapid, efficient, and sensitive detection of nucleic acids directly from clinical samples, improving diagnostic accuracy and reducing the time required for nucleic acid detection in various applications.
Implementation Method 1
hybridizing the target nucleic acid to a detection probe
Implementation Method 2
digesting the resultant duplex using the duplex-specific nuclease
Data Source
AI summary
The present invention provides methods for detecting a target nucleic acid in a sample by, for example, incubating the target nucleic acid with a detection probe containing a nucleic acid sequence complementary to at least a portion of the target nucleic acid and a nuclease enzyme that specifically cleaves double-stranded nucleic acids. Hybridization between the detection probe and the target nucleic acid thereby leads to cleavage of the detection probe, releasing a portion of the probe attached to a detectable agent. The portions of the digested probes attached to the detectable agent can be separated from unbound probe and detected in order to determine the presence of the target nucleic acid in the sample. Thus, the invention enables rapid and accurate analysis of a sample for the presence of desired nucleic acid biomarkers.