DNA Detection Probe With Embedded RNA Bases For Signal Amplification
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Solution Overview
Problem
Existing nucleic acid detection technologies face challenges with low sensitivity and inconvenient detection methods, particularly due to the lack of signal amplification in probe hybridization-based techniques.
Innovation Solution
A combination product comprising a ribonuclease HII and a single-stranded probe with embedded RNA bases, which divides the complementary region into segments of specific lengths to enhance signal amplification and sensitivity, compatible with isothermal amplification methods like LAMP, NEAR, and RPA, and incorporates a reporting system for direct result output.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If existing probes are used in probe hybridization-based detection, then the detection can be performed, but the sensitivity is low and detection is inconvenient due to lack of signal amplification
Solution Approach 1:
The probe is segmented into multiple regions by embedding RNA bases, which divide the complementary region into segments of specific lengths (0-13 DNA bases each). This segmentation enables the probe to be cleaved into fragments that can serve as primers for signal amplification, thereby improving detection sensitivity while maintaining operational simplicity through automated cleavage and amplification processes
Solution Approach 2:
The probe is pre-designed with embedded RNA bases that will be cleaved by ribonuclease HII to generate short DNA fragments (≤13 bases) before the detection reaction. These pre-prepared fragments serve as primers for subsequent signal amplification, enabling sensitive detection without requiring complex in-situ primer synthesis during the detection process
2Measurement precision
If the probe complementary region is divided into segments of ≤13 bases by embedding RNA bases, then the cleaved probes have increased percentage in complementary strand and intensified signal, but the product fragment is shorter and easier to dissociate
Solution Approach 1:
The probe complementary region is segmented into multiple short segments (each ≤13 DNA bases) by embedding RNA bases at specific positions. This segmentation ensures that after ribonuclease HII cleavage, the resulting DNA fragments are short enough to efficiently dissociate from the complementary strand and bind to new probes, thereby intensifying the detection signal while maintaining optimal fragment length for repeated cycling
Solution Approach 2:
The probe design changes the physical-chemical parameters of the complementary region by inserting RNA bases, which alter the hybridization properties and cleavage characteristics. This parameter change enables the probe to generate optimal fragments (≤13 bases) that balance signal intensification with efficient dissociation and reuse, resolving the contradiction between signal intensity and fragment length
3Adaptability or versatility
If the probe is used with isothermal amplification methods such as LAMP, NEAR, or RPA, then the system has good compatibility and can achieve result output without additional operations, but the probe must ensure specificity for detection results
Solution Approach 1:
The probe design with embedded RNA bases and specific segment lengths (≤13 bases) makes it universally compatible with multiple isothermal amplification methods (LAMP, NEAR, RPA). The same probe structure works across different amplification systems, enabling multi-functional detection without requiring method-specific probe modifications, while maintaining high specificity through the designed segment architecture
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 solution increases the sensitivity of DNA detection by intensifying signals, facilitates easier dissociation of probe fragments, and ensures specificity in detection results, allowing for efficient use in various isothermal amplification methods without additional operations or reactions.
Implementation Method 1
one or more RNA bases are embedded in a complementary region of the probe and divide the complementary region of the probe into at least two segments, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 DNA bases
Implementation Method 2
the probe is a single-stranded probe, and has a sequence which can be partially or entirely complementary to a target DNA molecule to be detected
Data Source
Figure 1A~1B
Figure 2
Figure 2
AI summary
The present disclosure relates to the field of biotechnology, in particular, to a combination product for detecting DNA. The product includes ribonuclease H II and a probe; the probe is a single-stranded probe and has a sequence which can be partially or entirely complementary to a target DNA molecule to be detected, and one or more RNA bases are embedded in the complementary region of the probe and divide the complementary region of the probe into at least two segments, each of which independently has DNA bases and base substitutions of less than or equal to 13 in total.