Permuted Nucleic Acid Probe Library for Hybridization Signal Amplification
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Solution Overview
Problem
Existing nucleic acid hybridization assays face limitations in sensitivity due to the interference of labels with hybridization characteristics and increased non-specific binding, which reduces the amount of label attached per unit mass of the probe, leading to poor hybridization and high background noise.
Innovation Solution
Modifying DNA sequences prior to or during labeling to promote network formation by fragmenting and ligating the DNA to create a permuted probe library, allowing multiple labeled probe molecules to attach to a single target sequence, thereby increasing assay sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If labels are attached to probe molecules to enhance detection sensitivity, then the amount of detectable signal increases, but hybridization characteristics are interfered with and non-specific binding increases
Solution Approach 1:
The probe library is segmented into multiple different probe sequences that collectively cover the target sequence. Each probe in the library binds to a different portion or aspect of the target, allowing the system to achieve high detection sensitivity through multiple binding events while maintaining specificity because each individual probe still requires specific hybridization to its complementary sequence.
Solution Approach 2:
The invention changes the parameter of probe diversity by using a library of different probe sequences rather than a single probe sequence. This parameter change allows the system to attach multiple labels to each target molecule through different probe binding events, enhancing signal strength while the specific hybridization requirements of each probe sequence maintain binding fidelity and reduce non-specific binding.
2Measurement precision
If more label moieties are incorporated into probe molecules to increase signal strength, then detection sensitivity improves, but hybridization characteristics are interfered with and non-specific binding increases
Solution Approach 1:
Instead of concentrating multiple labels on a single probe molecule, the segmentation principle distributes the labeling across multiple different probe molecules in the library. Each probe molecule carries fewer labels, minimizing interference with hybridization characteristics, while the collective effect of multiple probe-target binding events generates strong signal through the accumulation of labels from different probes.
Solution Approach 2:
The invention uses multiple copies of different probe sequences that all target the same or overlapping regions of the target nucleic acid. Each probe copy contributes additional labeled binding events to the signal, and the redundancy of multiple probe sequences ensures that specific hybridization is maintained even as total label count increases.
3Ease of operation
If a single probe sequence is used for hybridization, then the assay is simple to perform, but the amount of label captured per target is limited, reducing detection sensitivity
Solution Approach 1:
The probe library provides multi-functionality by containing multiple different probe sequences that can all bind to the target nucleic acid. This universal approach allows a single library preparation to achieve multiple binding events per target molecule, capturing more labels and enhancing detection sensitivity while maintaining operational simplicity because the entire library is applied together in a single hybridization step without requiring separate procedures for each probe.
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 permuted probe library enhances assay sensitivity by forming complexes with multiple labeled probe molecules attached to each target sequence, increasing signal strength and reducing non-specific binding, allowing for the detection of smaller target sequences that would be undetectable with standard probes.
Implementation Method 1
The ability of nucleic acids to bind their complementary sequences is the basis of assays for the detection of specific nucleic acid sequences
Implementation Method 2
modifying the DNA sequence prior to or during labeling so as to promote network formation during hybridization
Data Source
AI summary
This invention describes methods for generating nucleic acid probes that improve the sensitivity of hybridization assays. The sensitivity increase results from structural modifications of nucleic acids that promote network formation during hybridization with the result that a single target molecule becomes attached to a complex of many probe molecules. The structural modification involves fragmentation of the probe nucleic acid followed by joining the fragments together such that their order and orientation and number is altered from the original probe molecule. The result is the generation of permuted probe libraries. Individual members of permuted probe libraries can be isolated, amplified and perpetuated. Libraries can be prepared with additional sequences not present in the target and the fraction of the library made up by such sequences controlled. Probes for different targets can incorporate different non-target sequences in hyper-molar quantities permitting sensitive detection of multiple hybridization targets in the same sample.