Sequential Hybridization and Quenching for Nucleic Acid Detection
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Solution Overview
Problem
Existing nucleotide barcoding methods for detecting and differentiating multiple analytes in biological samples require multiple detection cycles and involve high temperature and potentially toxic chemical agents for probe removal, which can damage the sample and are time-consuming.
Innovation Solution
A method involving sequential hybridization of probes with detectable moieties and quenchers that allow for in situ detection without the need to remove the probes, using quenching to suppress signal from previously detected moieties, thereby reducing sample damage and simplifying the process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If high temperature and chemical agents are used to remove hybridized probes, then probe removal is achieved, but sample damage occurs
Solution Approach 1:
The patent extracts the harmful function of probe removal by using a quencher molecule that selectively binds to the detectable moiety (fluorophore) on the first probe, extracting the signal without extracting the probe itself from the sample. This allows signal elimination without the need for harsh physical or chemical removal processes that would damage the sample.
Solution Approach 2:
The quencher acts as an intermediary substance that mediates between the detectable moiety and the detection system. By introducing the quencher that binds to the fluorophore, the patent creates a mechanism to suppress the signal from the first probe without directly removing it, thus avoiding sample damage while achieving the desired signal control.
2Adaptability or versatility
If multiple detection cycles are performed sequentially, then multiple analytes are detected, but time consumption increases
Solution Approach 1:
The patent merges the detection of multiple analytes into a single simultaneous detection cycle. By using multiple probes with different detectable moieties that can be detected in one cycle, and using quenchers to selectively suppress signals from previously detected probes, the system achieves high multiplicity detection without requiring sequential removal and re-detection cycles, thus reducing total time consumption.
Solution Approach 2:
The patent applies preliminary action by pre-conjugating quenchers to probes that will be used in subsequent detection cycles. This allows the quenching mechanism to be prepared in advance, enabling rapid signal suppression without requiring time-consuming removal steps between detection cycles, thereby maintaining high versatility while reducing time loss.
3Object-generated harmful factors
If detectable moieties are permanently removed or inactivated, then signal suppression is achieved, but probe integrity is compromised
Solution Approach 1:
The patent introduces dynamics to the probe system through the reversible binding interaction between the quencher and the detectable moiety. The quencher binds to the fluorophore to suppress the signal, but this binding is reversible and does not permanently damage the probe. This dynamic interaction allows signal suppression while maintaining probe integrity and allowing the probe to remain functional for subsequent cycles.
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 provides a cost-and time-efficient approach for high multiplicity in situ assays with high specificity and accuracy, eliminating the need for probe removal steps and minimizing sample damage.
Implementation Method 1
a second probe, wherein: the second probe comprises: (i) a hybridization sequence H2 that hybridizes to a hybridization region H2′ of the nucleic acid molecule, the first probe, or a splint, (ii) a quencher Q1, and (iii) a detectable moiety D2, and Q1 quenches a detectable signal from D1
Data Source
AI summary
The present disclosure provides methods for detecting a nucleic acid molecule involving the use of a signal code sequence which corresponds to said nucleic acid molecule, comprising sequential hybridization of detectably labeled probes to allow detection of a signal code sequence. In particular, the present disclosure provides a method of sequential decoding comprising hybridization-directed quenching of detectable moieties that have already been imaged, allowing the newly added probe to be detected without the need to remove the previously imaged detectable moiety, thus providing an approach that reduces or eliminates the need for damaging probe stripping.