Chemical Ligation Dependent Probe Amplification for Degraded Nucleic Acid Detection
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Solution Overview
Problem
Current nucleic acid detection methods require extensive upstream processing steps, increasing time and cost, and are often inefficient with degraded samples, particularly in paraffin-embedded tissues.
Innovation Solution
The method employs non-enzymatic chemical ligation reactions using ligation probes with complementary reactive moieties that spontaneously ligate in the presence of target nucleic acids, allowing for rapid and specific detection without the need for exogenous ligases, and can amplify and detect nucleic acids from impure or degraded samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional nucleic acid detection methods are used, then detection sensitivity can be achieved, but the number of processing steps increases and time consumption increases
Solution Approach 1:
The patent combines target capture and signal generation into a single ligation step. The ligation probe contains both the capture sequence that binds to the target nucleic acid and the reporter sequence that generates the detectable signal, eliminating the need for separate capture and detection steps required in traditional methods
Solution Approach 2:
The ligation probe serves multiple functions simultaneously: it acts as a capture agent for target recognition, a ligation substrate for signal amplification, and a reporter carrier for detection. This multi-functionality reduces the number of reagents and steps needed in the detection protocol
2Measurement precision
If traditional nucleic acid detection methods are used, then detection sensitivity can be achieved, but processing time increases
Solution Approach 1:
The ligation reaction proceeds continuously without requiring intermediate purification or processing steps. The probe ligation to the target and subsequent signal generation occur in a single continuous reaction, maximizing the efficiency of the detection process
Solution Approach 2:
Multiple functions (target capture, ligation, and signal generation) are merged into a single reaction step, eliminating the sequential processing required in traditional methods and thereby reducing total processing time
3Measurement precision
If enzymatic ligation methods are used, then specific detection can be achieved, but cost increases and complexity increases
Solution Approach 1:
The patent replaces the biological enzymatic ligation system with a chemical ligation system. The ligation probe contains chemically reactive groups that enable direct chemical bond formation with the target, eliminating the need for enzymatic catalysts and associated buffer conditions
Solution Approach 2:
The ligation probe is designed with intrinsic chemical reactivity that enables self-ligation to the target sequence without requiring external enzymatic assistance. The probe's own chemical structure provides the mechanism for bond formation, simplifying the overall method
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 significantly simplifies the detection process, reduces the number of processing steps, and enables efficient detection of nucleic acids from degraded samples, such as those in paraffin-embedded tissues, while maintaining specificity and sensitivity.
Implementation Method 1
non-enzymatic chemical ligation reactions using ligation probes with complementary reactive moieties that spontaneously ligate in the presence of target nucleic acids
Implementation Method 2
The ligated product may be amplified and detected by capillary electrophoresis, microarray analysis, or any other suitable method
Implementation Method 3
The ligated product may be amplified and detected by capillary electrophoresis, microarray analysis, or any other suitable method
Data Source
AI summary
The present invention provides compositions, apparatuses and methods for detecting one or more nucleic acid targets present in a sample. Methods of the invention include utilizing two or more oligonucleotide probes that reversibly bind a target nucleic acid in close proximity to each other and possess complementary reactive ligation moieties. When such probes have bound to the target in the proper orientation, they are able to undergo a spontaneous chemical ligation reaction that yields a ligated oligonucleotide product. In one aspect, the ligation product is of variable length that correlates with a particular target. Following chemical ligation, the probes may be amplified and detected by capillary electrophoresis or microarray analysis.