Padlock Probe Blocking Oligonucleotide for Low-Background Ligation
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Solution Overview
Problem
Current methods for controlling the ligation of padlock probes in nucleic acid detection are complex, costly, or sacrifice ligation efficiency, leading to non-specific templated and non-templated ligation, which increases background signal and reduces assay specificity and sensitivity.
Innovation Solution
A method using a blocking oligonucleotide that hybridizes to the target-binding regions of a padlock probe, preventing ligation until it is displaced, allowing controlled ligation only when the probe is near its target nucleic acid, thereby reducing background signal.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current methods for controlling padlock probe ligation are used, then ligation can be achieved, but non-specific templated and non-templated ligation occurs, increasing background signal and reducing assay specificity and sensitivity
Solution Approach 1:
A blocking oligonucleotide is introduced as an intermediary molecule that temporarily binds to the padlock probe, preventing non-specific ligation. The blocking oligo acts as a mediator that must be displaced by the target nucleic acid before ligation can occur, thereby eliminating non-specific background signal while allowing specific target-dependent ligation to proceed
Solution Approach 2:
The blocking oligonucleotide is pre-bound to the padlock probe before the assay begins, performing a preliminary blocking action that prevents non-specific ligation events. This preliminary action ensures that only target-specific displacement events can trigger subsequent ligation, thereby improving assay specificity before the main detection process begins
2Reliability
If blocking oligonucleotide is used to control ligation, then non-specific ligation is reduced, but the complexity of the assay increases
Solution Approach 1:
The blocking oligonucleotide serves multiple functions simultaneously: it blocks non-specific ligation, acts as a displacement target for specific target recognition, and provides a template for subsequent ligation after displacement. This multi-functionality reduces the need for separate control mechanisms, thereby limiting the increase in assay complexity while maintaining high specificity
3Reliability
If blocking oligonucleotide is used to control ligation, then background signal is reduced, but the time required for the assay increases
Solution Approach 1:
The assay employs periodic thermal cycling that includes phases for blocking oligo displacement and phases for ligation. This periodic action allows the blocking oligo to be efficiently displaced during high-temperature denaturation phases, followed by ligation during cooler phases, thereby reducing background signal while managing assay time through optimized thermal cycling rather than prolonged continuous incubation
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 method effectively reduces non-specific ligation, enhancing the specificity and sensitivity of nucleic acid detection by ensuring ligation occurs only at the correct site and time, thus improving the accuracy of detection assays.
Implementation Method 1
the blocking oligonucleotide hybridizes to the target-binding regions of a padlock probe
Implementation Method 2
ligation of the 5' phosphate group of a second oligonucleotide to the 3' hydroxyl group of a first oligonucleotide
Data Source
AI summary
The present invention relates to improvements in the use of padlock probes whereby the ligation of the padlock probe may be controlled, and in particular to a method of detecting a target nucleic acid in a sample which comprises the use of a padlock probe complexed with a blocking oligonucleotide. The blocking oligonucleotide binds the target-binding regions of the padlock probe and holds them apart in a manner which prevents their ligation, until the padlock probe is in the vicinity of the target nucleic acid molecule, at which point the padlock probe is released from the blocking probe so that it can bind its target, thereby reducing background signal. Also provided is a kit comprising a padlock probe and blocking oligonucleotide, which can be used in the methods of the invention.