Split Barcode Region Probes for False-Positive Ligation Control
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Solution Overview
Problem
Existing oligonucleotide probe-based methods for in situ analysis suffer from low sensitivity, specificity, and detection efficiency, often requiring laborious optimization and leading to false positive signals due to chimeric probe ligation.
Innovation Solution
A method involving split barcode region probes, where the barcode sequence is divided between two probes, allowing for specific ligation and detection of target nucleic acids, followed by signal detection and amplification, thereby reducing false positives.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional oligonucleotide probe-based methods are used for in situ analysis, then the detection process is simple, but the sensitivity, specificity, and detection efficiency are low
Solution Approach 1:
The probe is divided into multiple segments: a first region that hybridizes to the target nucleic acid, a second region that hybridizes to an adjacent target sequence, and a barcode region. This segmentation allows for improved specificity through controlled ligation of the first and second regions to the target, while the barcode region enables efficient detection and amplification, thus resolving the contradiction between detection specificity and detection efficiency
Solution Approach 2:
The barcode region is pre-designed and integrated into the probe structure before hybridization. This preliminary configuration of the barcode sequence allows for subsequent efficient signal amplification and detection without requiring complex optimization during the detection process, thereby improving detection efficiency while maintaining high specificity through the hybridization-based ligation mechanism
2Reliability
If traditional probe ligation methods are used, then the assay procedure is straightforward, but false positive signals occur due to chimeric probe ligation
Solution Approach 1:
By segmenting the probe into a first region, second region, and barcode region, the invention enables specific ligation of the first and second regions to adjacent target sequences. This segmentation prevents chimeric ligation because the barcode region remains attached to the correctly ligated probe segments, allowing for easy identification and filtering of false positive signals through barcode verification
Solution Approach 2:
The barcode region acts as an intermediary element that mediates between the hybridization-specific first and second regions and the detection system. It provides a verifiable identifier that confirms successful specific ligation, thereby improving signal accuracy while the modular design keeps the assay procedure manageable
3Reliability
If high-fidelity ligases are used to improve ligation specificity, then false positives are reduced, but the cost and optimization time increase
Solution Approach 1:
The probe segmentation into functionally distinct regions (first region, second region, barcode region) allows the use of simpler, faster ligases because the specificity is enforced by the modular design and barcode verification rather than relying solely on ligase fidelity. This reduces optimization time while maintaining high ligation specificity
Solution Approach 2:
The barcode region provides a feedback mechanism that verifies successful specific ligation. By detecting and amplifying the barcode sequence, the system confirms that the first and second regions have correctly ligated to adjacent target sequences, thereby achieving high ligation specificity without requiring complex ligase optimization
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
Enhances detection specificity by filtering out incorrect ligation products, maintaining high sensitivity and accuracy even with low-fidelity ligases, and enabling efficient in situ analysis of nucleic acids.
Implementation Method 1
a first probe comprising a first hybridization region and a first portion of a barcode region, and (ii) a second probe comprising a second hybridization region and a second portion of the barcode region, wherein the first and second hybridization regions are complementary to target sequences in a target nucleic acid molecule
Implementation Method 2
contacting the biological sample with a detectable probe that hybridizes to a sequence of the barcode region or a complement thereof
Data Source
AI summary
In some aspects, the present disclosure relates to methods for reducing the detection of false positive ligation events. In some aspects, the method comprises use of a double split (or “split split”) probe. The methods herein have particular applicability in reducing the detection of false positive ligation events when using ligases that have high ligation efficiency but low specificity (e.g., SplintR® ligase). Also provided are kits comprising probes for use in such methods.