Barcoded Ligation Assay for Nucleic Acid Detection
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Solution Overview
Problem
Current methods for detecting nucleic acid sequences, especially in samples like FFPE tissues, face challenges in sensitivity and specificity, particularly in measuring low-abundance RNAs and distinguishing between similar sequences, and often require complex sample preparation and transfer steps.
Innovation Solution
The method involves using detector oligos that hybridize specifically to target nucleic acid sequences, with downstream and upstream detectors being ligated to form a product that can be labeled with barcodes, allowing for sensitive detection and identification of target sequences, even in small sample volumes, and can be performed in situ on slides without the need for solid-phase immobilization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current detection methods are used for nucleic acid sequences in FFPE tissues, then detection can be performed, but sensitivity and specificity are insufficient especially for low-abundance RNAs
Solution Approach 1:
The detection method segments the target nucleic acid sequence into multiple regions (e.g., 5' end, 3' end, internal regions) and uses multiple detector oligos that hybridize to different segments. This segmentation allows the system to detect low-abundance RNAs with higher sensitivity while maintaining specificity, as multiple segmented detections reduce false positives from similar sequences
Solution Approach 2:
The method employs partial hybridization where detector oligos hybridize to only specific regions of the target sequence rather than requiring complete sequence matching. This partial action approach enables detection of low-abundance RNAs by focusing on conserved regions while tolerating variations in other areas, improving sensitivity without sacrificing specificity
2Measurement precision
If complex sample preparation and transfer steps are used, then detection can be performed, but the process becomes more complex and time-consuming
Solution Approach 1:
The method merges multiple detection capabilities into a single hybridization assay. Instead of requiring separate sample preparation, purification, and detection steps, the detector oligos are designed to directly hybridize to target sequences in the sample matrix, combining multiple functions into one streamlined process that reduces complexity while maintaining detection precision
Solution Approach 2:
The detector oligos are designed with self-complementary sequences that allow them to automatically form detectable structures (such as hairpins or dimers) upon hybridization to the target. This self-service mechanism eliminates the need for external labeling or complex detection systems, simplifying the overall process while maintaining high detection capability
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 enables precise detection of nucleic acid sequences with high sensitivity and specificity, allowing for gene expression profiling in minute focal areas of FFPE samples, correlating molecular data with morphology, and reducing the need for extensive sample preparation or transfer.
Implementation Method 1
The samples are contacted with detector oligos to hybridize specifically to the target sequences
Implementation Method 2
If both the DDO and UDO are specifically hybridized to the DR and UR of a target sequence, they can be ligated
Implementation Method 3
the barcodes can be added by enzymatic or chemical methods, such as ligases or 'click' chemistry addition
Implementation Method 4
the barcodes can be added by enzymatic or chemical methods, such as ligases or 'click' chemistry addition
Data Source
AI summary
Barcoded ligation assay products from individual samples.