Barcoded Ligation Assays for Low-Bias Nucleic Acid Detection
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Solution Overview
Problem
Existing nucleic acid detection methods suffer from 3′-bias, inability to measure splice variants, fusion genes, and low sensitivity, especially in small sample sizes, and require complex sample preparation and solid-phase immobilization.
Innovation Solution
The method employs detector oligos that hybridize specifically to target sequences, are ligated to form a product, and are configured to resist nucleases, allowing sensitive detection in a single reaction container, with optional barcoding for sample identification and amplification, suitable for microfluidic platforms and FFPE samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If existing nucleic acid detection methods are used, then detection can be performed, but 3'-bias occurs and sensitivity is low especially in small sample sizes
Solution Approach 1:
The detector oligo is divided into multiple functional segments: a 5' end region that hybridizes to the 5' end of the target nucleic acid, a 3' end region that hybridizes to the 3' end of the target, and a linker region connecting them. This segmentation allows simultaneous binding to both ends of the target, eliminating 3'-bias and enabling detection of splice variants and fusion genes through ligation of the segmented detector.
Solution Approach 2:
The detection approach transitions from linear scanning (传统方法从5'到3'或反之) to a two-dimensional approach where the detector binds simultaneously to both 5' and 3' ends of the target nucleic acid. This dimensional change in the detection strategy allows direct measurement of the entire target sequence regardless of length or structure, eliminating the 3'-bias inherent in sequential detection methods.
2Measurement precision
If complex sample preparation and solid-phase immobilization are used, then detection can be achieved, but the process becomes complex and time-consuming
Solution Approach 1:
The method extracts and eliminates the need for solid-phase immobilization and complex sample preparation steps by using solution-based detector oligos that directly hybridize to target nucleic acids in liquid phase. The detector oligos are designed to bind specifically to target sequences without requiring immobilization on solid supports, thereby simplifying the overall workflow while maintaining detection precision.
Solution Approach 2:
The detector oligo performs multiple functions simultaneously: it binds to the 5' end, binds to the 3' end, and provides a ligation site for joining the 5' and 3' regions. This self-service capability eliminates the need for separate reagents and steps for each function, thereby reducing procedural complexity while achieving comprehensive detection.
3Adaptability or versatility
If traditional detection methods are used, then nucleic acids can be detected, but inability to measure splice variants and fusion genes limits versatility
Solution Approach 1:
The detector oligo is designed as a universal tool that can detect various types of nucleic acid structures including wild-type sequences, splice variants, and fusion genes. By incorporating both 5' and 3' hybridization regions with a linker, the same detector design can accommodate different target configurations, making the system universally applicable to diverse nucleic acid detection needs without requiring separate specialized reagents.
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
Enables sensitive detection of nucleic acids at the whole-transcriptome and single-cell level, with spatial precision and correlation to morphology, eliminating the need for solid-phase immobilization and allowing profiling of small focal areas in FFPE samples.
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
Data Source
AI summary
Barcoded ligation assay products from individual samples.