Polynucleotide Ligation via Bridging Probes
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Solution Overview
Problem
Existing methods for selectively forming ligation products, particularly with target polynucleotides like cell-free DNA, suffer from low efficiency and cumbersome workflows, making it difficult to isolate target polynucleotides from heterogeneous mixtures.
Innovation Solution
A method involving the formation of a polynucleotide complex using single-stranded adaptors and bridging probes, where the target polynucleotide is joined to extension sequences, and specific hybridization and ligation occur to form ligation products, followed by degradation of the bridging probes, allowing for selective ligation and enrichment of target polynucleotides without solid-phase enrichment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If existing methods are used for selectively forming ligation products with target polynucleotides, then the workflow is established, but the efficiency and yield are low and the workflow is cumbersome
Solution Approach 1:
The patent introduces bridging probes as intermediary molecules that facilitate the ligation reaction between adaptors and target polynucleotides. These bridging probes contain sequences complementary to both the adaptor and the target, acting as a mediator to enable efficient and selective ligation product formation while simplifying the overall workflow
Solution Approach 2:
The method employs extension sequences that are pre-attached to the target polynucleotide before the ligation step. These extension sequences serve as preliminary structures that enable specific hybridization with bridging probes, thereby improving ligation efficiency and selectivity without requiring complex enrichment steps
2Reliability
If solid-phase enrichment is used to isolate target polynucleotides, then enrichment is achieved, but the workflow becomes cumbersome
Solution Approach 1:
The patent extracts the enrichment function from complex solid-phase procedures and integrates it directly into the ligation reaction itself. By designing bridging probes with specific sequences that hybridize to target polynucleotides, the method achieves selective enrichment of targets during the ligation process, eliminating the need for separate solid-phase enrichment steps
Solution Approach 2:
The method merges the enrichment and ligation steps into a single unified reaction. The bridging probes simultaneously perform the functions of enriching target polynucleotides through specific hybridization and facilitating ligation product formation, thereby simplifying the workflow while maintaining reliable target isolation
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 method enhances the efficiency and yield of ligation product formation, enabling selective enrichment of target polynucleotides, such as cell-free DNA, without the need for cumbersome solid-phase based enrichment steps, improving the overall process efficiency and accuracy.
Implementation Method 1
a 5' end of the first bridging probe specifically hybridized to a 3' end of the first single-stranded adaptor via sequence complementarity, and a 5' end of the single-stranded sample polynucleotide specifically hybridized to a 3' end of the first bridging probe via sequence complementarity
Implementation Method 2
ligating (i) the 3' end of the first single-stranded adaptor to the 5' end of the single-stranded sample polynucleotide and (ii) the 3' end of the single-stranded sample polynucleotide to the 5' end of the second single-stranded adaptor
Data Source
AI summary
In some aspects, the present disclosure provides methods for forming ligation products comprising single-stranded polynucleotides without the need for enriching the single-stranded polynucleotides. Ligation products formed by various aspects of the present disclosure can be useful for various applications, including but not limited to sequence analysis. In some embodiments, the ligation products comprise cell-free polynucleotides. In some aspects, the present disclosure provides reaction mixtures, kits and complexes consistent with the methods herein.