Hooked Probe for Rapid Nucleic Acid Ligation
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Solution Overview
Problem
Current high-throughput sequencing library construction methods, particularly probe liquid phase hybridization and capture technologies, face challenges such as long hybridization times, high contamination rates, and high costs, as well as inefficiencies in enriching small-fragment cfDNAs, limiting their applicability and economic viability.
Innovation Solution
The introduction of a hook probe that allows for rapid hybridization and capture of nucleic acid fragments by adding a stretch of known tool sequence, enabling efficient ligation and subsequent reactions, thereby simplifying the process, reducing time, and lowering costs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If probe liquid phase hybridization and capture technology is used for library construction, then target region enrichment is achieved, but hybridization time becomes very long and contamination rate increases
Solution Approach 1:
The probe is segmented into two functional regions: a target-specific region for hybridization and a hook region for ligation. This segmentation allows the probe to perform both capture and adapter addition functions simultaneously, eliminating the need for separate hybridization and ligation steps, thereby reducing total processing time while maintaining enrichment effectiveness
Solution Approach 2:
The invention merges the hybridization capture function and the adapter addition function into a single probe structure and single reaction step. The hook region contains the known tool sequence that serves as an adapter, so when the probe hybridizes to the target, it simultaneously adds the adapter sequence, combining multiple functions into one operation
2Reliability
If probe liquid phase hybridization and capture technology is used, then target region enrichment is achieved, but contamination by non-target regions increases to at least 40%
Solution Approach 1:
The probe design implements local quality by making the target-specific region highly specific to the target sequence while the hook region provides a universal ligation interface. This localized functionality ensures that only true target sequences are captured and processed, reducing non-specific binding and contamination from non-target regions
Solution Approach 2:
The hook region acts as an intermediary that facilitates specific ligation only after successful hybridization of the target-specific region. This intermediary mechanism ensures that the known tool sequence is added only to correctly bound targets, preventing contamination from non-specific interactions
3Reliability
If conventional hybridization and capture methods are used, then target capture is achieved, but random loss of target sequence occurs during washing and elution
Solution Approach 1:
The adapter (known tool sequence) is added to the target sequence during the hybridization step itself, before the washing and elution steps occur. This preliminary action ensures that the target sequence is already stabilized with the adapter, preventing subsequent loss during processing steps
Solution Approach 2:
By merging the adapter addition with the hybridization step, the target sequence is captured and stabilized in a single operation. The hook region's known tool sequence becomes part of the hybridization complex, so the target sequence is protected from loss during subsequent washing and elution because it is covalently linked to the probe through ligation
4Reliability
If blocking nucleic acids and streptavidin beads are used in hybridization methods, then specificity is enhanced, but reagent costs increase significantly
Solution Approach 1:
The invention extracts and eliminates the need for expensive blocking nucleic acids and streptavidin beads by using a simplified probe structure with integrated target-specific and hook regions. The specificity is achieved through the probe's own design rather than requiring additional blocking reagents, thereby reducing reagent costs while maintaining hybridization specificity
Solution Approach 2:
The probe structure is self-sufficient, with the target-specific region providing specificity and the hook region providing the adapter function. The probe serves its own purposes without requiring additional blocking reagents or bead-based systems, making the process more cost-effective while maintaining reliability
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 facilitates rapid and cost-effective nucleic acid sequencing library construction, suitable for various sample types, enhancing process efficiency, and expanding the range of applications beyond high-throughput sequencing.
Implementation Method 1
the target-specific region comprising a sequence complementary to at least part of a single strand of a nucleic acid fragment to be ligated
Data Source
AI summary
Provided are a hooked probe, a method for ligating a nucleic acid and a method for constructing a sequencing library. The hooked probe comprises a target specific area and a hooked area ligated thereto; the target specific area comprises a sequence complementarily paired with at least part of the single chain of the nucleic acid fragment to be ligated; the hooked area comprises a sequence unpaired with the nucleic acid fragment; the end of the hooked area is a ligatable end; and the ligatable end can ligate the end of the single chain of the nucleic acid fragment.