Hybrid Capture Probe Segmentation for Short-Fragment Target Enrichment
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Solution Overview
Problem
Current nucleic acid hybrid capture technologies face challenges with low capture efficiency and on-target rates, particularly for small target regions and short fragment libraries, requiring longer synthesis times and complex operations, which are not suitable for applications like cancer tumor mutation detection and MRD screening.
Innovation Solution
A novel probe design method that includes selecting probe sequences with specific annealing temperatures and lengths, and using a pool of probes with complementary pairing regions to enhance binding stability and density, allowing for rapid hybridization and capture of short fragments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional probes of 120 nt are used for hybrid capture, then the probe can bind to target sequences, but the capture efficiency and on-target rate are low for small target regions and short fragment libraries
Solution Approach 1:
The probe is divided into multiple functional segments: a target-specific binding region (20-80 nt) that hybridizes to the target sequence, and complementary pairing regions at the 5' and 3' ends that form dual structures with other probes. This segmentation allows the probe to achieve both high binding affinity and high capture efficiency through cooperative binding of multiple probes to the same target fragment.
Solution Approach 2:
The probe design combines different functional elements into a composite structure: the target-specific single-stranded DNA region provides sequence specificity, while the complementary pairing regions form double-stranded structures that enhance stability and enable probe-probe interactions. This composite structure optimizes both the on-target rate and capture efficiency simultaneously.
2Stability of the object's composition
If the probe sequence length is increased to improve binding stability, then the hybridization annealing temperature is appropriate, but the synthesis difficulty and cost increase
Solution Approach 1:
The probe design optimizes the length parameter of the target-specific binding region to 20-80 nt, which is shorter than conventional 120 nt probes but sufficient to achieve appropriate hybridization annealing temperatures and stable binding. This parameter optimization reduces synthesis difficulty and cost while maintaining the required hybridization stability through the cooperative binding mechanism of multiple probes.
3Productivity
If the probe length is shortened to increase binding capacity, then more probes can bind to short target fragments, but the hybridization annealing temperature decreases significantly
Solution Approach 1:
Multiple short probes with complementary pairing regions merge through probe-probe hybridization to form extended bound structures on the target fragment. This merging allows the system to achieve high binding capacity (multiple probes per target) while maintaining appropriate hybridization temperatures, as the cooperative binding of multiple probes compensates for the shorter individual probe length.
4Reliability
If conventional hybrid capture is performed, then target sequences can be enriched, but the operation time is long (days) and the process is complex
Solution Approach 1:
The probe design enables dynamic cooperative binding where probes can rapidly associate and dissociate, allowing multiple probes to bind to the same target fragment. This dynamic binding behavior accelerates the hybridization kinetics and reduces the time required to achieve sufficient target enrichment, while the complementary pairing regions provide the stability needed for reliable capture.
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
The new probe design significantly improves capture efficiency and on-target rates, reducing operation time from days to hours while simplifying the process, making it suitable for applications like low-frequency mutation detection and metagenomic sequencing.
Implementation Method 1
a biotin-labeled probe specifically binds to a target region in a solution
Implementation Method 2
the probe labeled with biotin and liquid phase reaction conditions of hybrid capture have a significant impact on the capture efficiency of this system
Data Source
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AI summary
The present disclosure provides a novel hybrid capture probe for enrichment of a target nucleic acid sequence. Its principle is that a probe sequence is divided into three segments: a middle segment is a target sequence binding segment; a 5' end segment of one probe can be complementarily paired with a 3' end segment of another probe, and a 3' end segment of one probe can be complementarily paired with a 5' end segment of another probe. This novel probe can bind more robustly to a target sequence, and has better effects in hybrid capture and targeted enrichment at low starting amounts or small target regions (Panels) compared with traditional hybrid capture probes.