Bridge-Probe Library Formation for Low-Input DNA Capture
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Solution Overview
Problem
Current nucleic acid target capture methods are cumbersome, costly, and inefficient, particularly for low-input samples and bisulfite-treated DNA, with low on-target rates and low flexibility for multiplex-PCR amplicon-based target sequencing, and bisulfite conversion often damages nucleic acids, leading to low conversion rates and loss of methylation information.
Innovation Solution
A method involving hybridization of bridge probes to template nucleic acids, followed by exonuclease treatment and adaptor ligation, which enhances capture specificity and efficiency, allowing for high conversion rates and flexible, cost-effective target enrichment, even with damaged or low-input DNA samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bisulfite conversion is performed on NGS DNA library, then methylation analysis capability is improved, but nucleic acid damage increases and conversion rate decreases
Solution Approach 1:
The patent performs target capture and library construction before bisulfite conversion, rather than after. This preliminary action preserves methylation information in the original DNA while avoiding the damaging effects of bisulfite treatment on low-input samples. The method captures targets from intact DNA, then performs bisulfite conversion on the enriched material, maintaining both high conversion rates and methylation analysis capability.
Solution Approach 2:
The patent segments the workflow into distinct phases: (1) target capture from intact DNA, (2) library construction, and (3) bisulfite conversion. This segmentation allows each step to be optimized independently, preventing the compounding of damage that would occur if bisulfite conversion were performed earlier on low-input samples.
2Loss of information
If pre-bisulfite conversion capture is used, then methylation information is preserved, but high DNA input is required
Solution Approach 1:
The patent performs target capture from intact DNA before bisulfite conversion, preserving methylation information while requiring minimal input DNA. The capture probes hybridize to the original methylated DNA, enriching targets while maintaining methylation status, then bisulfite conversion is performed on the enriched material rather than the original low-input sample.
Solution Approach 2:
The patent changes the order of operations, performing capture before conversion rather than after. This parameter change in the workflow sequence allows the method to work with low-input samples while preserving methylation information, as the capture step occurs when DNA is still intact and abundant enough for effective enrichment.
3Manufacturing precision
If current target capture methods are used, then specific genes can be enriched, but protocol complexity and cost increase
Solution Approach 1:
The patent combines target capture with library construction into a unified workflow that occurs before bisulfite conversion. By merging these steps and performing them on intact DNA, the method eliminates separate post-conversion capture steps, reducing overall protocol complexity while maintaining target enrichment specificity and enabling methylation analysis.
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 method provides efficient, flexible, and cost-effective target enrichment with high nucleic acid sample complexity preservation, suitable for single-stranded, damaged, or fragmented DNA, and supports fast, easy, and practical analysis of methylation patterns.
Implementation Method 1
hybridizing a first target specific region of a first bridge probe to a first target sequence of a template nucleic acid molecule
Implementation Method 2
contacting the complex with a 3' to 5' exonuclease, wherein the 3' to 5' exonuclease cleaves the 3' end of the template nucleic acid molecule
Implementation Method 3
extending the 3' end of the template nucleic acid molecule using the adaptor sequence as a template, thereby generating a first extension product
Data Source
Figure 1A~1C
Figure 2A~2C
Figure 3A~3C
AI summary
The present disclosure provides targeted hybridization and/or proximity ligation of a probe for amplification and analysis of target sequences. The hybridization of the probe to the target sequences can be direct or through indirect association.