NEEDLS Droplet Sorting for Target DNA Enrichment
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Solution Overview
Problem
Current DNA enrichment methods require significant knowledge of the target sequence, a relatively pure sample, and a large amount of target sequence, limiting their efficiency and applicability in high-throughput sequencing applications.
Innovation Solution
The method involves diluting a DNA sample into multiple droplets, non-specifically amplifying DNA within the droplets, adding reagents for specific detection of the target sequence, and physically selecting droplets containing the target sequence, allowing for enrichment of target DNA molecules with minimal prior sequence information.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If hybrid capture, selective circularization, or PCR amplification methods are used for targeted enrichment, then target sequence enrichment can be achieved, but significant knowledge of the target sequence, relatively pure sample, and significant amount of target sequence are required
Solution Approach 1:
The method segments the DNA sample into multiple individual droplets, with each droplet containing a small number of template molecules. This segmentation allows parallel processing of many droplets, enabling enrichment without requiring significant prior sequence knowledge. The droplet compartmentalization isolates individual DNA molecules for amplification and detection
Solution Approach 2:
The invention introduces an intermediary detection step using sequence-specific probes that hybridize to target sequences within the droplets. This intermediary mechanism enables specific detection of target DNA without requiring extensive prior knowledge of the target sequence, as the probes can be designed to match known conserved regions
2Manufacturing precision
If current enrichment methods are used, then target DNA can be enriched, but the cost and effort of sequencing remain high due to low enrichment efficiency
Solution Approach 1:
The invention replaces traditional mechanical enrichment methods (hybrid capture, PCR) with a droplet-based digital approach. By partitioning the sample into thousands of droplets and performing parallel amplification and detection, the method achieves higher enrichment efficiency with reduced cost and effort, as the digital counting approach directly quantifies target molecules
Solution Approach 2:
The method changes the physical state and concentration parameters by diluting the DNA sample to achieve an average of less than 0.5 target molecules per droplet. This parameter change enables Poisson distribution-based enrichment, where the probability of droplet positivity directly reflects target abundance, significantly improving enrichment efficiency
3Loss of information
If whole-genome sequencing is performed without targeted enrichment, then complete genomic information can be obtained, but costs and effort increase significantly
Solution Approach 1:
The invention extracts and enriches only the relevant target DNA sequences from the complex genomic background by using sequence-specific detection in droplets. This extraction approach obtains complete information about the target regions of interest while discarding irrelevant genomic DNA, significantly reducing sequencing costs and effort compared to whole-genome sequencing
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 significantly reduces the cost and effort of DNA sequencing by increasing the concentration of target DNA relative to total DNA, enabling efficient enrichment and sequencing of target DNA molecules with reduced analytical burden, achieving a high degree of enrichment with minimal sequence information required.
Implementation Method 1
general amplification of the mixed DNA molecules in the multiple of droplets
Implementation Method 2
specific detection of droplets containing at least one of said target DNA molecule
Data Source
AI summary
The present invention pertains to an in vitro method in which a targeted DNA molecule containing a DNA sequence of interest is enriched by a) general amplification of DNA molecules in a multiple of droplets each containing less than 0.5 target DNA molecule on average (404), b) specific detection of the target DNA molecule in each of the droplets (405), and c) physically selecting droplets containing target DNA molecules (406).