Preparative Electrophoresis for Targeted Genomic DNA Purification
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Solution Overview
Problem
Current targeted sequencing methods require large amounts of nucleic acid reagents and are inefficient for analyzing long DNA molecules, making them unsuitable for studying large genomic regions, long-range genome structure, and rearrangements.
Innovation Solution
A method involving high-molecular-weight DNA entrapment in a gel matrix, followed by electrophoretic separation and treatment with specific DNA cleavase reagents like CRISPR/Cas9 to release targeted DNA fragments, allowing for efficient purification and isolation of specific DNA fragments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If hybridization capture or amplicon library construction methods are used for targeted sequencing, then specific genomic regions can be enriched, but large quantities of nucleic acid reagents are required
Solution Approach 1:
The invention extracts and isolates only the specific DNA fragments of interest from the complex genomic DNA mixture using preparative electrophoresis, eliminating the need for large amounts of hybridization probes or PCR reagents. The gel matrix physically separates and captures target fragments based on size, allowing direct extraction of purified DNA without requiring extensive reagent interactions.
Solution Approach 2:
The gel matrix serves as an intermediary medium that facilitates the separation and purification of DNA fragments. By embedding DNA in the gel and applying electrophoretic fields, the system mediates the separation process physically rather than relying on chemical interactions with large amounts of probes or primers.
2Reliability
If conventional DNA extraction and PCR methods are used, then targeted regions can be amplified, but DNA fragments greater than 50-100 kb cannot be reliably produced
Solution Approach 1:
The invention segments the complex process of DNA analysis into distinct phases: (1) extraction of intact high-molecular-weight DNA, (2) electrophoretic separation of specific size ranges, and (3) isolation of target fragments. This segmentation allows each step to be optimized independently, enabling reliable handling of very large DNA molecules without the limitations of PCR-based methods.
Solution Approach 2:
The invention replaces the biochemical amplification system (PCR) with a physical separation system (preparative electrophoresis). Instead of relying on enzymatic amplification that fails for very large fragments, the system uses electrophoretic mobility differences to physically separate and isolate intact large DNA fragments, achieving reliable results for fragments up to several megabases.
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
Enables the isolation of long DNA fragments up to several megabases in length, reducing the need for large reagent quantities and improving the analysis of complex genomic regions and rearrangements.
Implementation Method 1
entrapping the sample in a gel matrix wherein the HMW DNA has extremely low electrophoretic mobility
Implementation Method 2
subjecting the gel matrix with the entrapped sample to an electrophoretic field such that sample contaminants and lysis reagents are removed from the gel matrix
Implementation Method 3
subjecting the gel matrix with the entrapped purified DNA to treatment with DNA cleavase reagents configured to cleave at specific DNA sequences within the sample DNA
Data Source
AI summary
A sample containing particles having high-molecular-weight (HMW) DNA is entrapped in a gel matrix, and the gel matrix is exposed to a lysis reagent configured to release the HMW DNA from the particles. The HMW DNA may be purified by subjecting the gel matrix to an electrophoretic field that removes the HMW DNA from the particles, lysis reagents, and/or other sample constituents, from the gel matrix such that the HMW DNA remains. The gel matrix may be subjected with DNA cleavase reagents configured to cleave at specific DNA sequences within the HMW DNA to liberate defined segments of the DNA as fragments of reduced size. The gel matrix may also be subjected to an electrophoretic field, which moves and separates the DNA fragments from uncleaved DNA of the HMW DNA, which remains substantially immobile. The electrophoretically separated DNA fragments may be isolated from the gel matrix.