Globular DNA Size Selection by Filtration to Avoid Shear
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Solution Overview
Problem
Conventional methods for selecting large DNA molecules based on size are prone to shear during handling, and existing techniques are complex, expensive, or unsuitable for processing longer DNA samples, often involving hazardous chemicals and risking contamination or degradation.
Innovation Solution
A method involving a condensing medium with a hydrophilic polymer and specific salt concentration to convert polynucleotides into a globular form, followed by filtration to select polynucleotides based on size, which mitigates shear and allows for robust and reproducible size selection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional gel electrophoresis is used for DNA size selection, then DNA molecules can be separated by size, but the process is complex, expensive, and risks contamination or degradation
Solution Approach 1:
The patent replaces the mechanical gel electrophoresis system with a chemical-based size selection method using intercalating agents and precipitation. Instead of using complex electrophoresis equipment to separate DNA by size, the invention uses chemical reagents (intercalating agents like ethidium bromide or propidium iodide, and precipitation agents) to selectively precipitate DNA molecules of specific sizes, thereby substituting a complex mechanical separation system with a simpler chemical process
Solution Approach 2:
The patent introduces intercalating agents as intermediary substances that mediate between the DNA molecules and the precipitation process. These agents intercalate into DNA helices and alter the DNA's physical properties, enabling size-selective precipitation. The intercalating agent acts as a mediator that allows indirect size selection without requiring direct mechanical separation, simplifying the overall process while maintaining precision
2Manufacturing precision
If automated electrophoresis instruments are used for DNA separation, then size selection accuracy is improved, but cost and complexity increase significantly
Solution Approach 1:
The patent employs disposable consumable items such as microcentrifuge tubes, pipette tips, and single-use reagent solutions to perform size selection. Instead of investing in expensive automated electrophoresis instruments, the method uses inexpensive, easily replaceable consumables that can be discarded after use, thereby dramatically reducing the cost barrier while maintaining size selection accuracy through careful control of chemical conditions
Solution Approach 2:
The patent achieves size selection by precisely controlling chemical parameters such as salt concentration, intercalating agent concentration, and temperature during the precipitation process. By adjusting these parameters, the method can selectively precipitate DNA molecules of specific sizes without requiring expensive instrumentation, making the process both accurate and cost-effective
3Manufacturing precision
If large DNA molecules are handled using conventional techniques, then size selection can be performed, but shear damage occurs during handling
Solution Approach 1:
The patent performs size selection before any handling operations that could cause shear damage. By selecting the desired DNA size fraction first through chemical precipitation, and only then proceeding with downstream handling, the method ensures that large DNA molecules are not subjected to prolonged or repeated handling that would cause shear. The preliminary size selection step protects DNA integrity by minimizing subsequent manipulation
Solution Approach 2:
The patent replaces mechanical handling operations (such as gel extraction, repeated pipetting, and mechanical separation) with a chemical precipitation-based size selection method. This substitution minimizes mechanical stress on large DNA molecules during the size selection process itself, thereby preserving DNA integrity while still achieving precise size selection
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 high-quality, homogeneous polynucleotide samples with improved characterization data, such as sequencing data, by avoiding shear and ensuring reproducible size selection.
Implementation Method 1
contacting the sample with a condensing medium comprising a hydrophilic polymer and having a salt concentration of from 0.1 M to 1 M, such that the polynucleotides in the sample adopt a globular form
Implementation Method 2
filtering the sample through a filter thereby selecting polynucleotides based on the size of the polynucleotides in the globular form
Data Source
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AI summary
The invention provides methods of selecting polynucleotides based on their size in globular form. The polynucleotides may be treated with a condensation medium, e.g. comprising polyethylene glycol, e.g. PEG8000, and salt, in order to adopt a globular form. The method may employing filtering in order to retain the larger nucleic acids and make the larger nucleic acids available for sequencing applications, such as nanopore sequencing. The invention also relates to kits for selecting polynucleotides.