High-Purity DNA Fragment Preparation With Controlled Molecular Weight
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Solution Overview
Problem
Existing methods for preparing DNA fragment mixtures struggle to achieve high purity and safe reduction of molecular weight due to ineffective impurity removal and the use of hazardous chemicals like strong acids and bases.
Innovation Solution
A method involving the use of isopropanol for impurity removal and magnesium chloride for molecular weight reduction, including steps such as treatment with sodium chloride and sodium lauryl sulfate, precipitation with ethanol, and viral inactivation, to produce a high purity DNA fragment mixture with a controlled molecular weight range.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If ultrafiltration membrane is used for nucleic acid extraction, then extraction can be performed, but high-purity nucleic acids cannot be obtained due to ineffective removal of impurities such as RNA, protein, fat, and moisture
Solution Approach 1:
The patent changes the chemical parameters of the extraction system by introducing isopropanol and magnesium chloride at specific concentrations and ratios. Isopropanol is added to precipitate impurities while magnesium chloride facilitates DNA fragmentation and purification. These parameter changes enable effective removal of RNA, protein, fat, and moisture impurities that ultrafiltration alone cannot remove, thereby achieving high-purity DNA fragment extraction.
Solution Approach 2:
The patent introduces isopropanol as an intermediary substance to mediate between the DNA sample and impurities. Isopropanol acts as a precipitating agent that selectively precipitates impurities while allowing DNA to remain in solution or form a separable precipitate. Magnesium chloride serves as another intermediary that facilitates DNA fragmentation and enhances the selectivity of the extraction process, enabling effective separation of DNA from contaminants.
2Manufacturing precision
If strong acids and strong bases are used to reduce DNA molecular weight, then molecular weight reduction can be achieved, but the process becomes difficult and dangerous
Solution Approach 1:
The patent fundamentally changes the chemical parameters from extreme pH conditions to physiological conditions. Instead of using strong acids or bases, the patent employs magnesium chloride at controlled concentrations (typically 0.1-1.0 M) at neutral or near-neutral pH. This parameter change achieves DNA fragmentation through magnesium ion-mediated strand breakage while maintaining safety and ease of handling. The process becomes simple and safe while still achieving the desired molecular weight reduction to produce DNA fragments in the desired size range.
3Productivity
If conventional methods are used for DNA fragment preparation, then basic extraction can be performed, but high yield and purity with safe molecular weight reduction cannot be achieved simultaneously
Solution Approach 1:
The patent merges multiple functions into a single integrated process using magnesium chloride. Magnesium chloride simultaneously performs DNA fragmentation, impurity removal enhancement, and molecular weight control in one step. This merging of functions achieves high yield and purity while maintaining safety, as the magnesium chloride-based process eliminates the need for separate hazardous steps involving strong acids or bases. The combined approach improves productivity while ensuring reliable and safe DNA fragment preparation.
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
Effectively removes impurities like moisture, blood, and fat, and safely reduces DNA molecular weight to a narrow range of 1,000 to 10,000 KDa, enhancing the purity and consistency of the DNA fragment mixture.
Implementation Method 1
a step of treating milt with isopropanol to obtain milt with impurities removed
Implementation Method 2
a step of adding a sodium lauryl sulfate aqueous solution to the sodium chloride aqueous solution containing milt to obtain a primary milt lysate solution in which cells were decomposed and nucleic acids were extracted
Implementation Method 3
a step of precipitating the primary milt lysate solution with ethanol and separating the precipitate to obtain a secondary milt lysate solution
Implementation Method 4
a step of adding sodium chloride to the secondary milt lysate solution, and firstly inactivating virus at a high temperature of 80 to 100° C.
Implementation Method 5
a step of adding magnesium chloride to the virus-inactivated milt lysate solution to obtain a DNA fragment mixture with reduced nucleic acid molecular weight
Implementation Method 6
a step of secondarily inactivating virus by treating the DNA fragment mixture with reduced nucleic acid molecular weight with ethanol, and then obtaining a precipitate
Data Source
AI summary
The present invention relates to a method for preparing a high purity DNA fragment mixture using isopropanol and magnesium chloride, wherein impurities such as moisture, blood, and fat are effectively removed and the purity of the DNA fragment mixture increases using isopropanol, and a DNA fragment mixture having a relatively narrow molecular weight range of 1,000 KDa to 10,000 KDa can be prepared using magnesium chloride.