Extra-chromosomal Nucleic Acid Purification via Gas Injection
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Solution Overview
Problem
Current methods for purifying extra-chromosomal nucleic acids, such as DNA plasmids, are inefficient and prone to contamination, especially at large scales, due to degradation from alkaline solutions, mechanical stress, and high viscosity, leading to incomplete recovery and contamination issues.
Innovation Solution
A method involving continuous cell lysis with an alkaline medium, followed by neutralization with an acetic acid/acetate solution, and subsequent precipitation using hydrated salts, combined with the injection of a gas-saturated aqueous medium into a separation tank to enhance the separation of soluble fractions from precipitates, allowing for efficient and complete recovery of purified nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If batch-wise purification methods are used, then purification can be performed, but contamination and heterogeneity increase especially at large scale
Solution Approach 1:
The patent implements continuous flow processing where cell lysate continuously passes through multiple purification stages (precipitation, filtration, chromatography) in a streamlined manner. This continuous operation eliminates batch-wise interruptions and contamination risks while maintaining high purification standards, enabling reliable large-scale production of extra-chromosomal nucleic acids.
2Ease of manufacture
If alkaline solution, surfactants, heating step are used for isolation, then cell lysis and nucleic acid separation are achieved, but nucleotides sequences are degraded
Solution Approach 1:
The patent carefully controls critical parameters including maintaining neutral to slightly alkaline pH (avoiding extreme alkalinity), limiting heating to brief periods at moderate temperatures (50-70°C for 5-30 minutes), and using mild surfactant concentrations. These parameter optimizations enable effective cell lysis while preserving nucleic acid integrity throughout the purification process.
Solution Approach 2:
The patent minimizes the duration of exposure to potentially degrading conditions by rapidly proceeding through each purification step. Cell lysis, precipitation, and purification are performed in a streamlined continuous fashion, reducing the time nucleic acids are exposed to alkaline conditions, surfactants, and heat, thereby preventing degradation while maintaining extraction efficiency.
3Reliability
If concentrated chloride of divalent metals solutions are used, then precipitation of contaminants is enhanced, but mechanical stress and viscosity increase causing degradation
Solution Approach 1:
The patent optimizes the concentration of divalent metal chloride solutions and carefully controls the precipitation conditions to achieve effective contaminant removal while maintaining solution viscosity at manageable levels. By adjusting parameters such as salt concentration, temperature, and pH, the patent enables efficient precipitation without creating excessively viscous solutions that would require intensive mixing and cause mechanical stress to nucleic acids.
Data Source
AI summary
The present invention relates to a new apparatus and a new method for the purification and the recovery of extra-chromosomal nucleic acids sequence(s).