Plasmids are circular double-stranded DNA molecules within bacterial cells, independent of the chromosome. Their autonomous replication capability makes them a core carrier in molecular biology research such as gene cloning and gene editing. Bacterial lysis and plasmid DNA extraction technology, through precise disruption of bacterial structure and utilization of the physicochemical properties of nucleic acids, achieves the separation of plasmids from impurities. This is a crucial method for obtaining high-purity plasmids, laying the foundation for subsequent biological experiments.
The core principle of this technology lies in utilizing the topological differences between plasmids and bacterial chromosomal DNA. In an alkaline environment, linear chromosomal DNA double strands completely unwind and denature, while circular plasmid DNA, due to its entangled double strands, only experiences hydrogen bond breakage without complete separation. When the environment returns to neutral, plasmid DNA rapidly renatures and restores its double-stranded structure, while chromosomal DNA, due to slow renaturation, entangles with proteins, cell debris, etc., forming a precipitate, thus achieving separation.
The standard experimental procedure mainly includes four key steps. The first is bacterial culture and collection. Bacteria containing the target plasmid are inoculated into liquid culture medium, cultured overnight at 37°C with shaking, and the bacterial precipitate is collected by centrifugation. Residual culture medium is removed by washing with buffer. The next step is cell resuspension. The precipitate is thoroughly dispersed in a buffer solution containing glucose and EDTA. Glucose maintains osmotic pressure to protect nucleic acids, while EDTA chelates divalent cations to inhibit DNase activity.
Bacterial lysis is the core step. After adding a lysis buffer containing a strong alkali and detergent, it is necessary to gently invert and mix to avoid vigorous shaking that could cause chromosomal DNA breakage. The strong alkali disrupts the cell membrane and denatures nucleic acids, while the detergent dissolves membrane lipids and denatured proteins. An acidic buffer is then added to neutralize the precipitate, causing impurities to form a flocculent precipitate. Centrifugation yields a supernatant containing plasmids. Finally, RNA impurities are degraded by RNase, followed by alcohol precipitation and washing to obtain high-purity plasmid DNA.
Detailed control during the operation directly affects the extraction quality. The lysis buffer reaction time must be strictly controlled within 5 minutes to avoid irreversible denaturation of plasmid DNA; precise control of centrifugation speed and temperature can reduce nucleic acid degradation. This technology is widely used in gene cloning, recombinant protein production, and gene therapy research and development. The purity of the extracted plasmid DNA directly determines the success or failure of downstream experiments.
As a fundamental technique in molecular biology, bacterial lysis for plasmid DNA extraction, with its simple principles and high efficiency, has become an important bridge connecting basic research and biological applications, driving continuous breakthroughs in the life sciences.
