Purification Process of Supercoiled Plasmid DNA

What is a Plasmid?

Plasmid DNA contains independently replicating genetic structures and is a standard tool in genetic engineering. It is widely utilized in molecular biology fundamental research, agricultural transgenic detection, medical diagnosis, and gene therapy.

Plasmid DNA is a type of circular double-stranded DNA molecule capable of autonomous replication outside a bacterial chromosome. It typically exists in three configurations: supercoiled, open-circular, and linear. Different types of plasmid DNA molecules vary in size, ranging from small ones of around 103 kDa to larger ones of up to 105 kDa.

In the initial purification of plasmid DNA solutions, three configurations are often mixed, along with a certain amount of proteins, RNA, endotoxins, and host genomic DNA. These impurities can affect subsequent applications, necessitating further refinement of plasmid DNA. The following classical three-step method is commonly used to obtain a large amount of highly pure plasmid DNA. First, RNA is removed using Focurose 6FF to avoid animal-derived enzyme contamination caused by RNaseA and other inconveniences. Then, plasmid DNA is separated to eliminate open-circular and linear forms. Finally, high-purity plasmid DNA is obtained through Q-column purification.

• During plasmid purification, Size Exclusion Resin is primarily employed to separate RNA from the components. This technique is based on the significant differences in molecular weight among plasmid DNA, host DNA, RNA, and other smaller molecules in the lysate. The filler material used is often Focurose 6FF, and the separation of plasmid DNA and RNA is directly influenced by the type and concentration of the salt solution used. Stadler et al. [1] compared the separation efficiency of plasmid DNA and RNA under different salt solution types and concentrations. The results showed that a sodium chloride concentration of 0.5 mol/L is needed to achieve separation, while 2 mol/L ammonium sulfate is optimal for their separation.

• Open-circular plasmid DNA has properties highly similar to those of supercoiled DNA molecules. The generation of open-circular DNA generally occurs during fermentation and lysis processes due to the breakage of one strand of supercoiled DNA. The pre-breakage state exhibits a higher degree of exposed bases and surface charge. Plasmid fillers can efficiently differentiate between these subtle differences using unique thiol affinity, making them suitable for separating different configurations of plasmids. The optimal purification conditions may slightly vary for plasmids of different sizes and sequences.

• Ion Exchange Resin is a separation technique based on the differences in charge properties of different molecules in a mixture. In addition to purifying the target molecule, it can also remove endotoxins and trace impurities.

References:

[1] Stadler J, Lemmens R, Nyhammar T. J. Gene Med, 2004, 6: S54-S66.

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