Background of the Invention spin

Plasmids are double-stranded supercoiled DNA molecules that range in size from 1 kb to more than 200 kb. Plasmids are useful tools in genetic engineering. They are widely used as vectors to carry foreign DNA; such that the foreign DNA is amplified and isolated or expressed. Plasmid DNA has also been utilized in the development of vaccines and in gene therapy.

The analysis and in vitro manipulation of plasmid DNA is typically preceded by an isolation step in order to free the nucleic acid from unwanted cellular contaminants which may interfere with subsequent processing procedures. A mini-scale sample preparation from an overnight bacterial culture of 1-5 ml generates more than enough plasmid DNA (~ few micrograms) for many of these applications.

The most common plasmid DNA extraction protocols exploit reagents originally developed by Birnboim and DoIy (Birnboim, H. C. and DoIy, J., Nucl. Acids Res. 7, 1513

(1979)), to separate supercoiled plasmid DNA from bacterial genomic DNA, RNA and protein. These reagents, developed many years ago, work on the principle of sequential use of three buffers, commonly referred to as buffer I, II and III. They each have distinct compositions to bring about plasmid enrichment and separation from contaminants. Buffer I is used to resuspend the bacterial pellet obtained by an initial centrifugation step of an appropriate bacterial culture. Once resuspended, buffer II is added which contains SDS detergent and NaOH. These components lyse the bacteria and denature the genomic DNA (pH>12). Buffer III typically contains a chaotrope to further denature protein, the chaotrope also promotes binding of plasmid DNA to the silica matrix commonly used in spin columns. Buffer III also usually contains potassium acetate to rapidly neutralize the combined solutions. The addition of buffer III causes contaminants to "crash-out" of solution owing to the formation of insoluble complexes driven by rapid re-naturation of genomic DNA and the potassium salt of the detergent.

The insoluble flocculant material has traditionally been removed by a centrifugation step to "pack" the flocculant material at the bottom and side of a centrifugation tube (See, e.g. ILLUSTRA™ plasmidPrep Mini Spin Kit, GE Healthcare, Piscataway, New Jersey). The clarified plasmid-containing solution is subjected to a chromatographic separation. For mini-scale purification, the clarified solution is usually applied to a minispin column containing a glass fiber matrix or silica membrane. Plasmid DNA binds to the column in the presence of a chaotrope, while soluble impurities do not bind. After the soluble impurities are washed off, the plasmid DNA are eluted with an appropriate elution buffer.