Purification of Plasmid DNA

The plasmid DNA prepared as described above still has some broken RNA fragments and polysaccharides and may contain some bacterial DNA. Various approaches exist [3,4] for the purification of plasmid DNA. 

One of the simpler means to purify plasmid is the selective precipitation of plasmid DNA by PEG (polyethylene glycol). However, the yield of DNA obtained depends on the duration of incubation with cold PEG. The DNA sample (2 mL in TE) is added to 0.8 mL of PEG solution and incubated at 0°C for at least 1 hour, preferably overnight. The DNA is centrifuged at 12,000 rpm in an SS-34 Sorvall for 15 minutes at 40°C. The pellet is dissolved in 1 mL of TE and after extraction with phenol-chloroform and chloroform, the DNA is precipitated with 2 volumes of ethanol after adjusting concentration to 0.3 M sodium acetate with a 3 M solution. The DNA pellet is washed with 70% ethanol, dried, and dissolved in TE buffer at a concentration of 1 to 3 mg/mL in TE and stored at 4°C, or at —20°C for long-term storage. 

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Toyopearl HW-75 resin, with pores larger than 1000 A, have been used in place of ultracentrifugation steps for the purification of plasmid DNA. Ultracentrifugation is a time-consuming process and requires expensive chemicals, such as cesium chloride. Toyopearl HW-75 resin provides superior separation performance for plasmid DNA and also provides high yields (54). 

Blanche et al. [45] showed that the P-CAC technology is very promising for the purification of Plasmid DNA at preparative scale especially when resins with low binding capacities for the product of interest are used. The aim of the study was to purify the Plasmid DNA out of a clear lysate of E. coli. The lysate containing RNA, nicked DNA, as well as the Plasmid DNA was loaded onto the annular column filled with Poros 20 R2 beads as the stationary phase. The chromatographic process for the purification is shown in Fig. 7. 

Vo-Quang, T., Malpiece, Y., Buffard, D Kaminski, P. A., Vidal, D., and Strosberg, A. D. (1985) Rapid, large-scale purification of plasmid DNA by medium or low pressure gel filtration. Application construction of thermo-amplifiable expression vectors. Biosci. Rep. 5, 101-111. 

Interest in producing large quantities of supercoiled plasmid DNA has increased as a result of gene therapy and DNA vaccines. Due to the commercial interests in these approaches, the development of production and purification strategies for gene therapy vectors has been performed in pharmaceutical companies within a confidential environment. It is thus important to describe the downstream operations for the large-scale purification of plasmid DNA to attain a final product that meets specifications and safety requuements. " ... 

Figure 8.3 shows the process flow sheet for large-scale purification of plasmid DNA. Table 8.14 presents a comparison of laboratory methods and laige-scale pharmaceutical processes for plasmid DNA purification. 

Gnerrero-German et al. [288] developed a new bioprocess for the purification of plasmid DNA from E. coli ferments nsing a hollow-fiber tangential filtration (as a pretreatment of the feed solution) and tandem anion-exchange membrane chromatography as a second step. 

The latter class of bioorganic materials has gained momentum during the recent years, mainly because many functions have been implemented and potential applications realized [4]. The combination of nucleic acids with conjugated polymers [5] or electroactive macromolecules [6] has resulted in highly sensitive and selective probes, whilst equally important are polynucleic acids that are coimected covalently to thermoresponsive polymers such hybrids have been used successfully for the purification of plasmid DNA [7] and DNA-binding proteins [8]. 

Most of the published studies have used probes which detect all mRNA isoforms of a given RAR or RXR. It is also possible to generate isoform-specific riboprobes by limiting the template DNA to the specifically spliced region. In situ detection of individual RAR (RXR) isoforms poses problems, however, because of limitation in the nucleotide length of isoform-specific probes (e.g., 400-500 nt in the case of RAR isoforms), and in the very low abundance of individual mRNA isoforms Nevertheless, there have been recent reports of ISH detection of RARP isoforms in chick embryos (17,18) and RARy isoforms can be specifically detected on cryosections of mouse embryos (unpublished data) Detailed procedures for cloning, amplification, and purification of plasmid DNA, which are beyond the scope of this chapter, can be found in refs. 20 and 21... 

Branovic, K. et al. Application of short monolithic columns for fast purification of plasmid DNA. Journal of Chromatography, B Analytical Technologies in the Biomedical and Life Sciences, 2004 801(2) 331-337. 

Kepka, C. et al.. Integrated process for purification of plasmid DNA using aqueous two-phase systems combined with membrane filtration and hd bead chromatography. Journal of Chromatography, A, 2004 1057(1-2) 115-124. 

Kostal, J., A. Mulchandani, and W. Chen, Affinity purification of plasmid DNA by temperature-triggered precipitation. Biotechnology and Bioengineering, 2004 85(3) 293-297. 

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