Precipitation of RNA impurities with high salt in a plasmid DNA purification process: Use of experimental design to determine reaction conditions

Abstract

The use of high salt solution to precipitate RNA in a pharmaceutical-grade plasmid DNA purification process was investigated. Five antichaotropic salts were tested for their potential to precipitate RNA. Calcium chloride was by far the best precipitant with high RNA removal in a very short incubation time. Calcium chloride precipitation conditions were investigated at two stages of a plasmid purification process using experimental design techniques. The effect of up to five factors on RNA precipitation and plasmid recovery was assessed by statistical modeling. Optimized conditions for calcium chloride precipitation were then introduced to the plasmid purification process resulting in the efficient removal of most impurities (RNA, chromosomal DNA, proteins, and endotoxins).

Downstream Processing of Plasmid DNA for Gene Therapy and Genetic Vaccination

“…Therefore, the quantification of nucleic acids in the cleared lysate is necessary in order to determine the appropriate precipitation conditions. Nevertheless, selective precipitation of RNA could be demonstrated [29]. By using calcium chloride as a precipitation agent, the authors were able to remove a large quantity of the contaminating RNA and keep loss of product low.…”

Harnessing metal ion affinity for the purification of plasmid DNA

“…For affinity precipitation, the use of compacting agents, such as spermine (Mourich et al, 2003;Murphy et al, 1999), spermidine (Murphy et al, 1999;Stepanov and Nyborg, 2003), high concentration salt (e.g. 2.5-5 M LiCl (Mourich et al, 2003;Stepanov and Nyborg, 2003), 1.4 M calcium chloride (Eon-Duval et al, 2003) and 2.5 M ammonium sulphate (Diogo et al, 2000)) and polycation (Wahlund et al, 2004), to selectively isolate pDNA or impurities (e.g. RNA and endotoxin) from the alkaline cell lysate was reported.…”

A high-salinity solution with calcium chloride enables RNase-free, easy plasmid isolation within 55 minutes

“…Calcium chloride (CaCl 2 ) is an effective reagent that selectively removes RNA from a mixture of DNA and RNA (12,13). That is, RNA can be precipitated by centrifugation in the presence of CaCl 2 (RNase is not needed).…”