Full Length Research Paper

The technology of large-scale pharmaceutical plasmid purification by cetyltrimethylammonium bromide and Tritonx-114

Zhang, Q.¹, Yuan, W. F.¹, Sun, H. C.¹, Hou, S. H.² and Zhu, H. F.²*

¹Collge of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China.

²Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences, Beijing 100081, China.

*Corresponding author. E-mail: zqyy123@yahoo.com.cn. Tel.: +86 010 82106037. Fax: 86 010 82106855.

Accepted 27 February, 2009

   Abstract

Many methods for plasmid purification have been developed, and the whole process must be designed to remove the host RNA, protein, genomic DNA and endotoxin. Currently, plasmid is mostly purified by time-consuming chromatographies. As an alternative, a new plasmid purification technology with cetyltrimethylammonium bromide (CTAB) is described. After lysis with alkali, the CTAB was directly titrated into the supernatant for plasmid precipitation, then the coprecipitated pellets were dealt with 3 M KAc and TritonX-114. Quality detection showed that the purified plasmids were free from the contamination of host RNA. In 1 mg purified plasmid, the bacterial genomic DNA, host endotoxin and protein were less than 10 μg/ mg, 50 EU/ mg and 10 μg/mg, respectively. The ratio of OD₂₆₀/OD₂₈₀ was between 1.75 - 1.85, more than 90% of the prepared plasmid presented in the supercoiled form. Further test demonstrated that the pcDNAlacZ purified with CTAB and authoritative endotoxin-free plasmid Kit had the similar transfection efficiency in vivo and in vitro. CTAB can be used for plasmid purification; the main advantages of the DNAs purified with CTAB include the avoidance of animal-derived enzymes, toxic substance like chloroform and phenol. More attractive is that the whole process has the predominance of low cost.

Key words: Plasmid, CTAB, TritonX-114, genomic DNA, RNA, endotoxin, protein, transfection.