Fast and Accurate Method for Quantitating E. Coli Host-Cell DNA Contamination in Plasmid DNA Preparations

G.J. Smith, M. Helf, C. Nesbet, H.A. Betita, J. Meek, F. Ferre
1999 BioTechniques  
Plasmid DNA is being used successfully as a gene delivery vector in a variety of clinical applications. Similar to other pharmaceutical products for clinical use, the plasmid vectors must meet rigorous purity standards. One important contaminant is the DNA of the host cell used to produce the plasmids. We have developed a new method to accurately quantitate E. colihost-cell DNA in plasmid preparations. This method is based on kinetic PCR using the ABI P RISM ® 7700 with 23S rDNA as a target.
more » ... DNA as a target. This precise assay is significantly faster and has a lower limit of quantitation than the currently used Southern-based methods. Figure 4. Southern analysis of plasmid samples. (A) A Southern slot blot of E. coli standards (10 pg through 1 ng), plasmids p3-p7 and a negative control. Each sample was prepared in four different manners as indicated by the key. The Plasmid Safe DNase selectively digests the linear E. coli DNA. Samples refa and refb represent plasmid DNA calibrators (B) E. coli DNA standard curve. The standard curve was generated by densiometric analysis from the blot in Panel A. The equation of the curve is y = 1076.8 × + 35.363 and has an r 2 value of 0.9965.
doi:10.2144/99263rr03 pmid:10090994 fatcat:wuv2caxmp5agxpyibycqqpripe