A cautionary tale of cross-contamination among plasmids from commercial suppliers

Abstract

Many researchers have switched to purchasing their desired plasmids from commercial suppliers to save time and resources, as we did for 17 high-risk human papillomavirus plasmids. To our surprise, they were shown to be cross-contaminated with one another. Comparison between the production schedule and the pattern of contaminations proved that this contamination occurred during the production process, which was also shown for another two sets of commercial plasmids. Our experience indicates that the absolute purity of plasmids obtained from external sources cannot be guaranteed. Extreme caution should be exercised, especially when such plasmids are used for human gene therapies and DNA vaccines, where even a minute amount of contamination may pose significant risks to patients. METHOD SUMMARY Cross-contamination among commercial plasmids was discovered by performing real-time quantitative PCR reactions specific to individual plasmids on all plasmids, and the identities of the suspected contamination determined by Sanger sequencing of the PCR products and subsequent BLAST searches with the sequencing results as query sequences. The contaminated plasmids were transformed into Eschericia coli competent cells and single transformants were amplified by overnight culture for plasmid repurification. To minimize the possibility of cross-contamination, all equipment used for plasmid purification was thoroughly cleaned both before and after purification, and only one plasmid was repurified each day.