An Easy and Accurate Agarose Gel Assay for Quantit...
An assay for quantitation of plasmid copy numbers in bacterial cell cultures has been developed and validated.The method combines isolation of total bacterial DNA（including both plasmid and genomic DNA），running a series of two-fold dilutions of total DNA in an agarose gel followed by ethidium bromide staining，and subsequent scanning of the gel picture negatives.We have developed a novel set of rules for integration of the scan data that allows us to achieve high assay precision，accuracy，and sensitivity.The assay validation results were as follows：intra-and interassay precision with%CV of 8.2—9.9 and 7.1—9.8，respectively；ruggedness with%CV of 9.3—17.5；spike recovery of 80—102；and sensitivity of 1 plasmid copy per genome.
Key Words：plasmid copy number；bacteria；DNA；agarose gel；quantitation.
Recombinant proteins produced in bacteria are commonly used as therapeutics and vaccines.Heterologous genes，encoding for recombinant proteins，are generally introduced and maintained in bacterial cells as part of the recombinant plasmid，or expression vector.Accordingly，the general strategy for achieving high yield of recombinant proteins is to select for bacterial strains，bacterial vectors，and growth conditions that confer high plasmid copy number［1，2］.In reality，however，many proteins appear to be toxic to the host bacteria when overproduced.Several studies have shown that a significant increase in expression vector copy number may be associated with decline in the growth rate［3-7］，as well as decline in the efficiency of expression of cloned heterologous genes［5-8］.To achieve a high yield of each particular recombinant protein，an optimal copy number must be found（for review see［9，10］）.
Chiron Corporation，4560 Horton Street，Emeryville，California 94608-2916
Received February 22，2000(https://www.daowen.com)
此文发表于2000年“Anal.Biochem.”284：70-76.
The quality of cell banks for recombinant protein production is also critical.A common goal of process development in biopharmaceutical production is to establish and document the cell line stability.One of the approaches to genetic stability testing of the cell banks is the plasmid copy number assay.
For these reasons，it is of imperative importance to develop reliable and accurate methods for quantitation of plasmid copy numbers.Here we present a plasmid copy number quantitation assay that is an accurate，sensitive，and reproducible assay，and is also easy to perform.The assay can be used for estimation of the plasmid copy numbers in bacterial strains under different growth conditions，and at the different stages of the cell culture growth.The assay is also suitable for detecting any changes in plasmid copy number during the storage of the cell culture.
