A quantitative method for the determination of plasmid copy number in recombinant yeast.
A method is described for the determination of plasmid copy number (PCN) in the recombinant yeast Saccharomyces cerevisiae producing human coagulation Factor XIII. Southern hybridization of restriction endonuclease HindIII-digested total DNA from yeast transformant with the 32P-labelled URA3 gene as a probe detected two band: one corresponding to the chromosomal URA3 gene and the other corresponding to the recombinant plasmid. Thus, the present method using the genomic URA3 gene as an internal standard allowed an estimation of both the plasmid and the standard simultaneously. Since haploid yeast cells harbour one copy of genomic URA3 gene, the PCN can be determined as the ratio of the amount of plasmid to that of genomic URA3 gene. A linear relationship between the determined PCN and the plasmid content was observed ranging from 0.3 to 200 ng. The method is reproducible and independent of the extraction efficiency for the recombinant plasmid. The present method was successfully used to analyzed the PCN of a centromere-based plasmid YCp50 (= 2.7) and of a 2 microns-based plasmid pEMBLyex4 (= 112).
Fujimura
H
H
Laboratory of Drug Creation Research, Hoechst Japan Limited, Kawagoe, Japan.
Sakuma
Y
Y
Weimer
T
T
D004274:DNA, Recombinant; D005176:Factor XIII / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D006801:Humans; D010957:Plasmids; D011995:Recombination, Genetic; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D004274:DNA, Recombinant; D005656:Fungal Proteins; D005176:Factor XIII