An investigation into gene copy number determination in transgenic yeast; The importance of selecting a reliable real-time PCR standard

Abstract

Nowadays, Pichia pastoris is a well-known yeast for the production of recombinant proteins. The yield of protein production tightly depends on the copy number of the gene of interest into the host chromosome. Real-time PCR has been used as a high throughput method for molecular detection of gene copy number. In light of determining an absolute gene copy number, the reliability of the qPCR quantification standard is a major issue and it can be a potential source of errors in the final results. Since the literature on this issue is inconclusive, we set out to find a reliable quantification method that allows comparing results in different laboratories. We generated standard curves for two genomic loci (5′UTR AOX1 and ARG4) and for plasmid DNA carrying hGM-CSF coding sequence. These data was used to calculate the integrated hGM-CSFcDNA copy number in a recombinant P. pastoris clone. In our expriments the 5′UTR AOX1 gene showed a more accurate quantification standard, based on more efficient amplification and better reproducibility. The results obtained in this study showed that the differences in terms of structure and length between circular plasmid and linear gDNA could be the source of significant differences in the pattern of DNA amplification.