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.

如今，巴斯德毕赤酵母是用于生产重组蛋白的众所周知的酵母。蛋白质生产的产量严格取决于目标基因在宿主染色体中的拷贝数。实时PCR已被用作高通量方法，用于分子检测基因拷贝数。考虑到确定绝对基因拷贝数，qPCR定量标准品的可靠性是一个主要问题，它可能是最终结果错误的潜在来源。由于有关该问题的文献尚无定论，因此我们着手寻找一种可靠的定量方法，该方法可用于比较不同实验室的结果。我们为两个基因组基因座（5'UTR AOX1和ARG4）以及携带hGM-CSF的质粒DNA生成了标准曲线编码序列。这些数据被用于计算重组巴斯德毕赤酵母克隆中的整合的hGM-CSFcDNA拷贝数。在我们的实验中，基于更有效的扩增和更好的再现性，5'UTR AOX1基因显示了更准确的定量标准。这项研究获得的结果表明，环状质粒和线性gDNA之间在结构和长度方面的差异可能是DNA扩增模式出现重大差异的根源。