Determination of Copy Number of Foreign Gene in Genome of Pichia pastoris by Real-time Fluorescent Quantitative PCR

Objective To develop a method for determination of the copy number of foreign gene in genome of Pichia pastoris by real-time fluorescent quantitative PCR. Methods The standard curves of GAP and GFP genes were generated with the plasmids containing GAP and GFP genes respectively, and the genomic DNA of P. pastoris transformants containing GFP gene was analyzed by real-time fluorescent quantitative PCR. The copy number of GFP gene in each transformant was calculated with the Ct value of P. pastoris genomic DNA and the standard curve. Results The regression equation of Ct value and logarithm of original copy number of GAP gene was y = -3. 612x + 39. 28, while that of GFP gene was y = -3. 544x + 37. 99. The reaction efficiencies of GFP and GAP genes were 0. 89 and 0. 90 respectively. However, both the correlation coefficients of standard curves of the two genes were 0. 999, and both the curves showed good reproducibility. Of the ten P. pastoris transformants tested, nine contained one copy and one contained multiple copies of GFP gene. Conclusion A real-time fluorescent quantitative PCR method was successfully developed, which might be used for screening of P. pastoris transformants containing various copies of foreign genes.