Determination of copy number of Textilinin-1 gene in recombinant Pichia pastoris by fluorescence quantitative PCR

Abstract

Objective To detect the copy number of plasmin inhibitor gene Textilinin-1 in Pichia pastoris by fluorescence quantitative PCR. Methods The standard curves of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Textilinin-1 genes were generated with the plasmids containing GAPDH and Textilinin-1 genes, respectively, and the genomic DNA of Pichia pastoris transformants containing Textilinin-1 gene was analyzed by fluorescence quantitative PCR. The copy number of Textilinin-1 gene in each transformant was calculated according to the double standard curves. Results The reaction efficiencies of GAPDH and Textilinin-1 genes were 95.04% and 96.36%, respectively. The coefficients of determination for standard curves of two genes were both 0.999, and both curves showed good reproducibility. Of the 10 transformants tested, the copy number of Textilinin-1 gene was 2. Conclusion A fluorescence quantitative PCR method is successfully developed and it is useful for detecting the copy number of Textilinin-1 gene in recombinant Pichia pastoris. Key words: Fluorescence quantitative PCR; Pichia pastoris; Gene copy number