Polymerase Chain Reaction (PCR), Reverse Transcription (RT) and Quantitative PCR (qPCR)

All oligonucleotides designed and used in this work were synthesized by Integrated DNA Technologies (Coralville, IA, United States) and are listed in Supplementary Table S1.

To amplify the upstream and downstream fragments of the ERG3 and ERG4 genes, PCR analyses were performed using Pfu DNA polymerase (Agilent Technologies, Santa Clara, CA, United States). For E. coli recombinant clone selection and X. dendrorhous transformant confirmation, Taq DNA polymerase (Centro de Biotecnología, Facultad de Ciencias, Universidad de Chile, Chile) was used. PCR reactions were performed in a final volume of 25 μl using an Applied Biosystem 2720 thermocycler with the following program for standard PCR: initial denaturation at 95°C for 3 min (or 5 min in case of colony PCR or amplification with Pfu DNA polymerase), 35 cycles of denaturation at 94°C for 30 s, alignment of primers at 55°C for 30 s, and elongation at 72°C for 3 to 4 min followed by a final elongation step at 72°C for 10 min, and finally, the reaction was maintained at 4°C until analysis was performed.

Overlap extension PCR (OE-PCR) (Urban et al., 1997) was used to join the upstream and downstream DNA fragments of both studied genes, and the reaction was performed in a final volume of 25 μl with 1 U of Pfu DNA polymerase, PCR buffer 1X, each dNTP at 200 μM and 100 ng of each DNA fragment. The following program was used: initial denaturation at 94°C for 1 min, then 10 cycles of denaturation at 94°C for 30 s, DNA alignment at 55°C for 45 s, elongation at 72°C for 90 s, then a final elongation step at 72°C for 10 min, and finally the reaction was kept at 4°C until the next step. Then, the obtained reaction mixture was amplified by a standard PCR analysis with Pfu DNA polymerase and the same forward and reverse primers used to amplify the upstream and downstream DNA fragments, respectively.

RT reactions were performed in a final volume of 20 μl. First, 5 μg of total RNA in 11 μl of sterile water was mixed with 1 μl of oligo-dT (25 μM) and 1 μl of dNTPs (at 10 mM each) and incubated at 65°C for 5 min. Then, 1 μl of the enzyme Maxima Reverse Transcriptase (Thermo Fischer, Waltham, MA, United States), 4 μl of RT buffer 5X and 2 μl of DTT (0.1 M) were added. The mixture was incubated at 37°C for 52 min and then left at 70°C for 15 min, enabling it to cool at 4°C.

Real-time qPCR reactions were performed in a Mx3000P real-time PCR system (Agilent, Santa Clara, CA, United States) using Fast EvaGreen qPCR Master Mix (Bio-Rad Laboratories, Hercules, CA, United States). Samples were prepared in a final volume of 20 μl containing 1 μl of cDNA, 0.25 μM of each primer and 10 μl of kit reaction mixture. The efficiency of the primers used was greater than 95%, as determined by standard curves with a correlation coefficient R² ≥ 0.99. The obtained Ct (threshold cycle) values were normalized with respect to the corresponding value of the X. dendrorhous β-actin gene [GenBank: {"type":"entrez-nucleotide","attrs":{"text":"X89898.1","term_id":"1150539","term_text":"X89898.1"}}X89898.1] and expressed as fold change with respect to the control (Livak and Schmittgen, 2001).