Taq polymerase

cDNA was amplified with Taq polymerase (Thermo Fisher, USA) using the primers in Table 1.
Each reaction was prepared with 3 μL of 10x Taq buffer reaction, 2.5 μL of 25 mM MgCl₂, 0.1 μL of Taq polymerase, 0.5 μL of 10 mM dNTPs, 0.2 μL of the primer of interest, and 1.5 μL of cDNA for a final volume of 20 μL with water free of RNases. The amplification was performed in a thermocycler (PTC-200 Peltier Thermal Cycler ALO28386) as follows: 2 minutes at 94°C, followed by 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 60 s and 10 minutes at 72°C. The PCR products were separated on a 1.5% agarose gel at 90 to 100 volts.

Standard and nested PCR mix contained 1x Taq Polymerase buffer, 1.5 to 2.5 mM MgCl₂ (MgCl₂ concentration was optimized for every PCR), 200 μM deoxynucleotide triphosphate (dNTP), 2.5 U of Taq Polymerase (all from Thermo Fisher Scientific, Waltham, MA), and 200 nM of each primer (IDT Technologies). For standard PCR, 100 ng of DNA from samples or control cell lines or 5.5 × 10⁻⁵ ng from control plasmid (equivalent to the number of moles contained in 100 ng of genomic DNA) were used. Plasmids were linearized with restriction enzyme NdeI (New England BioLabs) before use and mixed with DNA from Daudi cell line in order to run the amplification reaction under identical mass/volume DNA concentration for both samples and controls. For the nested PCR, 0.5 or 0.05 μL (1 : 100 or 1 : 1000 dilution resp.) of product of first round PCR was used as template. All PCR reactions were carried out in a final volume of 50 μL. All primer sequences and cycling conditions used are detailed in Table 2. PCR products were analyzed by electrophoresis in 1.8% agarose gels stained with ethidium bromide and photographed under ultraviolet light using the Quantum ST4 System (VilberLourmat, Torcy, Marne-la-Vallée, FR). The viral identity of PCR amplified fragments was confirmed by sequencing.

E. curvula SSR markers previously developed by Garbus et al. (2017) (link) were used for genotyping of the mapping population; the primers are listed in Supplementary Table S1. PCR was performed in a final volume of 20 μl containing 1X Taq polymerase reaction buffer, 2.5 mM MgCl₂, 0.125 mM of each dNTP, 1 μM of each primer, 50 ng of genomic DNA, and 2 U of Taq polymerase (Invitrogen, Brazil). The PCR program consisted of an initial denaturation at 94°C for 4 min, 35 cycles of 94°C for 30 s, 58°C for 1 min, and 72°C for 5 min, and a final extension at 72°C for 5 min. The PCR was performed in a thermocycler (MJ Research). Samples were mixed (2:1, v/v) with denaturing loading buffer (95% formamide and bromophenol blue), denatured at 95°C for 5 min, chilled on ice, and resolved in 6% (w/v) silver-stained polyacrylamide gels.

Selected fragments were amplified by PCR using Taq polymerase (Invitrogen) in a Biometra T Professional Gradient 96 cycler. Amplification mixtures contained 10 pmol of each primer, PCR buffer (Invitrogen), 1.5 mM MgCl², 50 ng of genomic DNA, 200 µM dNTPs, 2.5 U Taq polymerase (Invitrogen), and water to a final volume of 25 µl. After denaturing at 94°C for 2 minutes, thermal cycling was performed for 35 cycles at 94°C for 30 seconds, followed by 30 seconds at a temperature set to 5°C less than the melting temperature of the selected primers, followed by 72°C for 30 seconds. Reactions were finished by a 5 minute incubation at 72°C. Amplification products were checked in 1.2% agarose gels stained with ethidium bromide to verify the presence of a single amplification product. Next, an aliquot (10 µl) of the amplification reaction was treated with 1 U of Exonuclease I (Fermentas) and 10 U of Shrimp Alkaline Phosphatase (Fermentas) for 45 minutes at 37°C and then for 30 minutes at 80°C to inactivate these enzymes. Subsequently two sequencing reactions were prepared, each with one of the primers used for the amplification of the product. Sequencing was carried out in an Applied Biosystems 3130 capillary sequencer using a Big-Dye terminator cycle sequencing kit, according to the instructions of the manufacturer.

HPV16 and HPV18 genotyping was performed by PCR for all HPV-positive tumors. Two sets of primers that amplify part of E7 region of each HPV genome were employed.²¹ (link) For the HPV16 genome, the amplicon presents 108 pb, and for HPV18 genome amplification, the amplicon presents 104 pb. PCR with HPV16 primers was performed in a final reaction volume of 25 μL, with 2 μL of purified DNA, 2.5 mM/L MgCl₂, 2 mM/L of each dNTPs, 2.5 μM/L of each oligonucleotide primer, 0.25 U Taq polymerase (Invitrogen, Brazil), and ultra pure water in sufficient quantity for the final volume. Cycling conditions included: preheating for 1 min at 94 °C, followed by 40 cycles of: 94 °C for 1 min, 1 min at 45 °C, and 1 min at 72 °C, with a final extension of 5 min at 72 °C. PCR with HPV18 primers was performed in a final reaction volume of 25 μL, with 5 μL of purified DNA, 2.5 mM/L MgCl₂, 2 mM/L of each dNTPs, 2.5 μM/L of each oligonucleotide primer, 1.25 U Taq polymerase, (Invitrogen, Brazil) and ultra pure water in sufficient quantity for the final volume. Cycling conditions included: preheat for 3 min at 94 °C, followed by 35 cycles of: 1 min at 94 °C, 1 min at 53 °C, and 1 min at 72 °C, with a final extension of 3 min at 72 °C.

To quantify the growth of P. cajani within the host tissue under different experimental conditions (E1, E2 and E3), the real-time PCR was carried out. Three pairs of sequence specific primers were designed from internal transcribed spacer (ITS) sequences of P. cajani using IDT Primer Quest software (eu.idtdna.com/Primerquest/Home/Index). ITS sequences (Acc nos. KJ010534-KJ010538) were aligned using BioEdit v. 7.2.5. and primers were designed from conserved region of the ITS sequences. To test the specificity of the primers pairs, PCR was carried out separately with the individual primers sets using pure gDNA of P. cajani in 50 μL reaction mixture containing: 5 μL of 10 X Taq polymerase buffer, 1.5 μL 50 mM MgCl₂, 1 μL of 10 mM each dNTP, 1 μM of each primer, 1 μL of 5u/1 μL Taq polymerase (Invitrogen, USA), about 100 ng of gDNA and H₂O up to 50 μL. Reaction conditions were: 94°C for 4 min, (94°C for 45 s, 60°C for 45 s and 72°C for 30 s) × 35 cycles followed by incubation at 72°C for 10 min. The amplified products were separated by 1.5% (w/v) gel electrophoresis and sequenced. The expected size of PCR amplicons was constantly generated from the real-time PCR analysis.