Pyromark annealing buffer

The biotinylated PCR products of the human PRNP gene were amplified by nonbiotinylated forward and biotinylated reverse primers. The volume of the total PCR mixture was 80 µL, including 2 µL Streptoavidin Sepharose High Performance Medium (GE Healthcare, Chicago, IL, USA), 40 µL PyroMark Binding Buffer (QIAGEN, USA), 8.5 µL high purity autoclaved water and 10 µL PCR products, and shaken for 15 min at 14,000 rpm. The PCR products were washed sequentially with 70% ethanol for 10 sec, PyroMark Denaturation Buffer (QIAGEN, USA) for 10 sec and PyroMark Wash Buffer (QIAGEN, USA) for 10 sec and then mixed with 24.2 µL Pyromark annealing Buffer (QIAGEN, USA) and 0.8 µL sequencing primer (2 µM). Finally, the samples were incubated in a PyroMark Q25 plate holder preheated to 80 °C for 2 min. Prepared samples were loaded into PyroMark Q24 (QIAGEN, USA) and operated in allele quantification (AQ) mode according to the manufacturer’s protocol.

Bisulfite pyrosequencing was performed as described previously [26 (link)]. Briefly, bisulfite-treated DNA was amplified using a PyroMark PCR kit (Qiagen, Hilden, Germany). PCR and cycling conditions were according to the kit manual. All pyrosequencing primers (PCR primers and sequencing primers) were based on the selected candidate 450 K array CpG probe using PyroMark Assay Design software (Qiagen). The amplification protocol was performed according to Collela et al. [27 (link)] using a universal primer approach. The biotinylated PCR products were captured using 1.0 μl streptavidin-coated sepharose high-performance beads (GE Healthcare, Little Chalfont, UK). The immobilized products were washed with 70 % alcohol, denatured with PyroMark denaturation solution (Qiagen), and then washed with PyroMark wash buffer (Qiagen). The purified PCR product was then added to 25 μl PyroMark annealing buffer (Qiagen) containing 0.3 μM sequencing primers for specific genes (all primers and their sequences are available on request). Finally, pyrosequencing was performed using the Pyromark Q24 MD system (Qiagen) according to the manufacturer’s instructions using the PyroGold Q24™ Reagent kit (Qiagen). Data were analyzed and quantified with the PyroMark Q24 software version 2.0.6 (Qiagen).

TAS-validated somatic mutations were independently PCR-amplified using biotinylated primers. Briefly, genomic DNA (10 ng) was mixed with Q5 high-fidelity DNA polymerase, 200 μM of each dNTP, 2 mM MgCl₂, and 0.5 μM of each primer (30 μl in total). The reaction mixture was subjected to PCR amplification under the following cycling conditions: 98 °C for 30 min; then, 33 cycles starting at 98 °C for 10 s, 64 °C for 10 s, and 72°C for 10 s; followed by 72 °C for 5 min. Annealing temperature was set to 64 °C to attain higher specificity than during TAS library preparation. The PCR product (30 μl) was mixed with 1.5 μl Streptavidin Sepharose High Performance medium (GE Healthcare, Chicago, IL, USA), 40 μl PyroMark Binding Buffer (Qiagen, Hilden, Germany), and 8.5 μl Milli-Q water, and shaken for 10 min. The resulting product was washed with 70% ethanol for 5 s, 0.2 N NaOH for 5 s, and 10 mM Tris–HCl (pH 7.6) for 10 s, and was then mixed with 38.5 μl PyroMark annealing buffer (Qiagen) and 1.5 μl sequence primer (10 μM). Finally, we performed pyrosequencing of the samples thus prepared by loading them into PyroMark Q96 (Qiagen) and operating it in the allele quantification (AQ) mode, according to the manufacturer’s protocol. Primers are listed in Supplementary Table S8.

PCR was performed using 2 μl bisulfite DNA, 1 x reaction buffer with 1 mM MgCl₂, 0.8 mM deoxynucleotide triphosphates, 1 unit of FastStart Taq DNA polymerase (Roche Diagnostics, Mannheim, Germany) and 400 mM each of PCR forward primer and a 1:9 mixture of PCR reverse primer and universal biotinylated PCR primer. PCR cycling consisted of incubation at 95°C for 4 min, 50 cycles of 95°C for 30 sec, 54°C for 30 sec and 72°C for 30 sec, followed by a final extension at 72°C for 1 min. Pyrosequencing was performed using the PyroMark annealing buffer (Qiagen) and PyroMark binding buffer (Qiagen), 3 μL Streptavidin Sepharose High Performance beads (GE Healthcare, Stockholm, Sweden) and 350 mM pyrosequencing primer on the PyroMark Q24 (Qiagen) according to manufacturer’s instructions. The PCR forward, reverse and sequencing primers, and pyrosequencing dispensation order were 5′-TTT GGA AGT TAG GAT TTT GG-3′, 5′-GGG ACA CCG CTG ATC GTT TAT CAA TAA AAA AAA AAC AAC CTC AA-3′, 5′- GTT TAT TTA GGG TTG TAA TGT TTT A-3′ and CTA CGA TCT GTC AGT CGT AG respectively for HOXA5, and 5′-GGA GTA AAA TAG GTG AAA GT-3′, 5′-GCC CTT CCC CAA CCT C-3′, 5′-GGT TTT TTT TTT TTA TTA CGT ATT-3′ and GTC AGT TGG TGA respectively for WNT5A. The sequence of the universal biotinylated PCR primer was 5′-GGG ACA CCG CTG ATC GTT TA-3′.

Pyrosequencing was performed using biotinylated amplicons of the bovine PRNP gene by PCR. Briefly, the genomic DNA (10 ng) isolated from cattle was amplified by PCR using a biotinylated forward primer and a non-biotinylated reverse primer. Detailed information on the primers and PCR conditions is described in Table 1. The PCR products (25 µL) were mixed with 2 µL Streptavidin Sepharose High-Performance medium (GE Healthcare, Madison, WI, USA), 40 µL PyroMark Binding Buffer (QIAGEN, USA), and 8.5 µL high-purity autoclaved water, and centrifuged for 15 min at 14,000 rpm. The resulting products were washed with 70% ethanol for 10 s, PyroMark denaturation buffer (QIAGEN, USA) for 10 s, and PyroMark wash buffer (QIAGEN, USA) for 10 s, and then mixed with 24.2 µL PyroMark annealing buffer (QIAGEN, USA) and 0.8 µL sequencing primer (2 µM). Finally, the prepared samples were loaded into the PyroMark Q24 (QIAGEN, USA) and operated in the allele quantification (AQ) mode according to the manufacturer’s protocol.