Ptc 100pcr

The tissues from patients with AGC were obtained via biopsy or surgery, fixed with 10% neutral buffer formalin for 24 h at room temperature, immersed in 60°C paraffin, embedded in a paraffin block and stored at 4°C. Genomic DNA was extracted from paraffin-embedded tissues of patients with AGC using the QIAamp DNA FFPE Tissue kit (Qiagen GmbH) according to the manufacturer's instructions. The polymerase chain reaction (PCR) primers for SNPs were designed using Sequenom Assay Design 3.1 software (Sequenom) and are listed in Table SII. A thermocycler (PTC-100PCR; MJ Research) and KAPA Taq HotStart DNA polymerase (Kapa Biosystems; Roche Diagnostics) were used for PCR amplification, the thermal cycling program employed was as follows: 94°C for 5 min, followed by 35 cycles of 30 sec at 94°C, then 30 sec of annealing at 60°C, 30 sec of extension at 72°C, and a final elongation step at 72°C for 10 min. The PCR products were sequenced using a 3730XL DNA Analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc.).

Genotyping of genomic DNA was performed using the predesigned TaqMan assay for MAPT rs1800547 (A/G), which defines the H1/H2 haplotypes and run on a Step One Plus Real-time PCR System (Applied Biosystems, Foster City, CA) as previously described.⁸ Twenty-seven patients with DNA available and 116 healthy controls were genotyped. All but 3 patients were of European Caucasian origin. In addition, in 7 patients with anti-IgLON5, we performed a mutational screening of MAPT exons 1, 9, 10, 11, 12, and 13, where pathogenic mutations have been previously identified in several neurodegenerative diseases including frontotemporal dementia or PSP.^{4 (link)} We performed a touchdown PCR amplification on a PTC-100 PCR (MJ Research; Watertown, MA) using 20–50 ng of total genomic DNA and using primer pairs as previously described.^{9 (link)} We used the BigDye Terminator v3.1 Cycle Sequencing Kit according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). Sequences were run on an ABI3100 automatic sequencer (Applied Biosystems, Foster City, CA) and analyzed using the DNA baser v4.36.0 software (dnabaser.com).