Abi prism 3130apparatus

We extracted DNA from 100 mg of frozen brain tissue with DNeasy Blood and Tissue
Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions.
We amplified the PrP gene (PRNP) coding sequence in a
50-μL final volume using 5 μL of extracted DNA, 1× AmpliTaq
Gold 360 PCR Buffer (Applied Biosystems, Foster City, CA, USA), 2.5 mmol/L
MgCl2, 1× 360 GC Enhancer, 200 μmol/L dNTPs, 0.25 μmol/L of
forward (5′-GCTGACACCCTCTTTATTTTGCAG-3′) and reverse
(5′-GATTAAGAAGATAATGAAAACAGGAAG-3′) primers (19 (link)), and 0.5 μL of AmpliTaq Gold
360 (Applied Biosystems), according to the following amplification protocol: 5
min at 96°C; 30 s at 96°C, 15 s at 57°C, 90 s at
72°C for 40 cycles, and 4 min at 72°C.
We purified amplicons by using an Illustra ExoProStar 1-Step clean-up kit (GE
Healthcare Life Sciences, Little Chalfont, UK). We conducted sequencing
reactions by using the BigDye Terminator v1.1 Cycle Sequencing Kit, purified
using BigDye XTerminator Purification Kit, and detected with the ABI PRISM 3130
apparatus (all Applied Biosystems). We analyzed sequences by using Seq Scape
version 2.5 (Applied Biosystems).

PCR amplification and sequencing were carried out as previously reported [41 (link)]. Briefly, primers were designed over the available sequence (GenBank accession number EU032305.1) for the amplification of the entire PRNP coding sequence (CDS). The PCR reaction included 1× Gold Buffer and 5 units of AmpliTaq Gold (Applied Biosystems, Foster City, CA, USA), 2.5 mM MgCl₂, 200 µM dNTPs, 0.25 µM of F1 (5′-CATTTATGACCTAGAATGTTTATAGCTGATGCCA-3′) and R1 (5′-TTGAATGAATATTATGTGGCCTCCTTCCAGAC-3′) primers and 50 ng of DNA. The PCR thermal profile started with an initial denaturation step at 94 °C (3 min) followed by 35 cycles of 30 s at 95 °C for DNA denaturation, 30 s for primer annealing at 57 °C and 1 min 30 s at 72 °C for primer extension, with a final extension step at 72 °C for 7 min. Sequencing reactions were performed with the primers T3 (5′-TTTACGTGGGCATATGATGC-3′) and T4 (5′-GGCTGCAGGTAGACACTCC-3′) using a BigDye Terminator Cycle sequencing kit v1.1 and an ABI PRISM 3130 apparatus (Applied Biosystems, Foster City, CA, USA). The sequences obtained have been aligned using Seq Scape v2.5.

DNA extraction, PCR amplification and sequencing were carried out as previously reported [90 (link)]. Briefly, PrP coding sequence was amplified using F1 (5’-CATTTATGACCTAGAATGTTTATAGCTGATGCCA-3’) and R1 (5’- TTGAATGAATATTATGTGGCCTCCTTCCAGAC-3’) primers, with standard PCR conditions. Sequencing reactions were carried out with primers T1 (5’-GGTCCTCATAGTCATTGCC-3’), T2 (5’-TGGTGGCTACATGCTGGG-3’), T3 (5’-TTTACGTGGGCATTTGATGC-3’) and T4 (5’-GGCTGCAGGTAGACACTCC-3’) using Big Dye Terminator Cycle sequencing Kit v1.1 (Life Technologies), subsequently purified using BigDye XTerminator Purification Kit and detected with ABI PRISM 3130 apparatus (Life Technologies). Sequences were analyzed using the software Seq Scape v2.5 (Life Technologies).