A codon 129 polymorphism in the PRIP gene

Source/Description: The polymerase chain reaction (PCR) and allele specific oligonucleotide (ASO) hybridisation were used to detect a common polymorphism within the open reading frame (ORF) of the human prion protein (PRIP) gene. This polymorphism has previously been reported as a mutation (1). The oligonucleotide sequences for amplification of the ORF of the PRIP contained EcoRI and BamHI linkers respectively and were 5'-AAGAATTCTCTGACATTCTCCTCTTCA-3' and 5'-AAGGATCCCTCAAGCTGGGA-3'. PCR mixtures (100 /J) contained 1 -2 fig genomic DNA, 250 ng of each primer and 2.5 units of Taq polymerase (Cetus) in the manufacturer's recommended buffer. Cycling conditions were: 95°C (1.5 mm), 50°C (1.5 min), 72°C (3 min) for 35 cycles. ASO sequences were 5'-CGGCTACATGCTGGG-3' (Met) and 5'-CGGCTACGTGCTGGG-3' (Val). Prehybridization (3h) and hybridization (16h) were in 6XSSPE, 0.1% BLOTTO, 0.5% SDS, 1 mM EDTA and 0.1 mg/ml denatured, sonicated, salmon sperm DNA at 35°C. Membranes were washed once at 35°C (15 min) and twice at 46°C (15 min) in 3M tetramethylammonium chloride, 50 mm Tris-HCl (pH 8.0), 2 mM EDTA and 0.1% SDS.