Exonuclease 1 fastap

Genomic DNA was extracted from peripheral blood leukocytes using standard salting-out method. Exons 4, 5, 13, 16, 17, 24, 26, 29, 32, and 36 of CACNA1A, exons 6, 14, 15, 16, 18, 20, 23, 24, and 26 of SCNA1A, exons 9, 16, 17, 18, 19, and 22 of ATP1A2, and exons 1, 2, and 3 of KCN1K18 (all with flanking intronic sequences) were sequenced using automated, fluorescent sequencing method. Genes and the exons were chosen on the basis of the frequencies of known migraine-causing mutations (Leiden Familial Hemiplegic Migraine Variation Database). In brief, the amplicons were amplified with Master Mix Kit and Hot Start Master Mix Kit (Qiagen) using genomic DNA from each patient. Primers were designed using Primer Premier software (Additional file 1). The annealing temperature of 58 °C was applied for all primers. Exon 26 of SCN1A and exon 3 of KCNK18 were amplified in overlapping fragments. Resulting amplicons were purified with Exonuclease I/FastAP (Fermentas) and sequenced using ABI PRISM 3130 Genetic Analyzer (Applied Biosystems) with the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems).

Patients (n = 11) and their unaffected family members (n = 5) were screened for the presence of DES mutations. Genomic DNA was isolated from peripheral blood leukocytes using standard methods. Intronic primers were used to amplify exons 1–9 of DES. Amplicons were purified with Exonuclease I/FastAP (Fermentas) and sequenced on an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems) using the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems).