3130 automated sequencer

Genomic DNA was extracted from blood samples using FlexiGene DNA Kit (Qiagen, Hilden, Germany). DM1 and DM2 expansion and CLCN1 mutations were excluded in the probands of each family. PCR fragments containing all the 24 coding exons and intron–exon junctions of SCN4A were amplified (primer sequences and conditions are available upon request). The fragments were directly sequenced using the same PCR primers and Big Dye Terminator Cycle Sequencing Kit in an automated sequencer 3130 (Applied Biosystems, Foster City, USA). Sequences were aligned using SeqScape software (Applied Biosystems) and compared to sequences NG_011699 and NM_000334 from NCBI. To confirm the results obtained, amplification and sequencing were repeated and the novel variant was checked in 200 Italian controls.
Microsatellite markers analysis was performed in order to verify the hypothesis of a founder effect for the c.644T>C mutation. Families 1, 2, and 3 were genotyped using markers D17S787, D17S944, D17S949, and D17S785 from ABI PRISM Linkage Mapping Set v2.5 (panels 23 and 24; Applied Biosystems) and markers D17S1792, D17S113, D17S584, D17S789, and D17S1786 annotated in NCBI (amplification conditions are available upon request). The fragments were resolved in an automated sequencer 3130 and the results were analyzed with GeneMapper Software 5 (Applied Biosystems).

Specific TOSV detection was carried out by sequencing PCR products as described previously [24 (link)]. The PCR products were purified using the ExoSAP cleanup procedure (Amersham Biosciences, France). All nucleotide sequences were obtained using the Big Dye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and the 3130 automated sequencer (Applied Biosystems, Foster City, CA, USA). Sequences were edited and aligned using DNA Baser Sequence Aligner v3.5.4 software (Heracle BioSoft SRL, www.DnaBaser.com) to obtain optimal sequence alignment files. A BLAST analysis at the NCBI database was made to retrieve sets of homologues exhibiting high scores to the L gene of TOSV.
To build phylogenetic tree, the maximum parsimony [26 (link)] method calculating bootstrap confidence values of 100 bootstrapping trials, using the MEGA 5.2 software, was used.

The specificity of the duplex PCR was confirmed by sequencing PCR amplicons of A. marginale and A. phagocytophilum using primers M4-OvMar-F/M4-Mar-R for msp4 gene and Msp2-3 F/Msp2-3R for msp2, respectively. Thirteen randomly chosen positive PCR products were purified using the ExoSAP cleanup procedure (Amersham Biosciences, Piscataway, NJ, USA). All nucleotide sequences were obtained using the Big Dye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and the 3130 automated sequencer (Applied Biosystems). The sequences were edited and aligned using DNA Baser Sequence Aligner v3.5.4 software (Heracle BioSoft SRL, www.DnaBaser.com) to obtain optimal sequence alignment files. A BLAST analysis was made in the NCBI database to retrieve sets of homologues exhibiting high scores with the partial msp2 and msp4 gene of A. phagocytophilum and A. marginale, respectively.