Abi 3500 xl automatic sequencer

The genomic DNA was extracted by the standard phenol–chloroform protocol from peripheral blood samples obtained from patients and their relatives. The exons and exon–intron boundaries were amplified by polymerase chain reaction (PCR) using primers designed by us (see Additional file 1). PCRs were performed in the presence of 50 ng of genomic DNA, 1× KCl buffer, 0.15 U Taq Polymerase, 200 μM each deoxynucleotide triphosphate (dNTP, Thermo Scientific), 0.5 pmol each primer and water to final volume of 10 μL. The PCR products were visualized on 1.5 % agarose gels. The amplicons produced by PCR were purified and were submitted to the bidirectional direct sequencing using the BigDye Terminator Cycle Sequencing Ready Reaction Kit version 3.1 (Applied Biosystems, Foster City, CA, USA), and were analyzed with an ABI 3500 XL automatic sequencer (Applied Biosystems^®) using standard methods.
Sequencing data were analyzed through the CodonCodeAligner version 4.1.1 software and the mutations found were compared with online data banks (Ensembl, HGMD^®, NCBI, dbSNP and LOVD^®). The novel mutation was valued by online prediction mutation software (http://www.mutationtaster.org/). In addition, 100 alleles from 50 control individuals were investigated for the novel mutation.

Eight nuclear microsatellite (nSSR) loci developed for E. horridum (EhA07, EhB09, EhC03, EhE01, EhE02, EhE11, EhG03, EhG07; Hmeljevski, Ciampi, Baldauf, Reis, & Forzza, 2013) were genotyped. For cpDNA, the transferability of plastid microsatellites (cpSSR) isolated from Vriesea gigantea Mart. ex Schult. & Schult. f. (Palma‐Silva et al., 2009) and Pitcairnia spp. (Palma‐Silva et al., 2011), as well as universal primers from Nicotiana tabacum L. (Weising & Gardner, 1999), were tested. Eighteen markers were tested, and five polymorphic loci were selected (VgCP2 and VgCP4 from V. gigantea; ccmp2, ccmp3, and ccmp6 from N. tabacum). Fluorescent‐labeled primers were used for genotyping reactions, following protocols described by Hmeljevski et al. (2013). Both nSSR and cpSSR alleles were resolved in an ABI 3500xL automatic sequencer (Applied Biosystems, Foster City, CA, USA) using GSLIZ600 as the size standard (Applied Biosystems). Locus amplification was performed individually, and the fragments were subsequently pool‐plexed for allele sizing. Allele scoring was made in GeneMapper Software 4.0 (Applied Biosystems).