Computational protocol: A catalogue of Triticum monococcum genes encoding toxic and immunogenic peptides for celiac disease patients

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Protocol publication

[…] Two hundred nanograms of purified poly A+ RNA were transcribed in the first strand synthesis using the BD Clontech SMART™ cDNA synthesis kit (Clontech Laboratories, Mountain View, CA, USA). After second strand synthesis the resulting cDNA was SfiI digested, column fractionated and ligated directly into a modified pBluescript plasmid (Stratagene, La Jolla, CA, USA). The vector contains the sequence of pTriplEx2 from the EcoRI and the SalI restriction site including the SfiIA and the SfiIB restriction sites, which were cloned between the corresponding EcoRI and SalI restriction sites into the pBluescript polylinker. Single fractions of the column-separated cDNA digest were ligated and transformed in electro competent Escherichia coli dH10b cells (Invitrogen, Carlsbad, CA, USA).About 960 randomly selected clones were sequenced, resulting in 918 expressed sequence tags (ESTs). Sequence reactions were performed using an Applied Biosystems (Weiterstadt, Germany) ABI Prism 3730xL sequencer using BigDye terminators either directly from both ends with SK and T7 primers on the plasmid DNA or with the SK primer after amplification of the insert DNA by using the T3 and T7 primer.The sequences were processed with Applied Biosystems DNA Sequencing Analysis Software and alignments of the deduced amino acid sequences were assembled, edited and displayed using the BioEdit Sequence Alignment Editor program 7.0.9 (Hall ). […]

Pipeline specifications

Software tools Sequencing Analysis Software, BioEdit
Application Sanger sequencing
Organisms Triticum monococcum, Homo sapiens, Triticum aestivum, Hordeum vulgare, Secale cereale
Diseases Celiac Disease