Computational protocol: Steatocystoma multiplex is associated with the R94C mutation in the KRTl7 gene

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Protocol publication

[…] Ethical approval was obtained from the ethics committee of Hainan Provincial Hospital of Skin Disease (Haikou, China) and written informed consent was also obtained. The genomic DNA was extracted from peripheral blood samples of 25 individuals within the family (10 affected and 15 unaffected family members) using a QIAamp blood-tissue kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. The exon 1 coding sequence of KRT17 (GenBank Entry: NM_000422) was amplified by polymerase chain reaction (PCR) using primer pairs designed with Primer3 (http://frodo.wi.mit.edu/primer3). The primer sequences were as follows: Forward 5′-ATGGAAACAGAGGAGCA-3′ and reverse 5′-CCTGACTCAGCTTGCTGT-3′. The DNA template (100 ng) was mixed in a solution containing 1X PCR buffer, consisting of 100 mM Tris-HCl (pH 8.3) and 500 mM KCl, 1.5 mM MgCl2, 50 µM dNTPs, 10 pmol of each primer, and 2.5 units of Taq DNA polymerase (Promega Corporation, Madison, WI, USA), in a final reaction volume of 30 µl. Amplification was performed in an initial denaturation at 95ºC for 1 min, followed by 35 cycles of denaturing at 95ºC for 40 sec, annealing at 58ºC for 40 sec and extension at 72ºC for 1 min, with a final extension at 72ºC for 3 min. This resulted in a 830 bp fragment with the use of Eppendorf Mastercycler Gradient PCR (Eppendorf, Hamburg, Germany). The PCR samples contained 5% dimethyl sulfoxide to amplify the two fragments. The PCR products were purified using a Promega Wizard® SV 96 PCR Clean-up kit (Promega Corporation), according to the manufacturer's instructions, eluted in 100 µl H2O and directly sequenced using an ABI Prism 377 DNA sequencer (Applied Biosystems Life Technologies, Foster City, CA, USA). The mutation was confirmed by performing identical PCR and sequencing in 100 unrelated control individuals. Geneious version R8 software (Biomatters, Ltd., Auckland, New Zealand) was used to compare sequences. […]

Pipeline specifications

Software tools Primer3, Geneious
Application qPCR
Diseases Steatocystoma Multiplex