Hotfire tag polymerase

The study was conducted according to the Declaration of Helsinki protocol and was approved by the local ethics committee (Medical Ethical Committee of the Univ. of Oldenburg, #2018-097; MHH ethical committee, #2576-2015). The consequences of the study were explained to the patients and unaffected controls. Written informed consent for clinical diagnostic testing and research applications was obtained from all participants before collecting blood samples and skin biopsies. Genomic DNA was extracted from EDTA blood samples as previously described [54 (link)] and used for molecular genetic analysis. To verify the mutation in exon 10 of RPGR, flanking intronic sequences and exon 10 were amplified by PCR using HotFire Tag Polymerase (Solis Biodyne, Tartu, Estonia) and analyzed by Sanger sequencing. Sequencing profiles of each family member were compared to the RPGR reference sequence NM_000328.3 (Snap Gene software, GSL Biotech LLC, San Diego, CA, USA).

Total RNA was extracted and isolated from cultured fibroblasts using NucleoSpin RNA kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instruction. Using 500 ng total RNA, cDNA was generated by applying random primers and SuperScript III Reverse Transcriptase (Invitrogen). RT-PCRs were performed using HotFire Tag Polymerase (Solis Biodyne, Tartu, Estonia) to amplify a fragment from exon 9 (5′-TCGCCACGGAAAATTAGGACTTG-3′) to 11 (5′-TCTGGCTGCATGAGGTCCTGTTC-3′). PCR products were analyzed on a 2% agarose gel and verified by Sanger sequencing.