Applied biosystems geneamp pcr system 9700

Tumour DNA was extracted from tumour specimens with NucleoSpin® Tissue (MACHEREY-NAGEL, Düren, Germany). Regions of interest for driver genes [23 (link), 27 (link)–30 (link)] were amplified by PCR with gene-specific primers (Supplementary Table 1) and TaKaRa Ex Taq® (TAKARA BIO, Shiga, Japan) (IDH1/2, H3F3A, and HIST1H3B) or AmpliTaq Gold 360 (Thermo Fisher Scientific, Waltham, MA) (TERTp, KRAS, HRAS, and NRAS) using Applied Biosystems GeneAmp PCR System 9700 (Thermo Fisher Scientific). PCR products were purified by ExoSAP-IT (Affymetrix, Santa Clara, CA), then sequenced with sequencing primer (IDH1) or PCR forward primer as a sequencing primer (IDH2, H3F3A, HIST1H3B, TERTp, and exons 2 and 3 of KRAS, HRAS, and NRAS) and BigDye® Terminator V1.1 Cycle Sequencing Kit (Thermo Fisher Scientific) using the ABI 3130xL Genetic Analyzer (Thermo Fisher Scientific).

Two male MADD patients, one relative from the affected pedigree and one normal control from an unrelated pedigree were included. This study was performed according to the tenets of the Declaration of Helsinki for research involving human subjects. The protocol was approved by the Ministry of Science and Technology of Taiwan and the Taipei Medical University-Joint Institutional Review Board (TMU-JIRB-N201506002). Whole blood (15 mL) from the study participants was drawn and collected in EDTA-containing tubes. Genomic DNA was isolated from the blood cells using a DNA purification kit (QIAamp DNA Mini kit, Qiagen, Valencia, CA, USA). Primer pairs covering 13 coding exons and the flanking intron splice sites were prepared and used to amplify DNA segments by polymerase chain reaction (PCR) using a DNA thermal cycler (Applied Biosystems GeneAmp PCR system 9700, Thermo Fisher Scientific, Foster City, CA, USA). The PCR products were purified and mixed with a dye terminator cycle sequencing kit (Applied Biosystems) and sequenced using an auto sequencer (Applied Biosystems 3730XL DNA Analyzer, Thermo Fisher Scientific). The putative mutations were tested for segregation in the family by direct sequencing.

cDNA was synthesized using SuperScript™ IV VILO™ Master Mix (Thermo Fisher Scientific) according to a modified version of the manufacturer’s instructions. First, 2 μl of SuperScript™ IV VILO™ Master Mix was added to 8 μl of extracted RNA (100 ng total RNA) in reaction tubes. Reverse transcription was conducted by heating the reaction mixture to 25 °C for 10 min, 50 °C for 10 min, and 85 °C for 5 min, before cooling to 4 °C with the Applied Biosystems GeneAmp PCR system 9700 (Thermo Fisher Scientific). As the majority of samples were small, the minimum amount of cDNA that could be detected by PCR probes was determined in advance, as was the dilution concentration. Synthesized cDNA was diluted 10-fold with 10 mM Tris-HCl (pH 8.0) and stored at −70 °C.

Tumour DNA was extracted from tumour specimens using NucleoSpin Tissue (MACHEREY–NAGEL, Düren, Germany). Regions of interest for driver genes^{3 (link),16 (link)–18 (link)} were amplified by PCR with gene-specific primers (Supplementary Table S1) and TaKaRa Ex Taq (TAKARA BIO, Shiga, Japan) (IDH1/2, H3F3A, and HIST1H3B) or AmpliTaq Gold 360 (Thermo Fisher Scientific, Waltham, MA) (TERTp) using an Applied Biosystems GeneAmp PCR System 9700 (Thermo Fisher Scientific). PCR products were processed by ExoSAP-IT (Thermo Fisher Scientific), then sequenced with sequencing primer (IDH1) or PCR forward primer as a sequencing primer (IDH2, H3F3A, HIST1H3B, TERTp) and a BigDye Terminator V1.1 Cycle Sequencing Kit (Thermo Fisher Scientific) using the ABI 3130xL Genetic Analyzer (Thermo Fisher Scientific).

Referent samples (12 buccal swabs) from potential living relatives were collected, recorded, and preliminarily labeled by local DNA experts. Living relatives were directly contacted by a local person (Mr Jure Lauc), who was present during the collection of each sample. Collection was performed with an Internal GENOS collection kit, and dried and labeled samples were transported to the laboratory. Upon arrival, the samples were relabeled and the relevant information entered into the Chain of Custody forms. The samples were stored at -80°C until DNA extraction was performed using the QIAmp DNA Mini Kit (14 ).
PowerPlex^® ESI kit (Promega Corp.) was used for further analysis. Similar amounts of DNA were used in all PCR reactions. Amplification was carried out as described previously (12 ). The total reaction volume was 10 μL. Additionally, PowerPlex^®Y23 kit (Promega Corp.) was used to simultaneously amplify 23 Y-STR loci according to manufacturer’s recommendations (13 ). PCR amplification was carried out by the Applied Biosystems® GeneAmp® PCR System 9700 (Life Technologies) according to manufacturer’s recommendations. Electrophoresis of the amplification products was performed on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The raw data were compiled and analyzed using 310 Data Collection Software and GeneMapper^TM 3.2.