Abi 3130xl automatic sequencer

DNA was extracted from index patients’ peripheral leucocyte with Maxwell16 Instrument according to the manufacturer’s instructions (Promega Corp., Madison, USA). The entire coding region and intron/exon boundaries of the MEN1 gene (GenBank entry NM_130799.2) were firstly investigated by sequencing germline DNA from all patients. DNA was PCR-amplified and sequencing reactions on both strands were performed with BigDye Sequencing Reaction kit v.1.1 (Applied Biosystems, Foster City, CA) and separated on ABI 3130XL automatic sequencer (Applied Biosystems). When we did not identify any MEN1 mutations or large deletions (see below), we carried out the sequence analyses of the DNA coding regions of CDKN1B/p27^Kip1 (NM_004064.4) and AIP (NM_ 003977.3) genes. In FIHP probands, in addition to the screening of the MEN1, CDKN1B and AIP genes, we directly sequenced the entire coding region and intron/exon boundaries of the CDC73 (NM_024529.4) and CaSR (NM_000388.3) genes. In kindreds carrying MEN1 mutation, the mutational analysis of the region of interest was extended to first degree relatives of the proband, independently of the presence of MEN1-related signs and symptoms. By analyzing the DNA of both parents of a mutation-positive sporadic case we could assess the de novo origin of the mutation.

RNA extraction from cell-sorted populations (2 × 10⁵–5 × 10⁶ cells) was performed using TRIzol (Life Technologies, Catalogue#15596). cDNA was amplified using Random Primers and SuperScript III Reverse Transcriptase (Invitrogen, Catalogue#48190011 and 18080-044). Both RNA extraction and cDNA synthesis were performed following the manufacturer's instructions. To perform CDR3 spectratyping26 (link), each obtained cDNA was divided into 23 parallel PCR reactions with a common Cβ reverse primer and 23 Vβ-specific forward primers (GoTaq DNA Polymerase from Promega, Catalogue#M7801, and primers from Life Technologies, Supplementary Table 2). Run-off reactions were done using dye-labelled Cβ primer. All primer sequences can be found on Supplementary Table 2. Run-off products were run on ABI 3130XL Automatic Sequencer (Applied Biosystems) together with GeneScan 500 ROX dye Size Standard (Applied Biosystems, Catalogue#401734) and consequently separated based on their nucleotide size. Gene Mapper software (Applied Biosystems) was used to obtain nucleotide length and area of each peak.

To examine the 16S rRNA gene, genomic DNA was isolated from the cell pellet of a 1-ml overnight liquid culture of strain KK2020170^T using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) per the manufacturer’s instructions. The 16S rRNA gene was directly PCR-amplified from the template (genomic DNA) using a universal primer set for bacteria (27F and 1492R) (Lane 1991 ; Kitahara et al. 2012 (link)). The resulting PCR product, which was well separated as a single band (approximately 1500 bp) by agarose-gel electrophoresis, was excised from the gel and purified using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). The amplicon was directly sequenced using an ABI 3130XL automatic sequencer (Applied Biosystems, Waltham, USA) according to the supplier’s procedure. A similarity search of the sequenced 16S rRNA gene was performed using international nucleotide sequence databases (DDBJ/ENA/GenBank). Phylogenetic trees were built by the neighbor-joining (NJ) and maximum-likelihood (ML) algorithms (Felsenstein 1981 (link); Fitch 1971 (link); Saitou, and Nei 1987 (link)) using MEGA v.7.0 (Kumar et al. 2016 (link)). Each topology of the two phylogenetic trees was validated by 1000 random bootstrap replicates.