Bigdye terminator cycle sequencing ready reaction kit

Fifteen milliliters of each parent (HY40E5 and Pnsb109) and randomly selected two offspring (HYOFA5 and HYOFB6) cultures were harvested in the stationary growth phase and pelleted by centrifugation at 2,000g (4 °C, 15 min). The genomic DNA of each sample was extracted using a DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) using the manufacturer’s protocols. The ITS2 rDNA region was amplified using the forward primer; PnITSF (ACT TTC AGC GGT GGA TGT CTA) and the reverse primer; PnITSR (CTT GAT CTG AGA TCC GGA ATT) following the PCR protocols from a previous study^{42 (link)}. Amplicons were separated on 1.3% w/v agarose gel and purified using the Qiaquick PCR purification kit (Qiagen, Hilden, Germany). The PCR products were then directly sequenced with a Big DyeTM Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA). Sequences of parents and offspring strains were aligned and compared with each other using Bioedit software (v 7.1.3.0).

Mutational analysis of exons 5–9 of the TP53 gene was conducted using PCR amplification and direct sequencing (Kim et al. 2014 (link)). The primers designed to amplify the exons and flanking introns of the TP53 gene were described previously (Kim et al. 2014 (link)). Briefly, PCR was performed using an Accu-PowerTM Premix (Bioneer, Daejeon, Korea) under the following amplification conditions: 94°C for 4 min followed by 50 cycles of 94°C for 1 min, 60°C for 30 s and 72°C for 30 s and final extension at 72°C for 15 min. Purified PCR products obtained using a QIAquick Gel Extraction kit (Qiagen) were used for sequencing with a Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems). The thermal cycler conditions were as follows: 96°C for 5 min followed by 24 cycles of 96°C for 10 s, 50°C for 5 s and 60°C for 4 min and final extension at 72°C for 5 min. The sequences were analyzed using an ABI 3500Dx system (Applied Biosystems). The TP53 sequences were compared to the GenBank database sequence (accession number NC_000017.9) (Cancer Genome Atlas Network 2012 (link)). Both forward and reverse strands were sequenced to confirm the full sequence length (bp) and nucleotide alterations (Kim et al. 2013 (link)).

To verify HCV genotype and determine possible inter- and intra-patient subtype differences, HCV core amplicons (approximately 429 bp) were further analysed. A semi-nested PCR was performed with Sc2 and Ac2 as first-round primers and S7 and Ac2 as second-round primers [33 (link)] . The PCR conditions were as described for genotyping above. PCR products were purified from 2% agarose gel using QIAquick gel purification protocol (Qiagen Ltd., Germany) according to the manufacturer’s instructions. Purified amplicons were cloned directly into pCR 2.1-TOPO plasmid vector (~ 3.9 kb) and used to transform chemically competent Escherichia coli. Positive clones were detected through purification by Miniprep protocol (Qiagen Ltd.) and digestion with Eco RI. LB Agar, LB Broth Base, pCR 2.1 TOPO vector and Escherichia coli were obtained from Invitrogen, Life Technologies, Paisley, Scotland; and Eco RI was from Roche Diagnostics GmbH., Mannheim, Germany.
For each isolate, at least two clones were sequenced on both strands using BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems). Sequencing products were purified by ethanol precipitation protocol. Electrophoresis and data acquisition were done on an automated ABI PRISM 310 genetic analyser (Applied Biosystems). Consensus nucleotide sequences obtained from the isolates were used in phylogenetic analysis.

Venous blood samples of all patients were collected at diagnosis with informed consent. The healthy controls were from center of Health Examination of Nanjing Drum Hospital. Genomic DNAs of IPF patients and healthy controls were extracted and purified from circulating leukocytes by using TIANamp Blood DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China) and Genomic DNA Purification Kit (Life Technologies Corporation, Carlsbad, CA, USA). Sequencing reactions were induced by using a BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA). DNA samples were genotyped to identify SNPs associated with IPF by polymerase chain reaction (PCR). PCR conditions were available according to the requirements. Primers were designed by software PRIMER3 and were as the following: rs868903—forward, TGCAACACCAGCTCACCAT; reverse, GTCCACAGCAGCATCACT; rs1881984—forward, ATAATGAGAGTTCAGGGGA; reverse, ACAAAGCTAAAAGACAAGG; rs2736100—forward, AACATTGCTACCCTTGTCC; reverse, CTCCTCGTGAGTCTCCACA; rs2853676—forward, GTTCTCTGTGCCCTGAAGG; reverse, TGAAAGTGGCTGATGTTGA; rs35705950—forward, CCGCCCCTTTGTCTCCACT; reverse, ACTTTGCCCTCGTCCCTCC.