Big dye termination chemistry system

Genomic DNA was extracted from 1 ml of EDTA blood by using FlexiGene® DNA Kit (QIAGEN, GmbH, Hilden, Germany) following the manufacturer’s protocol.
Primers for SNPs rs738409 (PNPLA3) were designed by using Primer3 software [46 (link)]. A 668-bp fragment of PNPLA3 gene was amplified by the polymerase chain reaction (PCR) technique using primers 5′-CGA TCT AGC CCC TTT CAG TC-3′ (forward) and 5′-GCA GAT TAA GTG AAC CAG CC-3′ (reverse). PCR reaction was carried out for 30 cycles consisting of denaturation for 30 s at 94 °C, annealing for 30 s at 62 °C and extension for 1 min at 72 °C. With regard to SNPs for VDR gene, the sets of primers used for amplification and their PCR cycling conditions were obtained from previously published studies [47 (link), 48 (link)].
The PCR amplified fragments were bi-directionally sequenced using Big-Dye Termination chemistry system (Applied Biosystems, Life Technologies Corp., Foster City, CA, USA). In order to discriminate between homozygotes and heterozygotes, the sequencing chromatogram was examined by using BioEdit Sequence Alignment Editor version 7.1.3.0.

NS3, NS5A and NS5B genomic regions were partially amplified by previously described RT-Nested PCR protocols specific for subtype 1a and 1b [23 (link),24 (link),25 (link)], covering positions involved in drug resistance. PCR products were bi-directionally sequenced using the Big-Dye Termination chemistry system (Applied Biosystems, Foster City, CA, USA).
HCV genotype and subtype were confirmed in each genomic region by phylogenetic analysis. BioEdit (v.7.2.5) software [26 ] was used for sequence alignment. Phylogenetic trees were constructed using the maximum-likelihood method in MEGA (v.6.0) [27 (link)], and visualized in TreeView v.1.6.6 [28 (link)]. Nucleotide sequences were deposited in GenBank under accession numbers MH733012–MH733240 and MK215006–MK215017.