Download Supplementary Text Cycling-Probe Real

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Supplementary Text
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Cycling-Probe Real-Time PCR to Quantify the Percent HCV-RNA Levels of NS5A-Y93H Mutant
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Strains Relative to the Total HCV-RNA Levels
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Cycling-probe real-time PCR was performed according to a previously reported method
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[4,5]. Briefly, a set of primers and 2 types of cycling-probe mixtures were synthesized with
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6-carboxyfluorescein (FAM) and 6-carboxy-X-rhodamine (ROX) labeling for each cycling probe by
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TaKaRa Bio Inc. Two types of oligonucleosides were also synthesized by TaKaRa Bio Inc. Re-
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al-time PCR was performed using the CycleaveRCR Core Kit (TaKaRa Bio Inc.) using the primer
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set, either of the 2 cycling-probe mixtures, and cDNA derived from the RNA samples. PCR ampli-
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fication and fluorescence detection were performed using the Thermal Cycler Dice Real Time Sys-
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tem II (TaKaRa Bio Inc.). The cycle conditions for the PCR were as follows: initial denaturation at
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95C for 30 seconds, followed by 45 cycles of denaturation at 95C for 5 seconds, primer annealing
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at 55C for 10 seconds, and extension and subsequent detection of fluorescence at 72C for 25 sec-
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onds.. To obtain the calibration curves, real-time PCR was similarly performed using either of the 2
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types of oligonucleoside mixtures instead of the cDNA derived from the RNA samples. HCV strains
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with an NS5A-Y93H mutant HCV-RNA level relative to the total HCV-RNA level of 1% or more
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were diagnosed as “with mutation”. A set of primers, cycling-probe mixtures, and oligonucleoside
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and oligonucleotide mixtures used for calibration are shown in Supplementary Table.
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Direct Sequencing and Ultra-Deep Sequencing in the NS3 and NS5A Regions of HCV Strains
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A fragment with a length of 546 bases (nt50-595) corresponding to the NS3 region of HCV
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was amplified by nested PCR using primer sets. Nested PCR was performed using PrimeScript II
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High Fidelity RT-PCR Kit (TaKaRa Bio Inc.), with primer annealing at 55C for 10 seconds and
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extension at 68C for 15 seconds over 50 cycles in the first PCR study and primer annealing at
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60C for 10 seconds and extension at 68C for 15 seconds over 50 cycles in the second PCR study.
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On the other hand, a fragment with a length of 379 bases (nt16-394) corresponding to the NS5A
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region of HCV strains was amplified using primer sets and PrimeScript II High Fidelity RT-PCR
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Kit or PrimeSTAR MAX DNA Polymerase (TaKaRa Bio Inc.) with primer annealing at 55C for 10
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seconds, and extension at 68C for 15 seconds over 35 cycles. These fragments were purified using
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the QIAquick PCR Purification Kit (Qiagen K.K.) or QIAquick Gel Extraction Kit (Qiagen K.K.)
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and sequenced using the BigDye Terminator v3.1 Cycle Sequence Kit (Applied Biosystems, CA,
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US) using the internal primers, according to the manufacturer’s protocol. Direct sequencing was
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performed using a 3130 Genetic Analyzer (Applied Biosystems), and the nucleotide sequences
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thereby obtained were assembled using ATGC ver.7 (GENETYX, Tokyo, Japan). The threshold of
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nucleotide mixture detection during sequencing was more than 10% of the minor peak relative to
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the major peak. The primer sets used in the PCR procedures are shown in Supplementary Table.
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Ultra-deep sequencing was performed using MiSeq (Illumina Inc., CA, US). This tech-
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nique revealed an average coverage depth of over 1,000,000 sequence-reads per base in the targeted
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region of the genome. Paired-end sequencing with multiplexed tags was also performed. The anal-
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yses were entrusted to TaKaRa Bio Inc.
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