PCR amplification of FOXL2 and TERT promoter hotspot loci was performed using the AmpliTaq Gold 360 Master Mix kit (Life Technologies, ThermoFisher Scientific) using previously described primers [22 (link),23 (link)]. PCR fragments were cleaned using ExoSAP It (ThermoFisher Scientific) and Sanger sequenced as previously described [22 (link)].
Amplitaq gold 360 master mix kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The AmpliTaq Gold 360 Master Mix kit is a ready-to-use solution for performing PCR amplification. It contains all the necessary components, including a thermostable DNA polymerase, buffer, and dNTPs, to facilitate efficient and reliable DNA amplification.
Automatically generated - may contain errors
Lab products found in correlation
Exosap it, by Thermo Fisher Scientific (3 mentions)
Dnaeasy blood and tissue kit, by Qiagen (1 mentions)
Simpliamp thermal cycler, by Thermo Fisher Scientific (1 mentions)
Abi bigdye terminator chemistry v3, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Chromas 2, by Technelysium (1 mentions)
3130xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
Qiaamp dna micro kit, by Qiagen (1 mentions)
Type it hrm pcr kit, by Qiagen (1 mentions)
Life touch thermal cycler, by Bioer (1 mentions)
Bigdye terminator cycle sequencing kit v3, by Thermo Fisher Scientific (1 mentions)
Abi 3130 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
100 bp dna ladder, by Thermo Fisher Scientific (1 mentions)
Proflex pcr system, by Thermo Fisher Scientific (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
8 protocols using amplitaq gold 360 master mix kit
1
Sanger Sequencing of FOXL2 and TERT
2
Profiling HRAS Mutations in Adnexal Tumors
3
Targeted Mutational Profiling in Metastatic Breast Cancer
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We conducted the assessment of TERT promoter hotspot loci, TP53 (exons 2 to 11), PIK3CA (exons 9 and 20), HRAS (exon 3), and BRAF (exons 11 and 15) hotspot mutations in 16 MBCs with insufficient DNA yield by Sanger sequencing. In addition, as TERT promoter region is usually poorly covered by exome sequencing, TERT promoter hotspot mutations were assessed by Sanger sequencing in the 27 MBCs subjected to WES. PCR amplification of the selected genes was performed using the AmpliTaq Gold 360 Master Mix kit (Life Technologies, ThermoFisher Scientific) using previously described primers16 (link),24 (link),60 (link) (Supplementary Table S4 ). PCR fragments were cleaned using ExoSAP It (ThermoFisher Scientific) and Sanger sequenced as previously described15 (link),16 (link).
4
Sanger Sequencing of FOXL2 and TERT
5
Identification of TERT Promoter Hotspot Mutations
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For the identification of hotspot somatic mutations in TERT promoter, primer sets that amplify the hotspot sites of the TERT promoter were designed as previously described22 (link) and are available in our previously published study23 (link). PCR amplification was performed from 100 ng of genomic DNA using the AmpliTaq Gold 360 Master Mix Kit (Life Technologies) on a SimpliAmp Thermal Cycler (ThermoFisher) as previously described23 (link). Sequencing was performed using purified PCR fragments (QIAquick PCR Purification Kit, Qiagen) on an ABI 3730 capillary sequencer using the ABI BigDye Terminator chemistry (v3.1, Life Technologies). Sequences of the forward and reverse strands were analyzed using 4Peaks (https://nucleobytes.com/4peaks/ ). All analyses were performed in triplicate.
RNA extraction from FFPE tissues was performed using RecoverAll Total Nucleic Acid Kit for FFPE (ThermoFisher) according to manufacturer’s guidelines. Quantitative RT-PCR analysis was performed using SYBR Green. GAPDH was used as housekeeping genes for normalization. mRNA fold expression change was calculated by the 2-ΔΔCT method as previously described24 (link). The following Primers set were used: GAPDH Foward 5′ -AGGTGAAGGTCGGAGTCAACG-3′ and Reverse 5′ -TGGAAGATGGTGATGGGATTT-3′ and TERT25 (link) Foward 5′ -GCCGATTGTGAACATGGACTACG-3′ Reverse 5′ -GCTCGTAGT TGAGCACGCTGAA-3′.
RNA extraction from FFPE tissues was performed using RecoverAll Total Nucleic Acid Kit for FFPE (ThermoFisher) according to manufacturer’s guidelines. Quantitative RT-PCR analysis was performed using SYBR Green. GAPDH was used as housekeeping genes for normalization. mRNA fold expression change was calculated by the 2-ΔΔCT method as previously described24 (link). The following Primers set were used: GAPDH Foward 5′ -AGGTGAAGGTCGGAGTCAACG-3′ and Reverse 5′ -TGGAAGATGGTGATGGGATTT-3′ and TERT25 (link) Foward 5′ -GCCGATTGTGAACATGGACTACG-3′ Reverse 5′ -GCTCGTAGT TGAGCACGCTGAA-3′.
6
Genomic DNA Extraction and Genotyping
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Genomic DNA was extracted from 300 μl of throat swab samples (for ILI cases and SARI cases) or 100 μl of anticoagulant blood(for healthy controls) using QIAamp DNA Micro kit (Qiagen) following the manufacturer's instruction. Extracted DNA integrity was checked by NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, U.S.A.), then kept at −40°C before testing. Genotype was achieved using Type-it HRM PCR Kit (Qiagen) with LightCycler 480 System (Roche, Zurich, Swiss) following the manufacturer's instruction. The High Resolution Melting (HRM) was carried out with the pair of primers 5′-GGAAACTGTTGAGAAACCGAA-3′ and 5′- CATACGCACCTTCACGGAGT-3′ (Zhang et al., 2013 (link)). In order to confirm the results of genotypes, 10% of the samples were randomly selected and sequenced. PCR was performed using AmpliTaq Gold 360 Master Mix Kit (Applied Biosystems) and the primers as described above. Then the PCR products were sequenced on 3130xl genetic analyzer (Applied Biosystems). The sequencing results were analyzed by Chromas 2.6 software (Technelysium, Brisbane, Australia).
7
Genomic DNA Isolation and Mitochondrial Sequencing
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We used the conventional phenol-chloroform method (Sambrook and Russell, 2001) to isolate the genomic DNA from liver or muscle tissues preserved in ca. 100% ethanol. Amplification was performed by PCR using an automated thermal cycler (Life Touch thermal cycler; Bioer Technology, Hangzhou, China). An AmpliTaq Gold 360 Master Mix kit (Applied Biosystems, Foster City, CA, USA) was used for PCR amplification. Aliquots of 0.1-0.2 μg of template DNA were added to a total volume of 20 μL of PCR mixture. The primers were M15997 and H16401 originally designed by Hirota et al. (2004) and similarly adopted by Sato et al. (2014) . The PCR conditions were as follows: initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 10 min. We also conducted PCR with a negative control, and confirmed no amplification on agarose gel electrophoresis. Sequencing of the PCR products was carried out using a Big-Dye Terminator Cycle Sequencing kit v3.1 (Thermo Fisher Scientific, Tokyo, Japan), followed by analyses on an ABI3130 genetic analyzer (Thermo Fisher Scientific).
8
Molecular Detection of EBV and β-Globin
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DNA was extracted from tonsillar tissue (DNeasy Blood and Tissue kit, QIAGEN) and stored at -20°C until used. DNA samples (100ng) were subjected to PCR using Taq DNA polymerase (AmpliTaqGold 360 Master mix Kit, Applied Biosystems), oligonucleotides GH20 (GAAGA-GCCAA-GGACA-GGTAC) and GH21 (GGAAA-ATAGA-CCAAT-AGGCA-G) to amplify β-globin gene, EBER-2S (CCCTA-GTGGT-TTCGG-ACACA) and EBER-2AS1 (ACTTG-CAAAT-GCTCT-AGGCG) to amplify the EBER-2 region of the EBV-genome. The PCR was performed in a ProFlex PCR System (Applied Biosystem). The standard cycle procedure was a 5-minute denaturation at 95°C followed by 35 cycles of 30-seconds of denaturation at 95°C, 1-minute of annealing at 60°C for EBER-2, 30-seconds at 55°C for β-globin and a 2minute extension at 72°C. Cycling was followed by a 7minute extension at 72°C. Visualization of gene amplification was done using an ethidium bromide stained 2% agarose gel in which 12μl of each PCR product was separated by electrophoresis; a 100bp DNA ladder was used as the parameter (Thermo Fisher Scientific). Image of the stained gels was captured using the BioDoc-It Imaging Systems (UVP,Inc).
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