Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue using a DNeasy Blood & Tissue Kit (Qiagen, Inc.). Prior to extraction, histological assessment was performed to ensure that the percentage of tumor in the specimens by area was >80%. Mutational analysis of KIT and PDGFRA was carried out by PCR amplification, followed by Sanger sequencing of the amplified products. Briefly, initial amplification was performed using Takara LA Taq polymerase (cat. no. RR02MA; Takara Bio, Inc.). The PCR amplification program was as follows: Denaturation at 94°C for 5 min, 45 cycles of denaturation at 94°C for 30 sec, annealing at 56°C for 45 sec, extension at 72°C for 20 sec, and finally, incubation at 72°C for 10 min. The sequencing reaction products were electrophoresed on an ABI3700 genetic analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc.). KIT exons 9, 11, 13 and 17, and PDGFRA exons 12, 14 and 18 were analyzed. The primer sequences used are shown in Table SI .
Abi 3700 genetic analyzer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI 3700 Genetic Analyzer is a high-throughput DNA sequencing instrument designed for large-scale genomic analysis. It utilizes capillary electrophoresis technology to separate and detect fluorescently labeled DNA fragments, enabling the determination of nucleotide sequences.
Automatically generated - may contain errors
Lab products found in correlation
Dneasy blood tissue kit, by Qiagen (1 mentions)
Genemapper 4, by Thermo Fisher Scientific (1 mentions)
Genemapper 5, by Thermo Fisher Scientific (1 mentions)
In fusion cloning, by Takara Bio (1 mentions)
T vector, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using abi 3700 genetic analyzer
1
Targeted Mutation Analysis of KIT and PDGFRA
2
Microsatellite Analysis of Rabbit Genetics
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Microsatellite analysis was performed on the newborn rabbits and recipient rabbits [18 (link)]. Eight microsatellite loci located on different rabbit chromosomes were first visualized with 3% agarose gel electrophoresis and further confirmed by capillary gel electrophoresis with fluorescently-labeled amplimers and laser scanning using an ABI 3700 Genetic Analyzer and GeneMapper 4.0 (Applied Biosystems, Foster City, CA, USA). All primers for the microsatellite loci are shown in Supplementary Table S4 .
3
Microsatellite Marker Haplotyping of FLCN Gene
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Microsatellite marker haplotypes analysis was performed using five microsatellite markers flanking the FLCN gene on an ABI 3700 Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA) (65 (link)). Data were analyzed using the genemapper 5.0 software (Applied Biosystems, Carlsbad, CA, USA).
4
Recombinant PvETRAMP11.2 and PvEXP1 Proteins
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Recombinant PvETRAMP11.2 (PVX_003565) and PvEXP1 (PVX_091700) proteins were designed based on the P. vivax Sal-1 strain sequence on the PlasmoDB website (www.plasmodb.org ) and amplified from the genomic DNA of P. vivax isolates from ROK. Here, we expressed and purified the PvETRAMP11.2 (amino acids [AAs], 23–74] and PvEXP1 (AAs, 23–148) proteins (Fig 1 ) lacking the SP using wheat germ cell-free (WGCF) expression [19 (link)]. Briefly, pvetramp11.2 and pvexp1 DNA fragments were amplified using the following primers: 5′-GGGCGGATATCTCGAG TTCTACAATAATGTTGTAGCAGGAAAG-3′ and 5′-GCGGTACCCGGGATCC TTATTGGATGTTGCTGCCTTT-3′, 5′-GGGCGGATATCTCGAG AATGTAAACGGGTTAGGTGCTG-3′ and 5′-GCGGTACCCGGGATCC TCATGACGTTGATTCGGTG-3′. The underlined primer sequence indicates it is homologous to the vector sequence. They were then cloned into the pEU-E01-His-TEV-MCS vector (CellFree Sciences, Matsuyama, Japan) by In-Fusion Cloning (Clontech, Palo Alto, CA, USA) and the cloned inserts were sequenced using an ABI 3700 Genetic Analyzer (Applied Biosystems, Inc., Foster City, CA, USA) by Genotech (Daejon, Korea). These proteins were expressed using a WGCF system and purified using a Ni-Sepharose column as described [20 (link)].
5
Designing Primers for Crab HSP90 Gene
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The specific primers of C. japonica HSP90 gene designed using consensus comparison of Brachyura sequences. Multiple sequence alignments were conducted using ClustalW in Mega 4.0. Specific primers of C. japonica HSP90 were designed from orthologue sequences of Chinese mitten crab (Eriocheir sinensis, GenBank accession No. ADE60732), swimming crab (Portunus trituberculatus, GenBank accession No. ACQ90225) and green mud crab (Scylla paramamosain GenBank accession No. ACN54679). Information of primer sequences was shown in Table 1 . The size of polymerase chain reaction (PCR) products were 442 bp for the HSP90 gene and 233 bp for the beta-actin gene. The condition of PCR mixture with a total volume of 50 µL contained 1×Taq polymerase buffer, 200 µM dNTP, 5 units of Taq polymerase, primers at concentrations of 10 µM and 0.5 M betaine. The PCR conditions were finally optimized as this protocol: one cycle of 94℃ for 5 minutes, 40 cycles of 94℃ for 30 seconds, 55℃ for 40 seconds, 72℃ for 1 minute and one cycle of 72℃ for 7 minutes. The amplified PCR products were cloned in the T-vector (Invitrogen, Inc., Carlsbad, CA, USA) and sequencing with an ABI 3700 genetic analyzer (Applied Biosystems, Foster City, CA, USA).
6
Germline Screening of Pathogenic Mutations
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All TP53 and other pathogenic and likely pathogenic mutations with variant allele frequencies above 30% were checked for the presence of their germline counterparts by Sanger sequencing of available peripheral blood. We directly sequenced the genomic DNA using a BigDye™ Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) and an ABI 3700 Genetic Analyzer (Applied Biosystems).
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