Full-length KIT cDNA was amplified with over 40 cycles of polymerase chain reaction (PCR) using appropriate primers (Table S1 ). The amplified full-length KIT cDNA was used as a template, and exons 8, 9, 11, 13, and 17 were analyzed using the direct sequencing method on an ABI 310 system (Applied Biosystems, Foster City, CA). In patients who were negative for KIT mutations, exons 12, 14, and 18 of the platelet-derived growth factor receptor A (PDGFRA) gene were further analyzed after PCR amplification of the full-length PDGFRA cDNA. GISTs that did not possess KIT or PDGFRA mutations were defined as wild-type GISTs. If no mutation was detected in either KIT or PDGFRA using cDNA, we also performed mutation analysis by conventional analysis using DNA6 (link),13 (link),19 (link) and confirmed whether wild-type GISTs were present.
Abi 310 system
Manufactured by Thermo Fisher Scientific
The ABI 310 system is a genetic analyzer designed for DNA sequencing and fragment analysis. It utilizes capillary electrophoresis technology to separate and detect fluorescently labeled DNA molecules. The system provides automated data collection and analysis capabilities.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using abi 310 system
1
Genetic Profiling of GIST Tumors
2
Genetic Analysis of GIST Mutations
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Using the amplified full-length KIT cDNA as a template, exons 9, 11, 13, and 17 were analyzed using the directional sequencing method on an ABI 310 system (Applied Biosystems, Foster City, CA). Sequence primer was shown in Table 1 and Fig. 2 ). 2 μl of PCR product was mixed with 4 μl of 50 times diluted primer, 1 μl of Big Dye terminator 3.1 Ready Reaction Mix (Applied Biosystems, Foster City, CA), 4 μl of Sequencing buffer and water. A total volume of 20 μl was performed sequencing reaction over 25 cycles of the following step (10 s of denaturation at 96 °C, 5 s of annealing at 50 °C and 1 min of extension at 60 °C). In GISTs which were negative for KIT mutations, exons 12, 14, and 18 of the PDGFRA gene were further analyzed after PCR amplification of full-length PDGFRA cDNA. GIST that did not possess KIT or PDGFRA mutation was defined as wild-type GIST.
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