Sequencing of Tumor Driver Genes

Tumour DNA was extracted from tumour specimens using NucleoSpin Tissue (MACHEREY–NAGEL, Düren, Germany). Regions of interest for driver genes^{3 (link),16 (link)–18 (link)} were amplified by PCR with gene-specific primers (Supplementary Table S1) and TaKaRa Ex Taq (TAKARA BIO, Shiga, Japan) (IDH1/2, H3F3A, and HIST1H3B) or AmpliTaq Gold 360 (Thermo Fisher Scientific, Waltham, MA) (TERTp) using an Applied Biosystems GeneAmp PCR System 9700 (Thermo Fisher Scientific). PCR products were processed by ExoSAP-IT (Thermo Fisher Scientific), then sequenced with sequencing primer (IDH1) or PCR forward primer as a sequencing primer (IDH2, H3F3A, HIST1H3B, TERTp) and a BigDye Terminator V1.1 Cycle Sequencing Kit (Thermo Fisher Scientific) using the ABI 3130xL Genetic Analyzer (Thermo Fisher Scientific).

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Makino Y., Arakawa Y., Yoshioka E., Shofuda T., Kawauchi T., Terada Y., Tanji M., Kanematsu D., Mineharu Y., Miyamoto S, & Kanemura Y. (2021). Prognostic stratification for IDH-wild-type lower-grade astrocytoma by Sanger sequencing and copy-number alteration analysis with MLPA. Scientific Reports, 11, 14408.

Publication 2021

NucleoSpin Tissue Ex Taq AmpliTaq Gold 360 Applied Biosystems GeneAmp PCR System 9700 ExoSAP-IT BigDye Terminator V1.1 Cycle Sequencing Kit ABI 3130xL Genetic Analyzer

Gene Genetic Gold Idh2 Primer Tissue Tumour Tumour dna

Corresponding Organization :

Other organizations : Kyoto University, Osaka National Hospital

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Variable analysis

independent variables

Primer type (gene-specific primers)
PCR enzyme (TaKaRa Ex Taq, AmpliTaq Gold 360)

dependent variables

Tumour DNA sequences (IDH1, IDH2, H3F3A, HIST1H3B, TERTp)

control variables

Tumour specimen
DNA extraction method (NucleoSpin Tissue)
PCR thermal cycler (Applied Biosystems GeneAmp PCR System 9700)
DNA sequencing method (BigDye Terminator V1.1 Cycle Sequencing Kit, ABI 3130xL Genetic Analyzer)

controls

No positive or negative controls were explicitly mentioned.

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