Computational protocol: The status of epidermal growth factor receptor in borderline ovarian tumours

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Protocol publication

[…] PCR products were then purified using the QIAquick PCR Purification Kit (Qiagen, Crawley UK) according to manufacturer's instructions. All purified PCR products were then sequenced in both orientations by Sanger sequencing using the manufacturers' recommendations. Sequencing reactions were run on 3500Xl Genetic analyser (life technologies), and then purified using Big DyeSAM Solution and Big DyeXterminator solution (life technologies), to eliminate the excess of labelled ddNTPs. The sequence alignments were done with the BioEdit Sequence Alignment Editor and analysed using SeqScape software 2.5 (Applied Biosystems). The DNA sequence of EGFR gene was obtained from GenBank (EGFR-001, transcript ID: ENST00000275493). We compared the KRAS sequence against (KRAS-001, transcript ID: ENST00000311936), and BRAF sequence against (BRAF-001, transcript ID: ENST00000288602) as in Gene Bank. Detected mutations/polymorphisms were confirmed by at least two independent PCR amplifications and repeated sequencing reactions. […]

Pipeline specifications

Software tools BioEdit, SeqScape
Application Sanger sequencing
Diseases Carcinoma, Neoplasms, Ovarian Neoplasms
Chemicals Tyrosine