title: Highly sensitive EGFR mutation detection by specific amplification of mutant alleles.
abstract: Mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene predict benefit from tyrosine kinase inhibitors in patients suffering from non-small-cell lung cancer. In this study, we developed a fast, simple, cost-effective and highly sensitive assay for detection of five clinically important EGFR mutations in exon 19 (2235_2249del and 2236_2250del), exon 20 (C2369T) and exon 21 (T2573G and c.2573_2574 TG > GT). We designed EGFR mutation detection assays by combining allele-specific PCR amplification with the detection of SYBR Green I fluorescence, and optimized PCR conditions to specifically amplify mutant alleles. These one-step assays were able to detect the mutations at levels as low as 1.5 mutant copies in a DNA sample. Commercially available probe-based allele-specific PCR exhibited relatively poor performance when detecting very low copies of mutated DNA, especially in exon 19 and 20. Our assays offered dramatically less reagent cost than that of the commercial kit and generated results in less than 90 min after DNA extraction. These protocols can also be applied to conventional thermal cyclers followed by gel electrophoresis detection.
authors.affiliation: Clinical Molecular Pathology Laboratory, Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Electronic address: n.leelatian@gmail.com.
authors.forename: Nalin
authors.initials: N
authors.lastname: Leelatian
authors.affiliation: Clinical Molecular Pathology Laboratory, Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Electronic address: pichpisithb@gmail.com.
authors.forename: Pichpisith
authors.initials: P
authors.lastname: Boonchoo
authors.affiliation: Clinical Molecular Pathology Laboratory, Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Electronic address: sitsom@gmail.com.
authors.forename: Sitsom
authors.initials: S
authors.lastname: Wijitburaphat
authors.affiliation: Clinical Molecular Pathology Laboratory, Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Electronic address: mytoey7@gmail.com.
authors.forename: Kanya
authors.initials: K
authors.lastname: Moolsuwan
authors.affiliation: Clinical Molecular Pathology Laboratory, Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Electronic address: pattara_o@yahoo.com.
authors.forename: Pattara
authors.initials: P
authors.lastname: Wongjaroen
authors.affiliation: Clinical Molecular Pathology Laboratory, Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Electronic address: priyakorn_c@hotmail.com.
authors.forename: Priyakorn
authors.initials: P
authors.lastname: Chinnasang
authors.affiliation: Clinical Molecular Pathology Laboratory, Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Electronic address: new42531@hotmail.com.
authors.forename: Komsan
authors.initials: K
authors.lastname: Anyamaneeratch
authors.affiliation: Department of Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Electronic address: rchruchi@gmail.com.
authors.forename: Ruchira
authors.initials: R
authors.lastname: Ruangchira-Urai
authors.affiliation: Clinical Molecular Pathology Laboratory, Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Electronic address: naravat@gmail.com.
authors.forename: Naravat
authors.initials: N
authors.lastname: Poungvarin
mesh_terms: D000483:Alleles; D004252:DNA Mutational Analysis / Q000379:methods*; D066246:ErbB Receptors / Q000235:genetics*; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000235:genetics; D050505:Mutant Proteins / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D050505:Mutant Proteins; C512478:EGFR protein, human; D066246:ErbB Receptors