Highly sensitive EGFR mutation detection by specific amplification of mutant alleles

Nalin Leelatian; Pichpisith Boonchoo; Sitsom Wijitburaphat; Kanya Moolsuwan; Pattara Wongjaroen; Priyakorn Chinnasang; Komsan Anyamaneeratch; Ruchira Ruangchira-Urai; Naravat Poungvarin

doi:10.1016/j.yexmp.2013.12.006

Highly sensitive EGFR mutation detection by specific amplification of mutant alleles

Exp Mol Pathol. 2014 Feb;96(1):85-91. doi: 10.1016/j.yexmp.2013.12.006. Epub 2013 Dec 25.

Authors

Nalin Leelatian¹, Pichpisith Boonchoo², Sitsom Wijitburaphat³, Kanya Moolsuwan⁴, Pattara Wongjaroen⁵, Priyakorn Chinnasang⁶, Komsan Anyamaneeratch⁷, Ruchira Ruangchira-Urai⁸, Naravat Poungvarin⁹

Affiliations

¹ Clinical Molecular Pathology Laboratory, Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Electronic address: [email protected].
² Clinical Molecular Pathology Laboratory, Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Electronic address: [email protected].
³ Clinical Molecular Pathology Laboratory, Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Electronic address: [email protected].
⁴ Clinical Molecular Pathology Laboratory, Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Electronic address: [email protected].
⁵ Clinical Molecular Pathology Laboratory, Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Electronic address: [email protected].
⁶ Clinical Molecular Pathology Laboratory, Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Electronic address: [email protected].
⁷ Clinical Molecular Pathology Laboratory, Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Electronic address: [email protected].
⁸ Department of Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Electronic address: [email protected].
⁹ Clinical Molecular Pathology Laboratory, Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Electronic address: [email protected].

PMID: 24370549
DOI: 10.1016/j.yexmp.2013.12.006

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Alleles
DNA Mutational Analysis / methods*
ErbB Receptors / genetics*
Humans
Lung Neoplasms / diagnosis*
Lung Neoplasms / genetics
Mutant Proteins / genetics*
Mutation / genetics*
Polymerase Chain Reaction / methods*

Substances

Mutant Proteins
EGFR protein, human
ErbB Receptors