title: Snapback primer mediated clamping PCR for detection of EGFR and KRAS mutations in NSCLC patients by high resolution melting analysis.
abstract: Assays for detecting somatic mutations are requested with higher sensitivity and more convenience. Here, we describe snapback primer mediated allele clamping enrichment polymerase chain reaction (SPACE-PCR), a novel form of PCR that amplifies minority alleles selectively from mixtures. We replaced regular PCR with SPACE-PCR before sequencing or genotyping assays to improve mutation detection sensitivity by up to 100-fold in detecting EGFR and KRAS somatic mutations. Combined SPACE-PCR with analysis of snapback primer by high resolution melting (SPACE-HRM), the high sensitive system that enables a closed-tube detection of mutations after isolating mutants has been established, as low as 1/10(5)-1/1000 mutant samples can be diagnosed. And finally, in a double-blind experiment of 150 cases of non-small-cell lung cancer (NSCLC) patients, compared with direct DNA sequencing and ADX-EGFR/KRAS mutation detection kit, up to 25% of the PCR-direct sequencing negative cases turned out to be positive in SPACE-HRM mutation tests; the specificity is 100%. Results demonstrated that the SPACE-HRM system we set up is a high sensitive assay that can be used for EGFR and KRAS allele enrichment and reliable detection. We anticipate that the method will be employed in multiple applications in the clinic, including diagnosis, scanner recurrence monitoring, and treatment management.
authors.affiliation: State Key Laboratory of Genetic Engineering, Fudan-VARI Genetic Epidemiology Center and MOE Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, China.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Sun
authors.affiliation: Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zheng Min Road, Shanghai 200433, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Department of Thoracic and Cardiovascular Surgery, Second Military Medical University, Changhai Hospital, Shanghai 200433, China.
authors.forename: Lixin
authors.initials: L
authors.lastname: Yang
authors.affiliation: Department of Central Laboratory, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zheng Min Road, Shanghai 200433, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Su
authors.affiliation: Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zheng Min Road, Shanghai 200433, China.
authors.forename: Gening
authors.initials: G
authors.lastname: Jiang
authors.affiliation: Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zheng Min Road, Shanghai 200433, China.
authors.forename: Ke
authors.initials: K
authors.lastname: Fei
authors.affiliation: State Key Laboratory of Genetic Engineering, Fudan-VARI Genetic Epidemiology Center and MOE Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, China.
authors.forename: Daru
authors.initials: D
authors.lastname: Lu
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics / Q000473:pathology; D004252:DNA Mutational Analysis; D066246:ErbB Receptors / Q000235:genetics*; D006801:Humans; D009154:Mutation; D009364:Neoplasm Recurrence, Local / Q000235:genetics / Q000473:pathology; D009691:Nucleic Acid Denaturation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D066246:ErbB Receptors; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins