Abstract
Epidermal growth factor receptor (EGFR) mutations are associated with response of tyrosine kinase inhibitors (TKIs) for patients with advanced non-small cell lung cancer (NSCLC). However, the existing methods for detection of samples having rare mutations(i.e. ~0.01%) have limits in terms of specificity, time consumption or cost. In the current study, novel wild-type blocking (WTB) oligonucleotides modified with phosphorothioate or inverted dT at the 5′-termini were designed to precisely detect 11 common deletion mutations in exon 19 of EGFR gene (E19del) using a WTB-PCR assay. And internal competitive leptin amplifications were further applied to enhance the specificity of the WTB-PCR system. Our results showed that WTB-PCR could completely block amplification of wild-type EGFR when 200 ng of DNA was used as template. Furthermore, the current WTB-PCR assay facilitated the detection of E19del mutations with a selectivity of 0.01% and sensitivity as low as a single copy. And, the results showed that the current WTB-PCR system exceeded detection limits afforded by the ARMS-PCR assay. In conclusion, the current WTB-PCR strategy represents a simple and cost-effective method to precisely detect various low-abundance deletion mutations.
Highlights
Lung cancer is the leading cause of cancer mortality worldwide, accounting for one third of all cancer-related deaths[1]
The potential advantage of this approach was that the mutant-gene specific TaqMan hydrolysis probes (MST) probes labeled with various fluorescent reporters could be used to precisely distinguish certain mutations located in the complementary region of wild-type blocking (WTB) oligonucleotides
To enable locked nucleic acids (LNA)/DNA chimeras to be used as WTBs, two novel types of WTB oligonucleotide were developed as part of this study
Summary
Lung cancer is the leading cause of cancer mortality worldwide, accounting for one third of all cancer-related deaths[1]. There are various methods available to analyze EGFR mutations; these methods include pyrosequencing, Sanger sequencing, amplification refractory mutation system (ARMS-PCR), allele-specific hydrolysis or dual hybridization probes, PCR restriction fragment length polymorphism (PCR-RFLP), high-resolution melting analysis (HRMA), generation sequencing (NGS), wild-type blocking PCR (WTB-PCR), and droplet digital PCR (dPCR)[11,12,13,14,15,16] Most of these methods, apart from more recently developed methods including WTB-PCR and dPCR, exhibit limitations in the detection of EGFR mutations[11,12,13,14,15,16]. Because the proportion of mutant gene of the afore-mentioned 6 positive samples exceeded the detection limits of the ARMS-PCR system
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